Partial replacement of fishmeal with Chaetomorpha algae improves feed utilization, survival, biochemical composition, and fatty acids profile of farmed shrimp Penaeus monodon post larvae

Shrimp are economically important candidates for aquaculture in Tanzania; however, their farming is constrained by reliance on costly feeds, attributed to the use of fishmeal as the main ingredient. This study determined the efficacy of green algae Chaetomorpha sp. on survival, feed utilization, biochemical composition, and fatty acids profile of the cultured Penaeus monodon as a potential candidate for replacing fishmeal. Four experimental feeds, F0, F1, F2, and F3, denoting fishmeal replacement levels of 0, 10, 20, and 30%, respectively, were made. Post-larvae weighing 0.49 ± 0.06 g were stocked at 15 individuals/m3 in 2 m3 hapa nets in three replicates and reared for 45 days. The F2 treatment showed a significantly higher (p < 0.05) survival rate than the other treatments. F2 and F3 improved growth significantly with F3 exhibiting the highest weight gain (8.02 ± 0.26 g) compared to the other treatments. The biochemical composition varied significantly (p<0.05) across treatment levels. The F2 treatment demonstrated the highest muscle somatic index (MSI) of 71.77 ± 2.32%. The F3 treatment exhibited significantly higher (p<0.05) crude protein (CP) than the other treatments. Crude fat (CF) in F0 and F2 treatments were significantly higher (p<0.05) than the other replacement levels. The F0 and F1 treatments exhibited higher proportions of saturated fatty acids (SFA) than the other treatments. In contrast, F2 and F3 treatments had high levels of polyunsaturated fatty acids (PUFA). The replacement of Chaetomorpha algae at 20 to 30% levels in feeds improved feed utilization, biochemical composition, and fatty acids profile of the farmed Penaeus monodon. However, the replacement of Chaetomorpha algae at 30% resulted in high saturated fatty acids but lowest survival rates. Thus, at a 20% replacement level, Chaetomorpha algae are suitable for partial replacement of fishmeal in P. monodon feeds.


Introduction
With suitable land and vast water resources, Tanzania has the highest potential for aquaculture in Africa. However, aquaculture in Tanzania is typically a subsistence activity practiced mostly by small-scale farmers (Chenyambuga et al. 2012). Shrimp farming in the country started in early 1989 (Ngoile 2012); however, as of now, there are only a few extensive aquaculture systems along the coast and one farm that engages in semi-intensive shrimp farming on Mafia island (MLF 2018), an indication of local communities' failure to adopt commercial farming. The failure may have partly been contributed by the high operating costs associated with shrimp farming, specifically related to those associated with the production of high-quality shrimp feeds due to costs associated with fishmeal − the primary source of protein, and the technical procedures for shrimp feed production. Currently, shrimp farming in the country is entirely dependent on imported feeds, which are generally expensive and challenging for most local farmers to access.
Protein is the key building block (FAO 2003) and the most expensive component of feeds (Shiau 1998). The commonly used source of protein in shrimp feeds is fishmeal (Smith et al. 2005). However, the decline in fish stocks (Akiyama et al. 1995), irregular availability, contribution to fisheries deterioration, the potential for adulteration, biological pathogens (Fox et al. 2004), high costs (Hardy 2006), and limited and unpredictable harvesting (Nagappan et al. 2021) have steered up research for its replacement. Consequently, several studies (e.g. Nasmia et al. 2022;Yıldırım et al. 2014) have been conducted to partially or completely replace fishmeal as the main protein source in fish feeds to reduce costs and improve feed utilization efficiency in farmed shrimps.
Green algae have been identified as a promising candidate for replacing fishmeal in shrimp feed formulations (Hanel et al. 2007;Ju et al. 2009;Nasmia et al. 2022;Putra et al. 2019;Cruz-Suárez et al. 2008), but research on the subject is limited. Remarkably, the use of Chaetomorpha algae on shrimp feeds formulation has not yet been reported.
Chaetomorpha algae are abundant along Tanzanian coastline and are rich in nutrients, and are commonly harvested by locals during low tide for animal feed formulation. Generally, macroalgae are rich in non-starch polysaccharides, vitamins, minerals (Wong and Cheung 2000), proteins and lipids (Da Silva and Barbosa 2008), and bioactive substances (Khan and Satam 2003). Bioactive substances in algae include carotenoids, alkaloids, polyphenols, phycocyanins, terpenes, and several enzymes (Maharana et al. 2015). According to Gokulakrishnan et al. (2015) and Kumar et al. (2021), algae are an excellent source of dietary protein.
Protein content in macroalgae varies from 10 to 47% of the dry weight across species, with red algae being superior to others (Cruz-Suárez et al. 2008;Setthamongkol et al. 2015). The protein content in green algae ranges from 9 to 26% in dry weight (Fleurence et al. 2018), while in Chaetomorpha algae it has been reported to be about 20.4% (Tsutsui et al. 2015). The species has also been reported to exhibit antioxidant properties (Gazali et al. 2019), making it a suitable candidate for promoting growth. These facts make Chaetomorpha algae a potential candidate for fishmeal replacement trials. Co-culture trials of Chaetomorpha algae with P. monodon have been reported to enhance growth performance and reduce feed conversion ratio (Tsutsui et al. 2015).
The current study investigated the efficacy of partial replacement of the costly fishmeal with cheap green macroalgae Chaetomorpha in Penaeus monodon rearing. A replacement level of up to 30% was chosen to maintain adequate protein levels in the formulated feeds; beyond this level, protein levels would be low, resulting in poor performance. The effect of the feeds that were formulated by incorporating Chaetomorpha algae at varying levels on P. monodon post larvae was determined by assessing survival, feed utilization, biochemical composition, and fatty acids profile. Furthermore, the most suitable replacement level was assessed to enable farmers to prepare feeds for their farming systems. The generated information is useful to the shrimp farmers globally in formulating low-cost feeds and hence increasing profit from the shrimp farming business.

