Kyle Olsen and John Chaston, Plant and Wildlife Sciences
Introduction
As a pseudo-ruminant, the alpaca is highly dependent on the bacteria in its intestinal tract to obtain the nutrients it needs. For example the starch-fermenting bacteria in the compartment 1 (C1) access nutrients from the normally indigestible starches the alpaca consumes. The bacteria release volatile fatty acids (VFAs) in return. The alpaca uses VFAs as a source of energy. In turn, the composition of the microbiome (bacteria in the intestinal tract) is affected by many factors involving its host. These factors could include host genome, diet, and location in the digestive tract. Our research was to survey many aspects of why the alpaca is able to extract more energy from ingested dietary fiber than almost all other animals. We also looked to perfect protocol for examining such traits as this analysis is done many different ways throughout the scientific community.
Methodology
We were able to examine the three main variances for this analysis found among the scientific community. The three main variances are kit type, taq type, and body site. Kit type is what extraction method they used to isolate the bacterial DNA from waste. Taq type is the master mix and taq polymerase they used to amplify the DNA with PCR. Body site is where they extracted the sample from. All of these vary among scientists and experiments throughout literature and we conducted analysis as to the differences between them in order to produce the most accurate data
Kit type
Our question was whether or not different extraction methods generated significantly different microbiota composition for the same sample. We started with 5 alpaca individuals who were treated equally with feed and environment. The animals were sacrificed and all samples were taken at the same time. We extracted three samples from each of the alpaca individuals. One of the samples was from Compartment 1 (C1), another from the ileum, and another from feces. This left us with 15 total samples to compare. We extracted the microbial DNA from all samples three different times using our three extraction methods (the PowerFecal® DNA Isolation Kit provided by Mo Bio Laboratories Inc; Fecal DNA MiniPrep from Zymo Research; and a non-commercial extraction method used on flies called saltingout). We then used AccuPrime Pfx Mastermix from Thermo Fisher Scientific to run PCR. Through 16s rDNA marker gene sequencing and the QIIME pipeline we analyzed the microbiota of each sample.
Taq type
Our question was whether or not different master mixes used in PCR generated significantly different microbiota composition for the same sample. We started with 5 alpaca individuals who were treated equally with feed and environment. The animals were sacrificed and all samples were taken at the same time. We extracted from three separate spots from the alpaca: Compartment 1, ileum and rectum. We extracted the microbial DNA from all 15 unique samples using three different extraction methods (the PowerFecal® DNA Isolation Kit provided by Mo Bio Laboratories Inc; Fecal DNA MiniPrep from Zymo Research; and a non-commercial extraction method used on flies called saltingout). We then used AccuPrime Pfx Mastermix from Thermo Fisher Scientific on all 45 unique experimental samples and also used 5 prime HotMastermix on all unique experimental samples to perform PCR. Through 16s rDNA marker gene sequencing and the QIIME pipeline we analyzed the microbiota of each experiment.
Body site
Our question was whether or not different intestinal sites were composed of significantly different microbiota. We started with 5 alpaca individuals who were treated equally with feed and environment. The animals were sacrificed and all samples were taken at the same time. We extracted three samples from each of the alpaca individuals. One of the samples was from Compartment 1 (C1), another from the ileum, and another from feces. We extracted the microbial DNA from all samples using the PowerFecal® DNA Isolation Kit provided by Mo Bio Laboratories Inc. We then used the AccuPrime Pfx Mastermix from Thermo Fisher Scientific to run PCR. Through 16s rDNA marker gene sequencing and the QIIME pipeline we analyzed the microbiota of each sample.
Once we finished isolating the ideal way to compose our analysis we were able to analyze the microbiome of over 100 alpaca individuals using our updated protocol. We compared grass fed alpacas vs alfalfa fed alpacas; alpacas vs mice vs otters; and individual alpacas amongst themselves.
Results
Upon cross-examining the microbiota associated with each kit we found that MoBio and Salting Out resulted in significantly different compositions for the same sample (p value of 0.048). However, MoBio does not produce significantly different microbiomes than Zymo if tested on the same sample (p value of 0.275). Lastly, Salting Out does not produce significantly different microbiomes than Zymo (p value of 0.419). Upon cross-examining the two Taq polymerase master mixes we found that microbiota found in the experiments run with AccuPrime were significantly different from experiments run with 5 prime. Even among the three different extraction methods used all results were the same. In the three extraction methods the results had p values of 0.001, 0.008, and 0.001 for MoBio, Zymo, and Salting Out respectively. Upon cross-examining the five fecal samples, five ileum samples and five C1 samples we found that the fecal and ileum microbiomes are both significantly different from the C1; having p-values of 0.012 and 0.009 respectively. However, the fecal and ileum microbiome are not significantly different from one another; having a p-value of 0.03.
Discussion
Looking at the results from the designed experiment to isolate the ideal robust DNA extraction method we can see that the ideal protocol would be to use a fecal sample if you were trying to examine any part of the intestine after C1, extract with MoBio, then amplify with AccuPrime. This is what we did for over 100 unique alpaca samples. Because the process of analyzing each subject takes time we are still in the process of finalizing our data. However, preliminary data suggests that finding a bacterial species that affects VFA production in any of these three digestive regions may provide a key to increasing health of low body condition (LBC) alpacas.
Conclusion
From our data we can reasonably conclude that Alpaca Microbiota does have an great effect on the ability for the host to digest dietary fiber. We isolated the ideal robust DNA extraction and amplification method for this type of analysis. The microbiota found in the alpaca varies greatly from that of mice or otter and further research has been planned to specific which individual bacterial genera contribute most to this great difference in fiber digestion.