1. Introduction
Animal feed's nutritional value is essential for quality and safe feed consumption and animal welfare. In addition to this fact and due to the great variability of the raw materials used to feed animals, it is necessary to develop strategies focused on tight controls of animal feed products. These should be together with the research and development of new, simple, economical, and robust methods for monitoring quality and safety parameters.
Any edible part of the plant that can be harvested or fed to animals, other than separate grains, is known as forage. It is one of the main feed products in animal husbandry and must therefore be subject to safety and quality controls. Among the most important quality parameters of forages, the following three can be highlighted. Fiber content is mainly provided by the fodder cell wall and represents its carbohydrate. Mineral content (ash) gives information about possible contamination with soil. and supplies micronutrients to the diet, it also provides information on the quality of the forage. Finally, the protein content, the importance of which lies in its influence on animal production.
Near Infrared spectroscopy (NIRS) techniques have always been valued and used for forage quality analysis [
1,
2,
3,
4,
5,
6] due to the speed of analysis, the simplicity of sampling, the non-invasive nature of the technique [
7,
8,
9] and the possibility to be implemented in the production line [
10]. In addition to the advantages of this technique, the possibility of developing miniaturized NIR systems, easy to use and specialized in quality control of raw forages used in animal feed, makes it possible to increase quality control (sampling). The use of easy to use portable NIRS instrumentation minimize time losses, because nonspecialized personnel are required to analyse samples on-site and a real-time response is achieved as soon as analysis is carried out. These mentioned characteristics are some of those required in precision farming.
Taking into account all these considerations, this work evaluates the feasibility of a low-cost (<$1,000) optoelectronic measurement system prototype to analyze forage samples. The core of the measurement system is a NIR spectrometer based on the Texas Instruments NIRscan Nano Evaluation Module (EVM). This module has a large sensing area and high resolution to analyse forage samples. To evaluate the feasibility of the prototype, different ways of presenting the sample, intact (raw) or ground, were studied.
This equipment has already been tested for its use in liquid sample analysis [
11], with a specific cuvette for liquid samples. For this purpose, alfalfa samples were analysed. Alfalfa is one of the main forages used for animal feeding, due to its high biomass production, protein, and fiber contents. In summary, this work contributes:
To confirm the efficiency of the proposed measurement system.
To find out what are the qualities of the equipment.
To look for aspects to improve and implement in the future.
The remainder of this paper is organized as follows:
Section 2 introduces the main features of the measurement system developed.
Section 3 focuses on methods for analysing dairy farm forage quality.
Section 4 reports on experimental and discussion.
Section 5 contains conclusions.
4. Results and Discussion
To understand a NIRS procedure, the characterization of the spectra data obtained with the NIRscan nano prototype is essential.
Figure 4 shows raw and SNV plus 1
st Savitzky Golay derivative spectra of alfalfa samples involved in the calibration procedure. Within the NIR wavelength range of the NIRscan nano prototype, we can identify some characteristic bands of forages [
7]. According to the bibliography, those bands observed at 1166 nm are related to the protein content of the samples [
19] and those observed around 1350 nm are related to the fibre content [
20]. And around 1400 nm there is a band that can give information about moisture content because at that wavelength OH bond overtone vibrations are observed [
7].
Figure 4 and
Figure 5 have been highlighted with a rectangle all the cited wavelengths and the referenced respective parameters. Hence, in
Figure 4B, which is an extended area between 1650 and 1700 nm of
Figure 4A we can observe that some of the collected spectra show a noisy signal at the end of the collected spectrum. This noisy wavelength range, as shown in
Figure 4C, can be minimized after applying scattering correction (SNV) and other math pretreatments, such as derivatives.
Figure 4.
Spectra of the whole samples: (A) raw spectra; (B) Extended area from 1650 to 1700nm; (C) Standard Normal Variate +1st Savitzky Golay derivative pretreatment.
Figure 4.
Spectra of the whole samples: (A) raw spectra; (B) Extended area from 1650 to 1700nm; (C) Standard Normal Variate +1st Savitzky Golay derivative pretreatment.
Figure 5.
Spectra of the ground samples: (A) raw spectra; (B) Extended area from 1650 to 1700nm; (C) Standard Normal Variate + 1st Savitzky Golay derivative pretreatment.
