## 4 Discussion
Whilst the chlorogenic acid trial was performed on a different species of saltwater shrimp (_Litopeneus vannanei_), the similarities of the effects add evidence to the possibility that chlorogenic acid is one of the active ingredients present in the sugarcane extract. [PERSON] et al. (2015) hypothesised that the increased antioxidant capacity and transcription of antioxidant enzymes observed was the likely mechanism that led to the improved production outcomes, including stress resistance and increased total biomass yield. Chlorogenic acid has been observed to be involved in the regulation of nuclear factor erythroid 2-related factor (_Nrf-2_) and inflammatory markers such as nuclear factor kappa-light chain enhancer of activated B-cells (_NF-kB_) in vertebrates [40]. However, the activation of _Nrf-2_ or regulation of _NF-kB_ have not been directly studied with chlorogenic acid-enriched diets in shrimp. Polygain(tm) (PRSE) extract contains a diverse range of compounds beyond polyphenols, including organic acids, minerals, phytosterols, fatty acids, and amino acids. Some of these have been quantified, such as chlorogenic acid, but many others have only been putatively identified in the extract or are known to be abundant in sugarcane and fermentation products, such as organic acids. Some of these qualitatively identified or even unidentified compounds could have a major role in the mechanism of action of this extract. For example, organic acids are abundant in sugarcane production streams [41]. Succinic acid has been shown to have a positive effect on growth and immune function in _Litopeneus vannanei_[42]. As a result of the variety of compounds present in the PRSE sugarcane extract, the potential of non-phenolic components playing an active role in the improved performance should not be overlooked. The most appropriate hypothesis at this stage is that a range of these components are acting synergistically to produce the beneficial results observed in this trial. Other studies on the addition of polyphenol-rich plant extracts to shrimp diets focus on the effect on shrimps' resistance to specific diseases. For example, an aqueous extract containing polyphenols from the leaves of spurred mangroves (_Ceriops tagal_) prevented infection with white spot syndrome virus (WSSV), when coated onto the diet at the rate of 10 g kg\({}^{-1}\), for whiteleg shrimp [28]; while an extract from Bermuda grass (_Cynodon dactylon_) added at 20 g kg\({}^{-1}\) to diets prevented infection by WSSV in black tiger shrimp [25]. However, the shrimp used in the current study were free of WSSV and most of the other shrimp pathogens tested. The pathogens present, hepatopancreas parvovirus (HPV) and gill-associated virus (GAV), were present at low levels and were not different between treatments (Figure 3). The mRNA expression of _dcr1_, which codes for the endoribonuclease Dicer-1 protein that plays an antiviral defence role in _P. monodon_[36], was also not different between treatments. While the presence of unknown pathogens cannot be ruled out, overall the pathogen data in this study suggest that differences in shrimp growth and survival were not limited to the PRSE-induced-inhibition of infectious disease. Further work on other damaging pathogens to the shrimp industry, such as _Enterocytozoon hepatopenaei_, and under challenge conditions would help in understanding the impact this extract may have on disease defence. Dietary polyphenols are associated with positive effects on animal health and growth performance, through a diverse range of other mechanisms not targeted in this experimental design, including interaction with gut bacteria, modulation of metabolic pathways, and as a direct immunomodulator [43]. However, for some of these factors, such as the microbiome, there may be species differences that may alter what would be expected to be observed in vertebrates.