Animal Reproduction (AR)
Animal Reproduction (AR)

Effectiveness of near-infrared spectroscopy as a non-invasive tool to discriminate spectral profiles of in vitro cultured oocytes from goats

Denilsa Pires Fernandes; Rafael Rossetto; Assis Rubens Montenegro; César Carneiro Linhares Fernandes; Pamela Angela Bravo; Maria Eugenia Moreno; Camila Muniz Cavalcanti; Guilherme Araújo Kubota; Davide Rondina

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Here, we aimed to discriminate between the spectral profiles of spent culture media after oocyte in vitro maturation (IVM) and culture (IVC) from goats of different ages subjected to repeated hormonal treatments. The profiles were discriminated using near infrared (NIR) spectroscopy combined with multivariate methods. A total of 19 goats (young = 10; old = 9) were subjected to serial hormonal stimulation (HS) with gonadotropins. Cumulus oophorus complexes (COCs) were collected using laparoscopic ovum pick-up (LOPU) and subjected to IVM and parthenogenetic activation. The initial embryos were subjected to IVC. Spent culture media were collected after oocyte IVM and on day 2 of IVC and analyzed using NIR spectroscopy. NIR spectral data were interpreted through chemometric methods, such as principle component analysis (PCA) and partial least square discriminant analysis (PLS-DA). The results of PCA analysis clearly showed a separation in the spectral profiles between the experimental groups (HS sessions; young and old animals) both after IVM and IVC. Overall, the main absorption bands were attributed to the C-H group second overtone, first overtone of O-H and N-H, and C-H combinations and may serve as molecular markers. On the other hand, the spectral data obtained using PLS-DA models provided a better classification of the groups. The results showed the possibility of discriminating young and old groups as well as the three HS sessions with high specificity, sensitivity, and accuracy using NIR spectra. Thus, the culture medium analysis using NIR spectroscopy combined with multivariate methods indicated the dissimilarities between the groups and provided an insight into the in vitro development of goat oocytes. This technique serves as an efficient, objective, rapid, and non-invasive method to discriminate spectral profiles.


near infrared, spectroscopy, culture medium, oocyte, goat


Ahlström A, Wikland M, Rogberg L, Barnett JS, Tucker M, Hardarson T. Cross-validation and predictive value of near-infrared spectroscopy algorithms for day-5 blastocyst transfer. Reprod Biomed Online. 2011;22(5):477-84. PMid:21397559.

Avelar SRV, Moura RR, Sousa FC, Pereira AF, Almeida KC, Melo CHS. Oocyte production and in vitro maturation in Canindé goats following hormonal ovarian stimulation. Anim Reprod. 2012;9:27-32.

Baldassarre H, Currin L, Michalovic L, Bellefleur AM, Gutierrez K, Mondadori RG, Glanzner WG, Schuermann Y, Bohrer RC, Dicks N, Lopez R, Grand FX, Vigneault C, Blondin P, Gourdon J, Bordignon V. Interval of gonadotropin administration for in vitro embryo production from oocytes collected from Holstein calves between 2 and 6 months of age by repeated laparoscopy. Theriogenology. 2018;116:64-70. PMid:29778922.

Baldassarre H, Karatzas CN. Advanced assisted reproduction Technologies (ART) in goats. Anim Reprod Sci. 2004;82-83:255-66. PMid:15271458.

Baldassarre H, Rao KM, Neveu N, Brochu E, Begin I, Behboodi E, Hockley DK. Laparoscopic ovum pick-up followed by in vitro embryo production for the reproductive rescue of aged goats of high genetic value. Reprod Fertil Dev. 2007;19(5):612-6. PMid:17601408.

Baldassarre H, Wang B, Kafidi N, Gauthier M, Neveu N, Lapointe J, Sneek L, Leduc M, Duguay F, Zhou JF, Lazaris A, Karatzas CN. Production of transgenic goats by pronuclear microinjection of in vitro produced zygotes derived from oocytes recovered by laparoscopy. Theriogenology. 2003;56(3-4):831-9. PMid:12517386.

Bik E, Ishigaki M, Blat A, Jasztal A, Ozaki Y, Malek K, Baranska M. Lipid droplet composition varies based on medaka fish eggs development as revealed by NIR-, MIR-, and Raman Imaging. Molecules. 2020;25(4):817. PMid:32070018.

Botros L, Sakkas D, Seli E. Metabolomics and its application for non-invasive embryo assessment in IVF. Mol Hum Reprod. 2008;14(12):679-90. PMid:19129367.

Chen H, Lin Z, Mo L, Wu T, Tan C. Near-infrared spectroscopy as a diagnostic tool for distinguishing between normal and malignant colorectal tissues. BioMed Res Int. 2015;2015:472197. PMid:25654106.

