1. Introduction
Extrusion is a common feed processing technique that has been increasingly developed and used in aquaculture in recent years [
1,
2,
3]. Compared with pelleted diets, extruded feed increases water stability and durability [
4,
5], decreases the content of anti-nutritional factors, enhances the utilization of nutrients, and reduces eutrophication in water as a result of lower evacuation of nitrogen and phosphorus [
6], all of which are due to the greater levels of heat, moisture and pressure used during the extrusion process [
5]. However, high temperature and strong pressure also have some disadvantages, such as destruction of vitamins and dietary enzymes, and extruded feed may be less used in the aquaculture industry because it is technologically difficult to produce and more expensive [
5].
Increasing evidence indicates that intestinal microbiota are closely linked to growth, nutrient absorption, and immunity in organisms [
7,
8,
9]. A stable intestinal microbial community can promote growth and disease resistance by balancing digestion and resisting pathogens, and it may even improve breeding efficiency [
10]. Thus, the diversity, composition, and functions of microbiota can serve as potential indexes of host health and help predict the influence of dietary additives [
11]. Previous studies of aquatic species have revealed significant effects of host genetics, developmental stage, health status, diet, and environmental and geographical factors on the gut microbial community [
10,
12], and diet has been reported to be a major factor involved in these effects [
10]. However, little is known about the relationship between feed processing techniques and intestinal microbiota.
The red swamp crayfish (
Procambarus clarkii) has become one of the most significant and popular commercial freshwater species in China due to its special food culture, rich nutrition, and delicious taste [
13]. The annual aquaculture production of crayfish has increased rapidly in recent years, with the number growing from 852,285 tons in 2016 to 2,393,699 tons in 2020. However, the lack of standard seed and feed processing techniques and increasing problems with disease have made the aquaculture of
P. clarkii difficult. To improve immunity and increase production to meet the growing demand for this species, researchers have studied its nutrition requirements and feeding management [
14,
15,
16,
17]. All of the previous studies of nutritional needs have focused on pelleted feed rather than extruded feed, and the effects of different feed processing techniques on crayfish are poorly understood. The effects of manufacturing processes can vary greatly depending on species [
1,
2,
6,
18], and the results of previous studies were not unanimous. Furthermore, developing suitable production technologies to keep feed costs as low as possible is needed [
1,
2,
5].
To address these issues, we subjected feed to two processing techniques to produce extruded and pelleted ones to investigate the effect of processing technique on growth, intestinal histology, and microbiota in P. clarkii. Our results can be used to improve the aquaculture of crayfish.
4. Discussion
Feed processing technology has received increasing attention in recent years. In most of these studies, feed efficiency was significantly improved when aquatic animals were fed extruded feed [
18,
21]. However, the effects of feed processing on growth performance were not unanimous. Several researchers reported a reduction of growth in organisms fed pelleted feed compared to extruded feed [
2,
6,
18], which was in contrast with [
2], who found that growth was unaffected when channel catfish (
Ictalurus punctatus) were fed feed processed by extraction or extrusion.
Our results showed that
P. clarkii fed the extruded feed had a better FCR than those fed pelleted feed (
Table 4). This result likely was related to the high gelatinization that resulted from extrusion, which increased the utilization of starch and improved the water-borne durability and nutrient intactness of the feed [
22]. The better FCR of the extruded feed also may be related to the compaction and retention of nutrients, which would minimize the amount of leaching into the water. This feature would lead to higher gut nutrient content and digestion, longer gastric evacuation time, and slower emptying rates of the gastric intestinal tract compared to that in fish fed pelleted feed (fish meal content was 6%, 0%, and 27%, respectively) [
2,
18,
23]. Furthermore, based on the research previously (fish meal content was 6%) [
2], the diet formulation in this study could meet the nutritional needs of crayfish, thus the improvement of nutrient digestibility and utilization rate by extruded processing wasn’t extremely obvious [
24]. Although the feeding economies of the two feeds differed significantly, growth performance of crayfish was not compromised by the feed processing techniques tested in this study.
The utilization of nutrients by aquatic animals is determined largely by the level of digestive enzyme activity in the intestine, which can be affected by exogenous and endogenous factors. Diet is one of the major exogenous factors [
25]. Modulation of digestive enzyme activity by feed type (e.g., pelleted feed and extruded feed) in a variety of aquatic species has been reviewed recently: the latter led to higher levels of digestive enzyme activity in some fish [
18,
26]. Enzyme release was probably related to gastric evacuation time, which was extended in the extruded feed group. In agreement with results of previous studies, we also found that higher levels of intestinal trypsin and amylase activity occurred in crayfish fed extruded feed compared to pelleted feed (
Table 5). These findings confirmed the beneficial effects of extruded feed on intestinal digestion processes and showed that increased digestive enzyme activity improved the FCR in the crayfish fed extruded pellets.