Figure 5.
Spectra of the ground samples: (A) raw spectra; (B) Extended area from 1650 to 1700nm; (C) Standard Normal Variate + 1st Savitzky Golay derivative pretreatment.
To evaluate the effect of the sample pretreatment on the spectra data set, ground samples were scanned with NIRscan nano.
Figure 5A shows the spectra data set. As can be seen, no differences in the representative bands are observed. Moreover, the extended wavelength range (1650-1700 nm, see
Figure 4B and
Figure 5B) shows that after milling the sample pretreatment the noisy wavelength range disappears. These data confirm that spectra quality depends on the sampling procedure (raw or ground). This distorted wavelength range is due to the huge and non-homogeneous particle size of alfalfa samples. It is worthy to mention, that after applying math pretreatments no differences were observed in the collected spectra of alfalfa samples (see
Figure 4C and
Figure 5C).
As observed in
Figure 4 and
Figure 5, between 1600 and 1700 nm the absorbance increases, and the SNR is lower than in other ranges. It is because the intensity measured at the detector is proportional to the number of DMD mirrors positioned to reflect the incident illumination towards it. As the number of pixels changes, the measured intensity is affected as well, resulting in an increase in noise levels.
Once the spectra were evaluated, the precision of the subsampling procedure for each scanned sample (raw and ground alfalfa) was evaluated [
21]. Five samples were randomly selected from the 57 analysed. The RMS value (Equation 1) was calculated for both intact and ground samples with the two ranges proposed (901−1700 nm and 901−1600 nm). Results are shown in
Table 2.
Table 2.
Root mean square error (RMS) values for paired subsamples of the same scanned sample.
Table 2.
Root mean square error (RMS) values for paired subsamples of the same scanned sample.
|
901−1700 nm |
901−1600 nm |
Sample |
Raw |
Ground |
Raw |
Ground |
1 |
60529 |
27209 |
43283 |
14560 |
2 |
58378 |
23853 |
33386 |
10886 |
3 |
54190 |
20057 |
34085 |
7029 |
4 |
54854 |
30655 |
36262 |
17678 |
5 |
48472 |
33325 |
34239 |
21939 |
Once the values have been calculated, two clear trends can be observed. As expected, the RMS values obtained for intact samples (raw) are higher than those obtained for ground ones. These results could be due to raw alfalfa heterogeneity. When comparing the entire range or suppressing the last 100 nm of the spectrum, we also observed a significant difference.
Table 2 shows that RMS values ranged from 900 to 1600 nm are lower than the full ranging values. These results highlight the influence of the sampling procedure on spectra data precision.
After characterizing the spectral signal, the next step was to develop a calibration model. To attempt calibration, it is necessary to build a data matrix including nutritive values (NDF, MC and CP) and spectra data. After that, prior to carrying out calibrations, as mentioned in the Material and Methods section, different mathematical pre-treatments were applied for the three parameters on raw and ground samples, both for the full range and the reduced range. Partial Least Square Regression is used to establish the correlation between spectra and assayed parameters.
Table 3 summarizes NIRS models' calibration statistics to quantify NDF. As can be seen in
Table 3, R
2 values are higher and SEC values are lower in chemometric models developed with the reduced wavelength range than in those developed using the full one. Related to the variability of the results depending on the math pretreatment, it is important to remark that SNV plus the second Savizky Golay derivative reached the best calibration statistics for raw and ground samples. Previous authors [
22] after evaluating different commercial portable NIRS instruments to analyze ground forages, for NDF obtained R
2 values ranging between 0.95-0.71 depending on the employed instrumentation. Regarding SEC values, their results were between 2.85-1.21. Being not possible to obtain SEC values lower than 1 because the standard error of the laboratory (SEL) for this parameter is higher than 1.3 [
22].
Table 3.
Calibration statistics of NIR multivariate models for Neutral Detergent Fiber quantification.
Table 3.
Calibration statistics of NIR multivariate models for Neutral Detergent Fiber quantification.
