The current study has been conducted to evaluate the in vitro antimicrobial properties of HI and TM fats and their effect as an alternative lipid sources in rabbit diets. The possible utilization of insect fats in animal diets has been poorly investigated so far, and few papers are available, focusing on antimicrobial activities in vitro or in vivo, but no data are present in literature on pathogen growth rate in presence of insect fats. Mustafa et al. [43] showed that the oil extracted from the melon bug (Aspongopus vidulatus) was able to inhibit the growth of bacterial species by using the agar well diffusion method, with only Gram positive bacteria (Staphylococcus, Bacillus and Enterococcus) being susceptible to crude oil extracts. Salmonella paratyphi was also tested and no inhibition was recorded, similarly to what was observed in the present research with other serovars. A recent study of Spranghers et al. [28] on the in vitro effects of fats extracted from HI (from prepupae fed to weaned piglets) pointed out that D-Streptococci and Lactobacilli were the only bacterial populations that reduced their load after being challenged with insect fats, whereas no effects were recorded on Gram negative bacteria. The data presented in our paper suggest that fats extracted from TM and HI are able to delay bacterial growth of both Gram positive and Gram negative pathogenic bacteria, even if the susceptibility changes with the considered species. Interestingly, no bactericidal effect was observed, thus indicating the possibility of bacterial cells to repair to damages that may be induced from FAs or monoglycerides [6].
The antimicrobial effect of TM fat was lower than that of HI fat. These results may be related to the different concentration of SFA. Indeed, HI fat used in this study was composed by 79% of SFA (25% in TM fat), and the major component was lauric acid, already reported as very effective against many bacterial species [6; 28]. On the other hand, the antibacterial effects of UFA reported in literature (Yoon et al., 2018) were not detected in this study, probably in relation to the prolonged incubation time and the temperature of 37 °C, which may have been responsible for the reduction/impairment of the activity of these molecules that characterize TM fat (75% of the FA v. 21% of HI fat). This hypothesis may also explain why soybean oil did not show any activity, considering its higher level in MUFA and PUFA among all fats used in this work.
The present observations highlighted the possibility of using insect fats in feed formulation, considering that they may be important for controlling growth of important microbial pathogens such as Listeria monocytogenes, Yersinia enterocolitica and Pasteurella multocida that may be important pathogens for rabbits [32;35] or part of the gut flora potentially contaminating rabbit meat during slaughtering [33;34]. Reducing microbial loads in rabbit gut, apart from animal welfare and safety implications, may also be important for food safety management. However, as already emphasized by Spranghers et al. [28], more studies need to be performed in vivo, in order to assess the activity of fat in the gut, considering that the bacterial activities and the digestive enzymatic systems of the rabbit (i.e. lipases) may neutralize the FAs, therefore limiting their activity.
As far as the in vivo trial is concerned, there was a lack of differences among groups on caecal fermentation traits in our study. Caecal pH and VFA content are the main variables characterizing the extent and the pattern of caecal fermentation, thus constituting an indirect estimate of caecal microbial activity. The dietary HI or TM fat inclusion led to a greater total VFA content in the caecum than that of control diet, thus potentially enhancing gut with a modification of the fermentation patterns and the composition of the caecal microflora.
Peeters et al. [44] previously observed that a high concentration of total caecal VFA in rabbits had a protective effect against enteropathogenic Escherichia coli infection. However, the molar proportion of different VFA was not affected by the total replacement of soybean oil with HI and TM larvae fat.
The present study is the first that investigate gut microbiota of growing rabbits fed diets supplemented with insect fats. The results of this study revealed an enrichment of different taxa according to the dietary treatment and a similar microbial diversity and richness between feces and caecum samples. Caecal microbiota is a primary determinant for rabbit health, whereas the fecal microbiota provides an accurate method for studying the evolution of rabbit gut microbiota from weaning to slaughtering [45–46]. Firmicutes, Bacteroidetes and Verrucomicrobia represented the dominant bacterial phyla in the control and insect fat-fed rabbits of the present study. These findings overall agree with previous researches that identified Firmicutes and Bacteroides as the main bacterial phylum in the gut microbiota [2; 46–48]. In relation to the genera composition, Bacteroides, Bacteroidales, Clostridiales, Lachnospiraceae, Ruminococcaceae and Ruminococcus families, mainly colonized the caecal and fecal microbiota of the rabbits fed soybean oil or insect fats in the current study. These findings are also in agreement with previous studies, which observed Bacteroides [46;48], Clostridiales [47] and Ruminococcus [46;49] as main the bacterial genera in caecal and fecal microbiota of rabbits.
Regarding the microbial composition, we did not observe any strong effects as a consequence of the dietary inclusion of HI and TM fats. However, a signature in the microbial population was observed. The fat of TM reduced some taxa such as Klebsiella, Lachnospira, Parabacteroides and Odoribacter. On the other hand, the dietary supplementation of HI and TM fats enriched the presence of Akkermansia, which is a maintaxa in the gut microbiota of rabbit [45]. It is well reported that Akkermansia can be considered a probiotic of new generation able to degrade the mucin in the gut with the production of beneficial molecules like SCFA, thus exerting a significant improvement in the gut barrier and in the maintenance of intestinal health [50–52]. In addition, it was suggested that Akkermansia have an important role in the hydrolysis of various dietary polysaccharides, contributing to increase cellulose digestibility as well as methane metabolism [53–55]. The increase of this taxon related with the insect fat inclusion suggests an optimal gut environment in our rabbits, even if this observation needs further investigation to be confirmed. In addition, a strong positive correlation between Akkermansia and NH3-N was observed.
Ruminococcaceae family was considered as an important producer of short-chain FAs (mainly butyrate, acetic, and succinic acids) through glucose metabolism and cellulose digestion [56–57]. It was reported that member of this family is an important component of the beneficial microbiota of several herbivores [58–59]. Their presence is related with an improvement of the immune system of the host via intestinal mucus degradation and a prevention of acidosis via lactate degradation [60]. The supplementation of HI fat also increased the presence of Ruminococcus (belonging to Lachnospiraceae family), that are butyrate producing bacteria. This ability was also confirmed by the strong positive correlation between L-Ruminococcus, acetic and propionic acid. The microbial signature drove by the inclusion of insect fat increases the presence of Bacteroides, Clostridium, Akkermansia and Ruminococcus and suggests that dietary HI and TM fats may exert a positive influence on the caecal microbiota of the rabbits. It should be pointed out that rabbit fed HI and TM fats showed no significant alterations at histopathological level and no differences on growth performance [12].