Improvements in intestinal histology are beneficial to feed utilization and health status because they increase the organism’s ability to prevent bacterial infection of the mucosal epithelium [
27]. The enteric and absorptive capacity of the intestine was closely linked to villus height, mucosal fold height, and mucosa thickness [
28]. Moreover, goblet cells have significant effects on the digestion and health of aquatic animals by synthesizing and secreting mucins, which lubricate and protect the intestinal epithelium [
29,
30,
31]. However, little is known about the effects of feed processing procedures on intestinal morphology. In this study, we found that the lamina propria thickness in crayfish fed pelleted feed was significantly increased compared to that of the extruded feed group (
Table 6). Extended intestinal lamina propria were also founded in intestinal damage induced by nutritional stress [
20]. Although the feed processing method did not significantly affect the numbers of goblet cells and lymphocytes and no pathomorphological changes were detected between crayfish fed the two diets (
Figure 1). Similarly, feed processing technique and size did not significantly affect histomorphology of digestive organs in fish [
32,
33].
Intestinal microbiota is closely related to growth, feed utilization, digestion, and nutrition absorption of aquatic species [
34,
35,
36]. Moreover, a stable and beneficial microbial composition has a favorable effect on immunity and health of the host [
18,
37]. Recent studies indicated that the variation of intestinal microbiota is more affected by host development and diet than by the geography and surrounding environment [
10]. Therefore, numerous studies have focused on the effects of diet on microbial composition [
11,
38].
To the best of our knowledge, no prior study has reported the effect of feed processing technique on intestinal microbiota for any aquatic animal. We found no significant difference in diversity (reflected by the Shannon and Simpson indexes) of crayfish, but the abundance of intestinal microbiota in the pelleted feed group was lower than that in the extruded feed group (
Table 7). The lower FCR observed in the extruded feed group might be related to the higher abundance of intestinal microbiota in this group, which could improve nutrient absorption [
39]. In accordance with the Chao1 index results, the extruded feed group had more OTUs than the pelleted feed group (
Figure 2), further confirming that the microbial composition was richer in crayfish fed this diet [
20]. The lower number of OTUs in crayfish fed pelleted feed suggested that these crayfish may ingest fewer nutrients due to leaching from the feed prior to ingestion [
40]. The PCoA plot showed that the intestinal microbiota of the two groups gathered into two independent clusters, which indicated that the feed processing technique altered the intestinal microbiota structure of the crayfish. Previous studies also reported that a flora imbalance caused by diet might be responsible for the variation in the structure of intestinal microbiota [
41,
42]. Thus, changes of the intestinal microbial flora induced by a specific feed processing technique might have negative impacts on crayfish.
In our study (
Figure 3),
Proteobacteria,
Tenericutes,
Firmicutes, and
Bacteroidetes were the dominant phyla in both groups. Previous studies reported that 90% of intestinal microbiota in many aquatic animals belong to
Proteobacteria,
Bacteroidetes, and
Firmicutes [
43,
44,
45], which indicates that these bacteria are closely associated with crucial functions of the intestine, including nutrient absorption, digestion, and immunity [
46].
Proteobacteria was the most dominant phylum in all crayfish examined in our study, which agrees with findings of previous studies of the intestinal microbiota of healthy
P. clarkii [
10,
12,
40] and other crustaceans, including various shrimp species [
47,
48,
49,
50]. Furthermore, the relative abundance of
Proteobacteria was significantly higher in crayfish fed the extruded feed diet than in those fed the pelleted feed.
Proteobacteria has also been identified as one of the most abundant phyla in shrimp species investigated, and this phylum plays a critical part in various biochemical functions, such as carbon and nitrogen cycling [
20]. Studies have also indicated that
Proteobacteria is likely involved in some biogeochemical processes and in the intestine of crustaceans [
51]. High abundance of
Proteobacteria may also pose potential risks for hosts, as higher numbers of
Proteobacteria were observed in shrimp infected with pathogenic bacteria or in those with poor growth [
52,
53].
At the genus level, Illumina high-throughput sequencing results demonstrated that the dominant genera were not consistent between the two feed groups. However,
Citrobacter was the most abundant genus in crayfish fed both diets.
Citrobacter rodentium is an attaching and effacing bacterial pathogen that shares pathogenic mechanisms with enteropathogenic and enterohaemorrhagic
Escherichia coli, which can cause various diarrheal diseases and death [
54,
55,
56]. In our study, the relative abundance of
Citrobacter in crayfish fed pelleted feed was higher than that in specimens fed with extruded feed, although the difference was not statistically significant. No significant differences in abundances of other dominant bacteria were detected.