Nucleotides are low-molecular-weight intracellular compounds which, besides being the building blocks of DNA and RNA, play important roles in physiological and biochemical processes [20,34,35]. They have also been reported to improve shrimp health by modulating their immune response [21,36,37]. The results from this study indicate that the use of nucleotides (Nucleoforce^®) improves growth performance and profitability in PWS and, especially, allows greater disease resistance. More specifically, in PWS receiving diets in which FM has been partially replaced with vegetable protein sources, nucleotide supplementation led to better FBW, PWG, FCR, TGC, ADG, and higher economic profits, as well as a healthier hepatopancreas and increased survival rates upon Vibrio harveyi challenge. On the other hand, in PWS receiving a commercial diet with regular FM levels, nucleotide supplementation allowed higher disease resistance and no inferiority in terms of performance, immune response, and profitability, without altering the organoleptic parameters.

The main positive impact of nucleotide supplementation in PWS observed in this study is probably the improvement seen in growth performance. As the trend in reducing FM usage continues, this beneficial effect would support the incorporation of nucleotides in such circumstances during PWS production. This is in line with the study by Andrino et al. [18], where the inclusion of 0.2% nucleotides produced significantly better specific growth rates (SGR) and feed conversion efficiency (FCE) compared to shrimp without dietary nucleotides after a 60-day feeding trial. In our previous study, despite not reaching statistical significance, the supplementation of 0.1% nucleotides led to better growth, especially in the group in which FM had been partially replaced with SBM, compared to the control diet [21]. Results from the present study confirm the efficacy of nucleotides in improving PWS growth performance up to commercial size, complementing the positive outcomes of the prior study [21]. The role of nucleotides and metabolites in aquatic organisms has been studied now for a little over two decades [35], and the positive effects of various nucleotide products on growth and feed utilization performance of fish and crustaceans are well established [34,38,39,40]. However, there are still very few reports explaining how nucleotides work or how they enhance the growth performance of PWS. Metailler et al. [41] and Hossain et al. [34] argue that the feed attractant properties of nucleotides promote the rapid intake of the feed which prevents the leaching of nutrients allowing the shrimp to benefit, especially by supporting growth performance, from the complete nutritional properties contained in the diet. In this study, we observed the effects of prolonged nucleotide administration on the growth of PWS, up until they reached their consumption size. We noted that the 110-day administration period with intensive culturing in an open pond system provides a clear positive trend towards increased L. vannamei growth compared to the 70-day results obtained in our previous study [21].

Nucleotides modulate the immune response, and this could be seen with the improved results in the immune parameters evaluated during this trial, namely THC and lysozyme activity. Unlike in previous experiments [21], here we did not see a significant effect in this regard. Nevertheless, the trends indicate that there was probably an effect that might have been significant, if only restricted to the initial and more vulnerable stages of early shrimp development.

In crustaceans, circulating hemocytes play an important role in immunological responses [42,43]. However, environmental stressors, especially in open pond systems, could induce apoptosis of hemocytes in the shrimp [44], leading to decreases in THC, and hypothetically, this could induce deterioration of the immune system [44,45]. Prior studies by Murthy et al. [46] and Shankar et al. [47] showed that THC significantly increased with nucleotide supplementation. This indicates that the administration of nucleotides might stimulate the release and activation of hemocytes. Hemocytes also produce cytotoxic molecules, such as lysozymes, that are involved in the degradation of microbes within and outside the hemocytes [48]. The benefits of the activation of lysozyme activity were confirmed after feeding the kuruma shrimp, Marsupenaeus japonicus, with nucleotide-rich baker’s yeast [49].

Infection with Vibrio harveyi is an important issue to be considered in shrimp production. Data from this study demonstrate a remarkable positive effect of nucleotides, as all supplemented groups achieved higher survival rates. Adding nucleotides to a diet with 10% FM resulted in significantly higher resistance to the challenge compared to 6% FM, but also to 10% FM, without nucleotides. The use of nucleotides facilitated a higher survival rate even in a diet with 8% FM when compared to 10% FM (i.e., with higher FM, but without nucleotides). This is consistent with prior observations in shrimp also challenged with V. harveyi under different conditions [21]. It has been reported that with the ability of nucleotides to induce the activation of specific and nonspecific immune systems, dietary nucleotides can enhance the resistance of aquatic organisms against various pathogens, including viral, bacterial, and parasitic pathogens [34]. A previous study by Andrino et al. [18]. revealed that L. vannamei survival was higher in the group fed with nucleotides compared to those fed without dietary nucleotides. The significantly higher survival rate of shrimp fed with dietary nucleotides in our study is in line with our previous study where all groups fed with nucleotide-supplemented diets reached significantly higher survival rates [21]. The protective effects of nucleotides are probably due to an accelerated immune response to disease and environmental changes.

