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Label-free imaging and biochemical characterization of bovine sperm cells.

Ferrara MA, Di Caprio G, Managò S, De Angelis A, Sirleto L, Coppola G, De Luca AC - Biosensors (Basel) (2015)

Bottom Line: A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination.In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS).We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols.

View Article: PubMed Central - PubMed

Affiliation: Institute for Microelectronics and Microsystems, National Research Council, Via P. Castellino, 111, 80131 Naples, Italy. antonella.ferrara@na.imm.cnr.it.

ABSTRACT
A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols.

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(A) 3D Principal Component Analysis (PCA) score plot comparing 900 X- and 900 Y-spermatozoa from 3 bulls; (B) Confusion matrix giving the classification for X-and Y-spermatozoa.
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biosensors-05-00141-f010: (A) 3D Principal Component Analysis (PCA) score plot comparing 900 X- and 900 Y-spermatozoa from 3 bulls; (B) Confusion matrix giving the classification for X-and Y-spermatozoa.

Mentions: Principal component analysis (PCA) was used to visualize spectral differences and cluster formation according to the cell type. Figure 10A shows the 3D score plot on 1800 spectra, 300 Raman spectra of X- and Y-spermatozoa from three different bulls, where the principal components PC2, PC3 and PC4 are plotted against each other. Each cell type is clearly separated from the other one as indicated by the different marked and colored scores in Figure 10A. The first principal component PC1 is not included in the data classification as essentially due to the background variation and not directly attributed to the cell differences. PC2, PC3 and PC4 loadings reveal the most feature-rich plot [3]. Particularly evident are the peaks in the spectral regions around 780, 1100 and 1580 cm−1 that match the DNA vibrational modes, and around 1350–1450 cm−1, revealing the different contribution of the sex-associated membrane proteins in the two cell types [3]. In order to discriminate and assign X- and Y-spermatozoa, a confusion matrix was built up using the leave-one-out classification approach [4,22]. The details of the prediction for the individual cell types are depicted in Figure 10B: 1631 out of 1800 spectra could be classified correctly. This results in the high prediction accuracy of 90.2%.


Label-free imaging and biochemical characterization of bovine sperm cells.

Ferrara MA, Di Caprio G, Managò S, De Angelis A, Sirleto L, Coppola G, De Luca AC - Biosensors (Basel) (2015)

(A) 3D Principal Component Analysis (PCA) score plot comparing 900 X- and 900 Y-spermatozoa from 3 bulls; (B) Confusion matrix giving the classification for X-and Y-spermatozoa.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493542&req=5

biosensors-05-00141-f010: (A) 3D Principal Component Analysis (PCA) score plot comparing 900 X- and 900 Y-spermatozoa from 3 bulls; (B) Confusion matrix giving the classification for X-and Y-spermatozoa.
Mentions: Principal component analysis (PCA) was used to visualize spectral differences and cluster formation according to the cell type. Figure 10A shows the 3D score plot on 1800 spectra, 300 Raman spectra of X- and Y-spermatozoa from three different bulls, where the principal components PC2, PC3 and PC4 are plotted against each other. Each cell type is clearly separated from the other one as indicated by the different marked and colored scores in Figure 10A. The first principal component PC1 is not included in the data classification as essentially due to the background variation and not directly attributed to the cell differences. PC2, PC3 and PC4 loadings reveal the most feature-rich plot [3]. Particularly evident are the peaks in the spectral regions around 780, 1100 and 1580 cm−1 that match the DNA vibrational modes, and around 1350–1450 cm−1, revealing the different contribution of the sex-associated membrane proteins in the two cell types [3]. In order to discriminate and assign X- and Y-spermatozoa, a confusion matrix was built up using the leave-one-out classification approach [4,22]. The details of the prediction for the individual cell types are depicted in Figure 10B: 1631 out of 1800 spectra could be classified correctly. This results in the high prediction accuracy of 90.2%.

Bottom Line: A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination.In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS).We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols.

View Article: PubMed Central - PubMed

Affiliation: Institute for Microelectronics and Microsystems, National Research Council, Via P. Castellino, 111, 80131 Naples, Italy. antonella.ferrara@na.imm.cnr.it.

ABSTRACT
A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols.

Show MeSH
Related in: MedlinePlus