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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) Average Raman spectra of 300 X- (purple line) and 300 Y-sperm cells (blue line) in the “fingerprint” spectral region; (B) Comparison between the Raman spectra of X- and Y-spermatozoa in the spectral region between 700–850 cm−1 and (C) 1400–1650 cm−1. (D) Measured peak area of the characteristic DNA bands at 726 and 785 cm−1 for X- and Y-spermatozoa.
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biosensors-05-00141-f009: (A) Average Raman spectra of 300 X- (purple line) and 300 Y-sperm cells (blue line) in the “fingerprint” spectral region; (B) Comparison between the Raman spectra of X- and Y-spermatozoa in the spectral region between 700–850 cm−1 and (C) 1400–1650 cm−1. (D) Measured peak area of the characteristic DNA bands at 726 and 785 cm−1 for X- and Y-spermatozoa.

Mentions: Figure 9A shows typical Raman spectra, between 600 and 1800 cm−1, of X- (purple line) and Y-bearing sperm cells (blue line) of a bull acquired from the nucleus region. Each spectrum is an average of 300 cells. Just before the acquisition the sample autofluorescence was bleached exposing the cell for about 40 s to the laser light and Raman spectra were recorded using an integration time of 20 s. The spectra were corrected by subtracting the background spectrum (quartz slide and PBS solution) and normalized. The acquired spectra are like molecular fingerprints representing contributions from various cellular components such as DNA, protein, lipids and carbohydrates, and a summary of the wavenumbers and their corresponding band assignment is given in Table 1. The sperm spectra show the characteristic cell features: a strong Amide I band around 1660 cm−1, an intense CH deformation band around 1450 cm−1, as well as the sharp band at 1005 cm−1 assigned to the amino acid phenylalanine. The two spectra look very much alike, however, X- and Y-spermatozoa vary in their composition and therefore also in their Raman spectra. The X-bearing spermatozoa show increased intensity of the peaks at 726, 785 and 1581 cm−1 (see Figure 9B,C), that are assigned to ring breathing modes in the DNA bases, as well as in the 1095 cm−1 mode of the symmetric stretching vibration of the DNA backbone. These features can be attributed to slightly higher DNA concentration in X- than in Y-bearing sperm cells. An additional difference between the two population of spectra and be observed in the spectral regions between 1400 and 1650 cm−1 (see Figure 9C) mainly assigned to the protein content. This is probably due to the presence of HY antigen absent on the membrane of X-sperm cells. Peak area measures of Raman bands at 726 and 785 cm−1 performed on 300 X- and 300 Y-spermatozoa are reported in the histograms of Figure 9D. More precisely, the measured mean variation of the Raman band areas is ∆A = 4.1% ± 0.4%, which is in good agreement with the expected differences in DNA content (3.8% in bull sperm).


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) Average Raman spectra of 300 X- (purple line) and 300 Y-sperm cells (blue line) in the “fingerprint” spectral region; (B) Comparison between the Raman spectra of X- and Y-spermatozoa in the spectral region between 700–850 cm−1 and (C) 1400–1650 cm−1. (D) Measured peak area of the characteristic DNA bands at 726 and 785 cm−1 for X- and Y-spermatozoa.
© Copyright Policy
Related In: Results  -  Collection

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

biosensors-05-00141-f009: (A) Average Raman spectra of 300 X- (purple line) and 300 Y-sperm cells (blue line) in the “fingerprint” spectral region; (B) Comparison between the Raman spectra of X- and Y-spermatozoa in the spectral region between 700–850 cm−1 and (C) 1400–1650 cm−1. (D) Measured peak area of the characteristic DNA bands at 726 and 785 cm−1 for X- and Y-spermatozoa.
Mentions: Figure 9A shows typical Raman spectra, between 600 and 1800 cm−1, of X- (purple line) and Y-bearing sperm cells (blue line) of a bull acquired from the nucleus region. Each spectrum is an average of 300 cells. Just before the acquisition the sample autofluorescence was bleached exposing the cell for about 40 s to the laser light and Raman spectra were recorded using an integration time of 20 s. The spectra were corrected by subtracting the background spectrum (quartz slide and PBS solution) and normalized. The acquired spectra are like molecular fingerprints representing contributions from various cellular components such as DNA, protein, lipids and carbohydrates, and a summary of the wavenumbers and their corresponding band assignment is given in Table 1. The sperm spectra show the characteristic cell features: a strong Amide I band around 1660 cm−1, an intense CH deformation band around 1450 cm−1, as well as the sharp band at 1005 cm−1 assigned to the amino acid phenylalanine. The two spectra look very much alike, however, X- and Y-spermatozoa vary in their composition and therefore also in their Raman spectra. The X-bearing spermatozoa show increased intensity of the peaks at 726, 785 and 1581 cm−1 (see Figure 9B,C), that are assigned to ring breathing modes in the DNA bases, as well as in the 1095 cm−1 mode of the symmetric stretching vibration of the DNA backbone. These features can be attributed to slightly higher DNA concentration in X- than in Y-bearing sperm cells. An additional difference between the two population of spectra and be observed in the spectral regions between 1400 and 1650 cm−1 (see Figure 9C) mainly assigned to the protein content. This is probably due to the presence of HY antigen absent on the membrane of X-sperm cells. Peak area measures of Raman bands at 726 and 785 cm−1 performed on 300 X- and 300 Y-spermatozoa are reported in the histograms of Figure 9D. More precisely, the measured mean variation of the Raman band areas is ∆A = 4.1% ± 0.4%, which is in good agreement with the expected differences in DNA content (3.8% in bull sperm).

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