Ethical statement
This study was carried out in accordance with the Tanzanian laws and guidelines of experimental animals' care. Shrimps were anaesthetized by immersing them in chilled water of 5 °C to reduce pain during death.

Experimental design
This experiment was carried out for 45 days in hapa nets (1.2 mm mesh size) measuring 2 m 3 with a stocking density of 15 individuals per meter cubed, making a total of 30 individuals per hapa. The hapa nets were installed in triplicates in an earthen pond measuring 4500 m 2 . Before stocking, the ponds were treated with lime at 30 g m −2 all at once and left to dry for 7 days, and filled with water that stayed for 5 days before stocking. On the day of stocking, water quality measurements (pH, salinity, temperature) were performed to control differences between acclimatization tanks and the culture ponds.

Preparation of the experimental feed
Several kilograms (wet weight) of the test ingredient, the green macroalgae Chaetomorpha sp., were collected from the shore near Pangani River Estuary, where several algae, including Chaetomorpha sp., proliferate during the dry season. The collected algae were sorted and washed to remove the unwanted materials, including other plants. The clean Chaetomorpha algae were dried, ground into powder, and analyzed for proximate composition, and was as follows: protein 235.2, carbohydrate 547.8, fat 109.9, ash 73.2, and moisture content of 39.1g/kg on a wet basis. All other ingredients including vitamins and mineral premix used in this experiment were purchased from animal feed vendors from Pangani and Dar es Salaam. Except for the oil, all other ingredients including the Chaetomorpha algae, the silver cyprinid (Rastrineobola argentea), rice husks, wheat flour, and shrimp heads were dried in an oven at 40 °C and mechanically ground into powder using a grinding machine. During the formulation, the vitamins and mineral premix were also added.
Four experimental feeds (F0 (control), F1, F2 and F3) with fishmeal replacement levels of 0, 10, 20, and 30%, respectively, were formulated using the Pearson Square formula of protein estimation, with the estimated protein differences based on the level of replacement as shown in Table 1. The ingredients in powder form were mixed with water in a food processor to form a dough. The dough was cooked for 10 min by steaming. Some of the cooked dough was made into crumbles that were suitable for post larvae feeding. The remaining dough was formed into pellets using a mechanical pelleting machine (size range: 0.1 mm, 0.3 mm, and 0.5 mm) and fed to the subsequent life stages of the shrimp. The produced feeds were oven dried at 90 °C for 1 min, reducing the moisture to around 18%, and then dried further at 60 °C to reduce the moisture to about 8%. The dried pellets were kept at room temperature ready for the feeding experiments. The formulated feeds were checked for proximate composition on dry basis through chemical analysis at Sokoine University of Agriculture (SUA) Food Science Laboratory, and the respective feed proximate compositions are shown in Table 1. The formulation was maintained throughout the experiment.