In the present study, the whole-body proximate composition indicated a higher protein deposition in the groups receiving dietary nucleotides compared to the group without dietary nucleotides. In addition, we found comparable values for other nutritional profiles, including total fat, moisture content, carbohydrate, and crude fiber content. The higher level of protein in the nucleotide-supplemented groups might be attributed to the ability of dietary nucleotides to influence protein biosynthesis by regulating the intracellular pool of nucleotides [34]. This is closely related to the role of nucleotides as the building blocks for DNA, which is further transcribed and translated to form protein in the shrimp body [34,50]. Numerically, the addition of nucleotides also increased the ash content within the whole body of the shrimp. This effect could be linked to the nature of the nucleotides used in this study, as a balanced concentrate of free nucleotides and active precursors, obtained from yeast. In a previous study, Tacon and Cooke [51] reported that the dietary inclusion of nucleotides in the form of yeast extract increased the ash content of rainbow trout carcass.

The main site for the absorption of nutrients in shrimp is the hepatopancreas [52]. This tubular system facilitates the rapid transport of cellular nutrients to the hemolymph, including dietary nucleotides. Thus, it is important to maintain the hepatopancreas in optimum condition. In the present study, the hepatopancreas of shrimp fed with 10% FM showed a normal structure without any cell structure damage. However, PWS fed with lower FM levels showed alterations such as hemocyte leaching and enlargement of the nucleus and vacuolation. Anti-nutritional factors in SBM might constitute an important factor responsible for the abnormal intestinal condition and reduced nutrient absorption [53]. A prior study by Yao et al. [54] indicated that the use of high SBM levels in feeds could induce intestinal damage and significantly decrease cell membrane thickness in the hepatopancreas of PWS. In the study herein, the inclusion of 0.1% nucleotides was capable of partially preventing the development of marked histological hepatopancreas alterations observed in the shrimp fed with 8% and 6% FM. The benefits of adding nucleotides to the diet may be due to their low molecular weight, which favors optimum digestion and absorption processes in the hepatopancreas.

Reports on the organoleptic evaluation of shrimp fed with functional or alternative ingredients are scarce. A study by Soller et al. [55] reported no statistical differences in the texture, appearance, aroma, or flavor between the shrimp fed diets containing menhaden fish oil and soybean oil. In our study, panelists were unable to distinguish shrimp from the different groups based on their color, aroma, flavor, or texture, which means that nucleotide supplementation does not alter these organoleptic parameters. It should also be highlighted that a better texture and sweeter flavor was achieved with nucleotides. Therefore, perhaps the use of nucleotides in diets might provide a positive influence on the sensory attributes of shrimp when FM is reduced.

Profitability is a key parameter in shrimp production, and feed-associated costs constitute a major variable in shrimp production from an economic standpoint. It is known, and can also be clearly appreciated in our study, that large-scale replacement of FM by vegetable protein leads to reduced profitability due, mainly, to impaired growth performance and health, despite savings in feed costs. That is why the use of high-quality alternative protein sources is preferred when replacing FM [56]. In addition, FM replacement strategies can also incorporate the use of complementary ingredients to obtain a more balanced nutrient profile and increase nutrient utilization [56,57]. Incorporating nucleotides does increase the cost, although not substantially, as the inclusion level is rather low. On the other hand, as mentioned previously, adding nucleotides leads to better disease resistance, which can enhance the benefits obtained in each shrimp production batch. As a result, in this study we managed to show that, overall, nucleotides supplementation generates an increased return on investment to the farmer. More specifically, adding nucleotides to PWS diets with reduced FM leads to a higher gross margin thanks to the markedly improved performance, and despite the slight increased investment required initially. Furthermore, profitability in 10FMN is quite similar to that of 10FM, but 10FMN provides better disease resistance.

Further studies are warranted in order to further validate the effects of nucleotides in this animal species as well as to support the approach suggesting their ability to counteract the negative effects of replacing FM by SBM in shrimp diets. This could be done in other countries with similar trends, like in Vietnam.