4.3 | Additional methodological considerations
Dietary metabarcoding techniques are being increasingly applied to
investigate the diets of herbivores and omnivores, including the diets
of pigeons, bears, monkeys, deer, zebras, and goats (Ando et al., 2013;
Quemere et al., 2013; De Barba et al., 2014; Kartzinel et al., 2015;
Gebremedhin et al., 2016; Erickson et al., 2017). Much higher resolution
is provided by the molecular analysis in comparison with the traditional
alkane analysis of scats (Zhang, Jin, Badgery, & Tana, 2017). Both
frequency of occurrence (FOO) data of food taxa and relative read
abundance (RRA) of sequences are commonly used to convert sequence reads
to dietary data. Both metrics have inherent biases when the number of
food taxa in samples is large. In the current study, the diet data were
presented using these two approaches to reduce any biases.
Accumulating evidence suggests that the number of sequence reads is
approximately
proportional to relative biomass of prey in a diet sample (Elbrecht, &
Leese, 2015; Evans et al., 2016; Hänfling et al., 2016; Clarke, Beard,
Swadling, & Deagle, 2017). However, the relationship between organism
biomass and number of sequences can be variable due to PCR amplification
bias of barcode primers (Albaina, Aguirre, Abad, Santos, & Estonba,
2016), and such biases were observed in our previous studies (Guo,
Zhang, Chen, & Zhang, 2018). The utilization of different sets of
barcode primers amplifying different gene regions could further improve
the taxonomic resolution of the dietary items. Besides the
consideration
of primers, the prey taxa are important factors associated with the
amplification bias. Our previous research showed that the sequencing
process consistently overrepresented some herbage taxa (e.g.,
Leguminosae and Compositae) and underestimated others (e.g., Gramineae)
relative to actual biomass. Nevertheless, a significant positive
correlation between the DNA sequence proportion acquired from the
sequencing procedure of feces samples and the actual biomass proportion
in the diet was observed in this study,
providing
support for quantitative investigation using metabarcoding dietary
analysis. Additionally, the determination of relative abundances of
target items could be improved through post-sequencing quantification
methods or correction factors (Matesanz et al., 2019).
Although metabarcoding is considered as a robust technique for assessing
a species’ presence and its relative abundance, there are some
shortcomings. Some common Poaceae species (such as Zea mays ) were
missing from the diet of lambs in the time-limited group, thus being
consistent with the findings from an earlier study (Guo, Zhang, Chen, &
Zhang, 2018). The lack of certain food taxa may have resulted from
methodological biases or from a draconian cutoff in the PCR protocols,
as proposed by Vesterinen et al. (2016) and Clarke et al. (2014). In
addition, incidental consumption of non-food DNA in the drinking water
and fungi in the digestive tract can hinder the identification of
certain groups. Furthermore, metabarcoding depends on single gene
regions to detect food items, and this may cause that numerous related
species to be indistinguishable (e.g., Lespedeza potaninii andLespedeza chinensis ).
Having
said this, the utilization of primer sets targeting composite barcodes
(e.g., ITS2 and trn H-psb A) should be taken into
consideration in future studies to further improve the taxonomic
resolution and obtain a more thorough analysis (Pang, Shi, Song, Chen,
& Chen, 2013). Likewise, establishment of reference databases targeting
regional location-based items has been suggested as an important
consideration of diet studies.
In conclusion, our results demonstrated that grazing systems could
affect the fatty acid composition and promote n-3 PUFAs deposition in
Tan sheep. Grazing and time-limited grazing feeding systems had lower
ratios of n-6/n-3 fatty acids in the muscle than
stall-fed modes,
a result that was more in line
with human health needs. Moreover, this research systematically studied
the correlations between herbage taxa and omega-3 fatty acids and
thereby has provided an initial view of the herbage effect on PUFAs of
lambs. The significant correlations found in this study provide
experimental evidence for future feeding research. The diet-PUFAs in the
meat quality regulation of sheep may be one of the key research points
for in-depth understanding of the flavor accumulation of beneficial
health-related PUFAs.