Raw alfalfa |
Wavelength Range: |
901-1600 nm |
901-1700 nm |
Math pretreatment |
R2 |
SEC |
R2 |
SEC |
1 4 4 SG |
0.898 |
1.554 |
0.883 |
1.670 |
2 4 4 SG |
0.784 |
2.184 |
0.786 |
2.289 |
SNV 1 4 4SG |
0.911 |
1.392 |
0.791 |
2.187 |
1 4 4 SG SNV |
0.840 |
1.910 |
0.145 |
1.398 |
SNV 2 4 4SG |
0.955 |
1.066 |
0.514 |
3.155 |
2 4 4 SG SNV |
0.726 |
2.558 |
0.540 |
3.238 |
Ground Alfalfa |
Wavelength Range: |
901-1600 nm |
901-1700 nm |
Math pretreatment |
R2 |
SEC |
R2 |
SEC |
1 4 4 SG |
0.756 |
2.749 |
0.598 |
2.830 |
2 4 4 SG |
0.842 |
2.258 |
0.623 |
3.371 |
SNV 1 4 4SG |
0.761 |
2.694 |
0.043 |
5.383 |
1 4 4 SG SNV |
0.796 |
2.421 |
0.510 |
3.946 |
SNV 2 4 4SG |
0.892 |
1.861 |
0.540 |
3.803 |
2 4 4 SG SNV |
0.730 |
2.860 |
0.524 |
3.321 |
Table 4 summarizes the calibration statistics for CP. Most math treatments reach R
2 values lower than 0.5 for raw alfalfa samples. This could be related to the heterogeneity of alfalfa forage, with two clearly different parts, the leaf and the stem. The leaf is part of the plant containing protein fraction. However, it is important to remark that using the reduced range, the spectra math pretreatment of SNV for scatter correction and the second Savitzky Golay derivative (the same math pretreatment as for NDF), R
2 values of 0.885, with a SEC of 0.377 were achieved. A typical SEL for reference CP analysis is around 0.210 [
22].
Table 4.
Calibration statistics of NIR multivariate models for Crude Protein quantification
Table 4.
Calibration statistics of NIR multivariate models for Crude Protein quantification
Raw alfalfa |
Wavelength Range: |
901-1600 nm |
901-1700 nm |
Math pretreatment |
R2 |
SEC |
R2 |
SEC |
1 4 4 SG |
0.742 |
0.510 |
0.884 |
0.428 |
2 4 4 SG |
0.262 |
0.911 |
0.608 |
1.262 |
SNV 1 4 4SG |
0.307 |
1.314 |
0.156 |
1.524 |
1 4 4 SG SNV |
0.678 |
0.842 |
0.257 |
1.378 |
SNV 2 4 4SG |
0.885 |
0.377 |
0.345 |
0.855 |
2 4 4 SG SNV |
0.328 |
0.812 |
0.318 |
1.368 |
Grounded Alfalfa |
Wavelength Range: |
901-1600 nm |
901-1700 nm |
Math pretreatment |
R2 |
SEC |
R2 |
SEC |
1 4 4 SG |
0.671 |
0.986 |
0.706 |
0.927 |
2 4 4 SG |
0.906 |
0.530 |
0.290 |
1.014 |
SNV 1 4 4SG |
0.773 |
0.816 |
0.790 |
0.650 |
1 4 4 SG SNV |
0.734 |
0.882 |
0.723 |
0.651 |
SNV 2 4 4SG |
0.862 |
0.660 |
0.216 |
1.145 |
2 4 4 SG SNV |
0.820 |
0.746 |
0.179 |
1.433 |
Developed models carried out with ground samples and using a reduced range (901-1600) showed statistics around 0.7 or higher, with SEC values between 0.530 and 0.986. Considering these results, it is worth mentioning that, even though the homogeneity of ground samples gives better calibration statistics, NIRscan nano reached acceptable values when scanning raw samples.
Feeding animals with minerals is a common practice, however, an abnormal mineral content (MC) there, is a big probability of contamination with soil, which is not desirable for animal feeding systems. To quantify MC in alfalfa forages, 24 different calibration models have been developed assaying different math pretreatments of spectra data. Statistics of proposed PLS models are shown in
Table 5. As stated before, the reduced range gave better calibration statistics than the full one. Comparing math pretreatments, scatter correction applied after the derivatization procedure increased R
2 values and reduced SEC. The highest R
2 and the lowest SEC values were 0.861/0.219 and 0.867/0.318 for raw and ground samples respectively.