Source of Penaeus monodon post larvae
The P. monodon post larvae were collected from the wild between October and November 2021, which is the peak breeding season in Pangani Estuary and Tanga coastal waters. Soon after collection, the larvae were stored in 75-l plastic buckets and supplied with oxygen using a portable aerator. The collected post larvae were transported to a local fish farmer hatchery in Pangani District located on the northern coast of Tanzania. At the hatchery, Table 1 Composition of experimental diets c Vitamin premix (per 100 g premix): thiamin hydrochloride, 0.15 g; retinyl acetate, 0.043 g; capantothenate, 0.3 g; riboflavin, 0.0625 g; niacin, 0.3 g; ascorbic acid, 0.5 g; biotin, 0.005 g; pyridoxine hydrochloride, 0.225 g; para-aminobenzoic acid, 0.1 g; folic acid, 0.025 g; α-tocopherol acetate, 0.5 g; cholecalciferol, 0.0075 g; menadione, 0.05 g; inositol, 1 g d Mineral premix (per 100 g premix): NaH 2 PO 4 , 10.0 g; CaC0 3 , 10.5 g; KH 2 PO 4 , 21.5 g; Ca(H 2 PO 4 ) 2 , 26.5 g; KCl, 2.8 g; AlCl 3 .6H 2 O, 0.024 g; MgSO 4 .7H 2 O, 10.0 g; MnSO 4 6H 2 O, 0.143 g; KI, 0.023 g; ZnSO 4 7H 2 O, 0.476 g; CuCl 2 2H 2 O, 0.015 g; CoCl 2 .6H 2 O, 0.14 g; Calcium lactate, 16.50 g; Fe-citrate,1 g the P. monodon post larvae were acclimatized for 7 days in 4 m 3 concrete tanks filled with brackish water from the Pangani River Estuary. To block out the sun's rays, the tanks were covered with a hard translucent plastic. During acclimatization, the water was fully aerated, and the behavior of the post larvae was observed, and the dead post larvae were removed from the system. Prior to stocking, post larvae were sorted and their weight and length measured using a portable digital scale and a digital vernier caliper, respectively.

Feeding experiments and data collection
Post larvae weighing 0.49 ± 0.06 g on average were stocked in 2 m 3 hapa nets at a stocking density of 15 individuals per meter cubed. They were fed by placing the feeds on the feeding trays. Feeds were administered in descending order, from five times a day in the first week to two times a day in the fourth week, at a daily feeding ratio of 12% to 6% biomass, respectively. During the experiment, 20 individuals were randomly collected from each hapa, every 15 days, and their weight and length were recorded. The sampled shrimps were kept in water before measurements, and all measurements were performed in a wet condition. After taking measurements, the shrimps were immediately returned to their respective hapas.

Determination of growth performance and biochemical composition
At the end of the experiment, ten individuals were obtained at random from each experiment unit, and the weight of each individual was recorded. The individuals were then frozen for 48 h to facilitate the peeling process, which was followed by the removal of the exoskeleton. The obtained flesh was stored at − 36 °C prior to biochemical composition analysis. Each biochemical value was determined in three replicates and expressed as % wet weight, based on the following: protein (Latimer 2016), lipid (Pombal et al. 2017), total ash (AOAC 2000), fiber (AOAC 2005), fatty acids (Kang and Wang 2005; Kokotou 2020), carbohydrates (Soga 2000), and dry mater and moisture (Jain and Singh 2000). Minerals including calcium (Ca) and phosphorus (P) were analyzed following the method by AOAC (1995). Caloric contents of experimental feeds were determined by multiplying conversion factors of 4.15, 9.4, and 5.65 for carbohydrate, lipid, and protein, respectively, as described by Phillips (1969) and expressed as kcal/g on wet weight basis. Feed utilization parameters, including feed conversion ratio (FCR), weight gain (W), and muscle somatic index (MSI), were analyzed using methods described by Kokotou (2020) and Sarlin and Philip (2016) as follows:

