Link between microbiota 
and piglet performance

The coevolution of intestinal microbiota and a specific host animal has formed unique host-microbe combinations. Since bacteria utilise different substrates, the composition of diet and the physiological functions of the host largely determines the composition of the microbiota in the gastrointestinal tract. Consequently, this offers a possibility to modulate the microbial community by feeding. Due to the complexity of intestinal bacterial communities and the interrelationships between its habitants the traditional culturing methods of microbes are not applicable for studying the relation between microbiota and animal performance. 
The advent of molecular biological techniques, such as DNA sequencing and quantitative real-time PCR (qPCR), have offered a better means to study these relations.

Piglet trial

Denkavit BV, Hankkija Oy and Alimetrics Ltd carried out a joint research project for investigating the microbial profiles in the hindgut of piglets and their possible relations to piglet performance. Rectal faecal samples of weaned piglets were collected individually but pooled per pen from a total of seven trials conducted in Finland, France and the Netherlands. Sampling time varied from 14 to 26 days post-weaning and the total amount of analysed samples was 80, representing 343 piglets. Performance data was registered per pen from each trial. The samples were initially analysed at the Alimetrics laboratory by percent guanine + cytosine (%G+C) profiling method and correlations between different %G+C fractions and feed conversion ratio (FCR) were calculated. Percent G+C profiling sort different bacterial taxa to different positions in density gradient centrifugation according to the characteristic guanine + cytosine content of their chromosomal DNA. Pooled DNA samples were subjected to high-throughput 16S rRNA gene sequencing to reveal possible differences between gut microbiota compositions in different countries, diets as well as links between FCR and the prevalence of specific microbes. Based on the data set from the sequencing study, 16S rRNA gene –targeted quantitative real-time PCR assays were designed and validated for the 13 most important species. In the final phase of the project, the abundance of the target species in the original piglet faecal samples (80) were determined by using the newly developed qPCR assays. This quantitative information was used in assessing the microbial taxa affecting the piglet performance. Additionally, the relationship between microbial numbers and FCR was evaluated mathematically. The end result was an algorithm, which can be used to calculate Performance-linked Microbial Index (PMI) values for piglets from faecal samples.

Superpools found

In order to obtain general understanding on the microbiota present in the piglet colon, a superpool was made of the 80 different samples representing 343 piglets of seven different trials conducted in three different countries. Figure 1 shows the %G+C profile of this superpool sample and the predominant genera present in faeces of piglets. Figure 2 shows the distribution of the most abundant genera in piglet faeces as revealed by 16S rRNA gene sequencing. Genus Lactobacillus was evidently the most abundant group of bacteria in the samples. Results also showed that the most prevalent lactobacilli species were Lactobacillus amylovorus and Lactobacillus reuteri (Figure 2). The second largest genus in the superpool was Eubacterium, which is a heterogeneous group of slow-growing Gram-positive obligate anaerobic bacilli. Species of this genus can produce butyric (especially E. rectale, which is also frequently found in the human colon), acetic, lactic and formic acids. The third largest genus in the superpool was Clostridium, which represents Gram-positive obligate anaerobic bacteria. There are approximately 100 species present in this genus, most of which are commensal intestinal bacteria. However, there are also certain well-known pathogens, which belong to this heterogeneous group of microorganisms (e.g. C. perfringens and C. difficile). Other abundant genera discovered in the superpool were Blautia and Faecalibacterium (especially species F. prausnitzii).

Effect bacteria on FCR

In animal production, feed conversion ratio (FCR; kg of feed consumed per kg of weight gain) is an important factor having a major impact on the economy of the business. Furthermore, FCR is in many cases regarded as an indicator of good intestinal health of the host. In practice, there is a lot of variation in performance even between the individual animals and therefore it would be essential to understand the connection between microbiota composition and piglet FCR. In this research project with piglets originating from 80 pens, both microbiota composition and performance data of the same pens were available. Table 1 shows the bacterial genera that correlated with FCR during the first 2-4 weeks after weaning. In order to take into account cross-correlation and interaction between microbes, linear regression is needed. Consequently, a stepwise estimation was performed, which finally made it possible to have a mathematical model based on five microbial species/clusters that were responsible for significant differences in the performance of piglets. Each microbe was given a frequency weight based on their prevalence. For example: PMI = X *bact. A + Y*bact. B – Z*bact. C etc. Figure 3 shows the relation between the Performance-linked Microbial Index (PMI) and the observed FCR. The coefficient of determination (R2) value was 0.55, which indicates good predicting power.

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Conclusions

It can be concluded from the data of this work that there is a correlation between the intestinal microbial composition of piglets and their performance. By relating the abundance of the five most relevant bacterial genera with FCR of piglets in the period after weaning, a Performance-linked Microbial Index (PMI) could be assessed. As an initial step, PMI was applied to understand 1) why some piglets were less efficient than others in digesting their feed and 2) what was the role of microbiota in this process. A concept of good microbiota of piglets is difficult to define. Therefore, the presented research provides valuable information regarding the balanced gut microbiota i.e. which microbial groups should be stimulated or inhibited to achieve a good FCR.