Table 5.
Calibration statistics of Mineral Content multivariate models
Table 5.
Calibration statistics of Mineral Content multivariate models
Raw alfalfa |
Wavelength Range: |
901-1600 nm |
901-1700 nm |
Math pretreatment |
R2 |
SEC |
R2 |
SEC |
1 4 4 SG |
0.524 |
0.503 |
0.211 |
0.572 |
2 4 4 SG |
0.619 |
0.492 |
0.129 |
0.861 |
SNV 1 4 4SG |
0.783 |
0.374 |
0.734 |
0.464 |
1 4 4 SG SNV |
0.502 |
0.579 |
0.312 |
0.491 |
SNV 2 4 4SG |
0.675 |
0.409 |
0.679 |
0.434 |
2 4 4 SG SNV |
0.861 |
0.219 |
0.687 |
0.444 |
Grounded Alfalfa |
Wavelength Range: |
901-1600 nm |
901-1700 nm |
Math pretreatment |
R2 |
SEC |
R2 |
SEC |
1 4 4 SG |
0.650 |
0.530 |
0.652 |
0.506 |
2 4 4 SG |
0.770 |
0.435 |
0.243 |
0.819 |
SNV 1 4 4SG |
0.570 |
0.586 |
0.670 |
0.519 |
1 4 4 SG SNV |
0.867 |
0.318 |
0.347 |
0.723 |
SNV 2 4 4SG |
0.604 |
0.566 |
0.591 |
0.579 |
2 4 4 SG SNV |
0.781 |
0.424 |
0.301 |
0.625 |
These NDF, CP and MC calibration model statistics, obtained with the NIRscan nano prototype, are similar to those acquired with commercial portable instruments using a wavelength range similar to this evaluated in this work [
22,
23]. SEC values are in accordance with laboratory results [
22] and the effect of the sampling procedure has been studied comparatively in this work. As a summary of the obtained results,
Table 6 selects the best models obtained for each sampling procedure (raw or ground alfalfa) and parameter. As can be seen, the second derivative is the best of the assayed pretreatments that provide satisfactory results for nutritive value quantification.
Table 6.
Statistical analysis of alfalfa nutritive values (N = 57).
Table 6.
Statistical analysis of alfalfa nutritive values (N = 57).
Parameter |
Sampling |
Math pretreatment |
Range (nm) |
R2
|
SEC |
NDF |
Raw |
SNV 2 4 4 SG |
900-1600 |
0.955 |
1.066 |
Ground |
SNV 2 4 4 SG |
900-1600 |
0.892 |
1.861 |
CP |
Raw |
SNV 2 4 4 SG |
900-1600 |
0.885 |
0.377 |
Ground |
2 4 4 SG |
900-1600 |
0.906 |
0.530 |
MC |
Raw |
2 4 4 SG SNV |
900-1600 |
0.861 |
0.219 |
|
Ground |
1 4 4 SG SNV |
900-1600 |
0.867 |
0.318 |
5. Conclusions and Future Work
This work reports on a miniaturized optoelectronic measurement system for decentralized agrifood quality control. Heterogeneous forage (alfalfa) has been selected as a model to evaluate the precision of instrumental measures (spectra collected) and the effect of sampling presentation (raw or ground) on calibration statistics. Results have revealed that homogeneous forage samples (those milled) allow reaching better calibration models than those scanned in their raw form (heterogeneous). However, for all sampling procedures, it has been possible to obtain satisfactory calibration to quantify nutritive parameters.
This technological proposal combined with proper chemometric tools offers an excellent alternative to on-site, real-time, and non-destructive analysis of agrifood products. In addition to providing owners and technicians with easy-to-use, the proposed measurement system offers a means of evaluating forage quality, taking more samples without incurring expenses, obtaining real-time results, and making quick decisions.
In the future, Internet access will be useful for sharing information between different NIRscan nano instruments. These instruments can be standardized and share calibration models to quantify nutritive parameters. Moreover, a common database of spectra collected with different instruments can be used as a reference to increase sample variability and improve calibration statistics.