Statistical analysis
Prior to statistical tests, data were checked for normality using the Shapiro test, followed by checking for homogeneity of variance using the Levene's test. The experiment was randomized in design with set of three replicates for each of the experimental diets and control diet. Data were statistically analyzed using one-way ANOVA followed by Duncan's multiple range test (DMRT) and was carried out for the feed utilization, survival rate, biochemical composition, and fatty acids within and across experimental diets. All statistical inferences were performed in R version 4.1.0 and based on a significant level of α = 0.05.

Survival rate and feed utilization
The results on survival rate (SR) and feed utilization are shown in Fig. 1a-e. Both survival and feed utilization were affected by replacement levels. The mean survival rate of shrimps fed with the F2 treatment (92.59 ± 7.14) was significantly higher (p < 0.05, Fig. 1a) than the other treatments. Shrimps fed with F3 treatment exhibited the lowest SR (89.62 ± 10.50). On the contrary, the highest weight gain (8.02 ± 0.26 g) was observed in shrimps fed with F3 treatment, being slightly similar to that of shrimps fed with F2 treatment (7.80 ± 0.49 g), but differing significantly (p < 0.05, Fig. 1b) to shrimps fed with F0 (5.78 ± 0.29 g) and F1 (5.72 ± 0.43 g) treatments. There was no direct correlation between SR and biomass across treatments. The highest biomass was observed in shrimps fed with F3, while the highest survival was observed in shrimps fed with F2.
Crude fat (CF) levels were significantly higher (p < 0.05) in shrimp fed with low treatment levels (F0 and F1) compared to those fed with high treatment levels (F2 and F3). The highest CF (3.81 ± 0.43%) was recorded in shrimps fed with F0 treatment, while the lowest (2.20 ± 0.37%) was recorded in shrimp fed with F3 treatment. Similarly, shrimps fed with F2 and F3 treatments had significantly higher (p < 0.05, Table 2) crude fiber levels than shrimps fed with F1 and F0 treatments.
In contrast, there were no significant differences (p > 0.05) in ash, dry matter, or phosphorus across treatment levels. Carbohydrate content was low in shrimp fed with F0 (3.24 ± 0.33%) and F1 (3.42 ± 0.44%) treatments, which differed significantly (p < 0.05) from shrimps fed with F2 (4.64 ± 0.11%) and F3 (4.08 ± 0.05%) treatments. Besides, the calcium composition of shrimps fed with F3 treatment differed significantly (p < 0.05) from the rest. The lowest calcium composition (62.76 ± 0.43 mg/100 g) was exhibited by shrimp The fatty acid composition of shrimps fed with different treatment varied significantly with treatment level (Table 3). Shrimp fed with F0 and F1 treatments exhibited a significantly higher (p < 0.05) proportion of saturated fatty acids (SFA) than shrimps fed with F2