Currà A, Gasbarrone R, Cardillo A, Trompetto C, Fattapposta F, Pierelli F, Missori P, Bonifazi G, Serranti S. Near-infrared spectroscopy as a tool for in vivo analysis of human muscles. Sci Rep. 2019;9(1):8623. PMid:31197189.

Currin L, Michalovic L, Bellefleur AM, Gutierrez K, Glanzner W, Schuermann Y, Bohrer RC, Dicks N, da Rosa PR, De Cesaro MP, Lopez R, Grand FX, Vigneault C, Blondin P, Gourdon J, Baldassarre H, Bordignon V. The effect of age and length of gonadotropin stimulation on the in vitro embryo development of Holstein calf oocytes. Theriogenology. 2017;104:87-93. PMid:28822904.

Damiani P, Fissore RA, Cibelli JB, Long CR, Balise JJ, Robl JM, Duby RT. Evaluation of developmental competence, nuclear and ooplasmic maturation of calf oocytes. Mol Reprod Dev. 1996;45(4):521-34.<521::AID-MRD15>3.0.CO;2-Z. PMid:8956291.

Fernandes CCL, Feltrin C, Martins LT, Gaudêncio Neto S, Aguiar LH, Silva AM, Oliveira CH, Silva LM, Silva CM, Bertolini M, Rondina D. Goat oocyte quality and competence to undergo IVM and embryo development after parthenogenetic activation from goats fed with different levels of cashew nut bran as source of dietary lipids. Theriogenology. 2014;82(2):332-7. PMid:24853280.

Freitas VJF, Souza-Fabjan JMG, Mermillod P, Melo LM, Teixeira DIA. Estado da arte e perspectivas da produção in vitro de embriões em caprinos. Rev Bras Reprod Anim. 2017;41:201-7.

Gibbons A, Pereyra Bonnet F, Cueto MI, Catala M, Salamone DF, Gonzalez-Bulnes A. Procedure for maximizing oocyte harvest for In Vitro embryo production in small ruminants. Reprod Domest Anim. 2007;42(4):423-6. PMid:17635781.

Gosselin R, Rodrigue D, Duchesne C. A bootstrap-VIP approach for selecting wavelength intervals in spectral imaging application. Chemom Intell Lab Syst. 2010;100(1):12-21.

Gromski PS, Muhamadali H, Ellis DI, Xu Y, Correa E, Turner ML, Goodacre R. A tutorial review: metabolomics and partial least squares-discriminant analysis–a marriage of convenience or a shotgun wedding. Anal Chim Acta. 2015;879:10-23. PMid:26002472.

Huang J, Romero-Torres S, Moshgbar M. Raman: practical considerations in data pre-treatment for Nir and Raman spectroscopy. Am Pharm Rev. 2010;13:116.

Ishigaki M, Hashimoto K, Sato H, Ozaki Y. Non-destructive monitoring of mouse embryo development and its qualitative evaluation at the molecular level using Raman spectroscopy. Sci Rep. 2017;7(1):43942. PMid:28272511.

Ishigaki M, Kawasaki S, Ishikawa D, Ozaki Y. Near-Infrared Spectroscopy and Imaging Studies of Fertilized Fish Eggs: In Vivo Monitoring of Egg Growth at the Molecular Level. Sci Rep. 2016;6(1):20066. PMid:26818027.

Ishigaki M, Taketani A, Sato H. Discrimination of fish egg quality and viability by Raman spectroscopy. Anal Methods. 2014;6(23):9206-11.

Kirdar AO, Chen G, Weidner J, Rathore AS. Application of near‐infrared (NIR) spectroscopy for screening of raw materials used in the cell culture medium for the production of a recombinant therapeutic protein. Biotechnol Prog. 2010;26(2):527-31. PMid:19938040.

Kirkegaard K, Svane ASP, Nielsen JS, Hindkjær JJ, Nielsen NC, Ingerslev HJ. Nuclear magnetic resonance metabolomic profiling of Day 3 and 5 embryo culture medium does not predict pregnancy outcome in good prognosis patients: a prospective cohort study on single transferred embryos. Hum Reprod. 2014;29(11):2413-20. PMid:25256566.

Koeman J, Keefer CL, Baldassarre H, Downey BR. Developmental competence of prepubertal and adult goat oocytes cultured in semi-defined media following laparoscopic recovery. Theriogenology. 2003;60(5):879-89. PMid:12935865.

Leary C, Sturmey RG. Metabolic profile of in vitro derived human embryos is not affected by the mode of fertilization. Mol Hum Reprod. 2020;26(4):277-87. PMid:32059054.

Lee LC, Liong CY, Jemain AA. Partial least squares-discriminant analysis (PLS-DA) for classification of high-dimensional (HD) data: a review of contemporary practice strategies and knowledge gaps. Analyst. 2018;143(15):3526-39. PMid:29947623.