Discussion
Fishmeal replacement has a significant impact on feed utilization at various treatment levels. The current study found different variations in response to Chaetomorpha algae treatment levels. For instance, weight increased with increasing treatment levels, indicating that Chaetomorpha algae supported weight gain and eventually the growth of shrimp. Algae have been reported to be effective growth promoters due to the presence of amino acids and other compounds which can promote growth (Nasmia et al. 2022). Despite the fact that experimental feeds had similar protein content (approx. 40 CP), shrimps differed in protein composition due to varying sources of nutrients in the experimental diets. Weight gain is a function of feed utilization and efficiency. The maximum weight gain was observed in shrimp fed with 30% fishmeal replacement level indicating the influence of the Chaetomorpha diet. Chaetomorpha algae are known to contain complex antioxidant compounds (Gazali et al. 2019), which contribute to protein turnover by reducing the oxidative damage in the skeletal muscles, and hence promoting growth (Xavier et al. 2020). The antioxidants also boost immunity (Datta 2003), resulting in a healthier state of being. The effectiveness of green algae in promoting the growth of farmed shrimps is reported elsewhere (examples include Hanel et al. 2007, Ju et al. 2009Nasmia et al. 2022;and Putra et al. 2019). The addition of 30 g kg −1 of Caulerpa lentillifera, for example, has been shown to improve the growth rate and weight gain of P. monodon (Putra et al. 2019). According to Nasmia et al. (2022), adding Caulerpa sp. flour to a polyculture system of whiteleg shrimp and Chanos chanos feed improved feed utilization efficiency and eventually weight gain and growth of the culture organisms. Feed conversion ratio (FCR) was influenced by treatment replacement levels. The FCR decreased with increasing treatment levels up to 20% replacement, beyond which only slight change was observed. These findings are consistent with those reported by Cruz-Suárez et al. (2008) who found that the green algae Ulva meal reduced FCR significantly. In contrast to our findings, an increase in FCR of shrimps as a result of including green algae, particularly Caulerpa sp., in their feed has been reported elsewhere (Putra et al. 2019;Nasmia et al. 2022).
Muscle somatic index (MSI) increased across replacement levels up to the F2 treatment level, with further increases in treatment levels showing insignificant changes. When MSI increases, it indicates that the fish's diet is well assimilated, and vice versa. This study's findings suggest that supplemented algae improved feed assimilation.
In the present study, the fishmeal replacement with Chaetomorpha algae had an impact on the survival rate (SR), which was influenced positively up to F2 treatment (20% replacement level), beyond which the SR was slightly negatively influenced. The presence of nutritional substances such as polysaccharides with the potential of improving shrimp immunity (Wells et al. 2017) may have contributed to the observed survival rate. The SR results reported in the present study are consistent with those reported by Nasmia et al. (2022), who found that SR of whiteleg shrimp (Litopenaeus vannamei) was high at 6% Caulerpa sp. inclusion level and was significantly low in the control. In this study, high 1 3 levels of replacement have shown to reduce the survival rates. However, the use of other algae in feeds, for example, the red algae Hypnea cervicornis and Cryptonemia crenulata meals (Da Silva and Barbosa (2008) have been reported to improve SR in L. vannamei at 39% inclusion level.
It is well known that the biochemical composition of the edible tissues of marine invertebrates, among other factors, is influenced by their nutritional habits (Srilatha et al. 2013). In the current study, partial replacement of fishmeal with Chaetomorpha algae has shown to improve the nutritional value of shrimps across treatments. The literature on algae feed improving farmed shrimp quality of shrimp flesh is scant. However, these findings are partly in agreement with those reported by Jeyasanta and Patterson (2017), who found higher protein, lipid, carbohydrate, fiber, and ash in shrimps fed with an experiment diet made from local materials, mainly trash fishes.
In the current study, shrimps fed with 30% replacement level showed highest protein gains, with intermediate replacement levels (10 and 20%) showing insignificant differences. These findings are in agreement with studies on fresh water shrimp culture by Reddy et al. (2008) and Ferdose and Hossain (2011), which reported protein gains of 72.99 to 74.89% and 74.85 ± 0.65%, respectively. Moreover, the diet composition influenced protein content, which probably contributed to the observed variations in protein composition.
In the present study, the carbohydrate values increased with increasing treatment levels. Shrimp fed with F2 and F3 treatments had high carbohydrate content, which was probably contributed by high carbohydrate content from the Chaetomorpha algae, which has been reported to contain up to 14% carbohydrate (Gazali et al. 2019). Carbohydrate values for shrimp fed at low treatment levels (F0 and F1) in the current study are similar to those reported by Pratap et al. (2018), who reported carbohydrate content of 3.38 ± 0.05% for P. monodon and Gunalan et al. (2013) who reported carbohydrate content of 3.2 ± 0.3% for P. vannamei. The findings of this study, however, contradict those of Abdel-Salam (2013), who reported a carbohydrate composition of 1.89 to 1.91% in farmed P. indicus.
Ash is an indicator of minerals in shrimp flesh; the higher the ash, the higher the mineral content, particularly calcium. In the present study, ash content was closely similar across treatments, with high treatment levels exhibiting slightly higher values, indicating the somewhat influence of Chaetomorpha algae in mineral composition. Chaetomorpha algae are known to contain ash content of up to 7.45 mg/g (Gokulakrishnan et al. 2015). The content of ash reported in this study is different from the findings of Islam et al. (2017), who reported ash content of 0.87 ± 0.06%, and Reddy et al. (2008) who reported ash content of 9.09%, but similar to those reported by Ali et al. (2017) in P. monodon. The trend of calcium content (62.29 and 69.72 mg/100 g) across treatments in the present study is a mirror image of that of ash content. In contrast, the phosphorus content (72.10 to 74.69 mg/100 g) remained slightly constant across treatment levels, similar to those reported by Abdel-Salam (2013), who reported phosphorus amounting to 74.32 ± 1.99 and 75.45 ± 1. 78 mg/100 g, for male and female shrimps, respectively, in farmed P. indicus. However, calcium contents (45.09 ± 1.30 and 39.96 ± 2.20 mg/100 g) reported by the same author contradict the calcium content observed in the present study.
Fiber content varied with treatment levels but increased with replacement levels, most likely due to the high fiber content of Chaetomorpha algae, amounting to 21% (Tsutsui et al. 2015). Additionally, the crude fiber found in this study is similar to that reported by Liu et al. (2021), but differs from 8.2% of crude fiber in P. indicus flesh as reported by Ravichandran et al. (2009).
In this study, all treatments exhibited lipid levels below 5%, similar to the lipid value of 4.72 ± 0.11% reported by Pratap et al. (2018) for P. monodon caught in the wild. Lipid levels decreased with increasing treatment levels in the current study, which is likely due to the low-fat content in algae, which is around 0.83-1% (Gazali et al. 2019) on dry weight basis. According to Abulude et al. (2006), the lipid composition of wild shrimp ranged between 5.0 and 9.0%, which is substantially higher than 1.70% and 2.69% reported by Pombal et al. (2017) value for natural and farmed P. monodon, respectively.
The fatty acids profile has been shown to be influenced by the partial replacement of fishmeal. In low replacement levels, SFA was abundant, while PUFA was abundant in high replacement levels. MUFA levels were closely similar across treatment levels. These findings are consistent with Banu et al. (2016), who found that PUFA predominated over SFA and MUFA in the shrimp muscle tissues. Chaetomorpha algae contains substantial amount of fatty acids (Kalasariya et al. 2021;Khgtimchbnko 1993;Yazici et al. 2007) which would have partly contributed to observed trend in this study.
Although algae replacement has been shown to improve feed utilization efficiency and growth, at high replacement levels, it may have a negative impact on shrimp growth due to the fact that many algae contain low protein levels. In this regard, the formulations proposed in this study may be limited to Chaetomorpha algae or algae with similar protein contents. Despite this, green algae, particularly Chaetomorpha algae, contains bioactive compounds such as carotenoids, phenol and their derivatives, vitamins, minerals, nonstarch polysaccharides, antioxidants, fatty acids, and amino acids Sami et al. 2021;Wong and Cheung 2000), making it a good candidate as feed ingredient for shrimps and other animals. Furthermore, green algae contain dimethyl sulfonyl propionate compound, which has been shown to act as a good attractant, increasing feed intake and thus resulting in improved growth performance of shrimp (Van Alstyne 2001). The composition of active compounds in algae, on the other hand, varies according to seasons and species (Cruz-Suárez et al. 2008).

Conclusions
The replacement of fishmeal with Chaetomorpha algae at various levels (0, 10, 20, and 30%) in the shrimp diet had varied impacts on the survival, feed utilization, biochemical composition, and fatty acids profile of P. monodon. The replacement of fishmeal with Chaetomorpha algae at 20 and 30% levels yielded closely similar results. However, 30% fishmeal replacement had a negative effect on survival rate and exhibited relatively high saturated fatty acids. Thus, at a 20% replacement level, Chaetomorpha algae are suitable for partially replacing fishmeal in P. monodon feeds. The 20% fishmeal replacement is also suitable when low saturated fatty acids shrimp muscle is of interest.

Data availability
The data that support the findings of this study can be made available by the corresponding author upon request.

Declarations
Competing interests The authors have no financial or proprietary interests in any material discussed in this article.