Leoni GG, Palmerini MG, Satta V, Succu S, Pasciu V, Zinellu A, Carru C, Macchiarelli G, Nottola SA, Naitana S, Berlinguer F. Differences in the kinetic of the first meiotic division and in active mitochondrial distribution between prepubertal and adult oocytes mirror differences in their developmental competence in a sheep model. PLoS One. 2015;10(4):e0124911. PMid:25893245.

Leoni GG, Succu S, Satta V, Paolo M, Bogliolo L, Bebbere D, Spezzigu A, Madeddu M, Berlinguer F, Ledda S, Naitana S. In vitro production and cryotolerance of prepubertal and adult goat blastocysts obtained from oocytes collected by laparoscopic oocyte-pick-up (LOPU) after FSH treatment. Reprod Fertil Dev. 2009;21(7):901-8. PMid:19698294.

Li X, Xu Y, Fu J, Zhang WB, Liu SY, Sun XX. Non-invasive metabolomic profiling of embryo culture media and morphology grading to predict implantation outcome in frozen-thawed embryo transfer cycles. J Assist Reprod Genet. 2015;32(11):1597-605. PMid:26463877.

Li XX, Cao PH, Han WX, Xu YK, Wu H, Yu XL, Chen JY, Zhang F, Li YH. Non-invasive metabolomic profiling of culture media of ICSI-and IVF-derived early developmental cattle embryos via Raman spectroscopy. Anim Reprod Sci. 2018;196:99-110. PMid:30001829.

Mariano RSG, Uscategui RAR, Nociti RP, da Camara Barros FFP, Feliciano MAR, Rodriguez MGK, Taira AR, Teixeira PPM, Vicente WRR. Aspiração folicular em ruminantes–revisão de literatura. Investigação. 2015;14:46-53.

McLennan HJ, Saini A, Dunning KR, Thompson JG. Oocyte and embryo evaluation by AI and multi-spectral auto-fluorescence imaging: livestock embryology needs to catch-up to clinical practice. Theriogenology. 2020;150:255-62. PMid:32088032.

Morand-Fehr P, Gall C. Nutrition and feeding of goats: application to temperate climatic condition; Goat production. London: Academic Press; 1981.

Muñoz M, Uyar A, Correia E, Díez C, Fernandez-Gonzalez A, Caamaño JN, Trigal B, Carrocera S, Seli E, Gomez E. Non-invasive assessment of embryonic sex in cattle by metabolic fingerprinting of in vitro culture medium. Metabolomics. 2014;10(3):443-51.

Nagy ZP, Jones-Colon S, Roos P, Botros L, Greco E, Dasig J, Behr B. Metabolomic assessment of oocyte viability. Reprod Biomed Online. 2009;18(2):219-25. PMid:19192342.

Paramio MT. In vivo and in vitro embryo production in goats. Small Rumin Res. 2010;89(2-3):144-8.

Pasquini C. Near infrared spectroscopy: fundamentals, practical aspects and analytical applications. J Braz Chem Soc. 2003;14(2):198-219.

R Core Team. R: a language and environment for statistical computing [Internet]. Vienna, Austria: R Foundation for Statistical Computing; 2019 [cited 2021 Jan 11]. Available from:

Rødgaard T, Heegaard PMH, Callesen H. Non-invasive assessment of in vitro embryo quality to improve transfer success. Reprod Biomed Online. 2015;31(5):585-92. PMid:26380864.

Rohart F, Gautier B, Singh A, Lê Cao K-A. MixOmics: an R package for ‘omics feature selection and multiple data integration. PLOS Comput Biol. 2017;13(11):e1005752. PMid:29099853.

Rubessa M, Ambrosi A, Denmark SE, Wheeler MB. Non-invasive analysis of gamete metabolites during in vitro embryo production using nuclear magnetic resonance. International Journal of New Technology and Research. 2016;2:54-8.

Seli E, Botros L, Sakkas D, Burns DH. Non invasive metabolomic profiling of embryo culture media using proton nuclear magnetic resonance correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril. 2008;90(6):2183-9. PMid:18842260.

Seli E, Robert C, Sirard M. OMICS in assisted reproduction: possibilities and pitfalls. Mol Hum Reprod. 2010;16(8):513-30. PMid:20538894.

Seli E, Sakkas D, Scott R, Kwok SC, Rosendahl SM, Burns DH. Noninvasive metabolomic profiling of embryo culture media using Raman and near-infrared spectroscopy correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril. 2007;88(5):1350-7. PMid:17923129.

Souza-Fabjan JMG, Locatelli Y, Duffard N, Corbin E, Touzé JL, Perreau C, Beckers JF, Freitas VJF, Mermillod P. In vitro embryo production in goats: slaughterhouse and laparoscopic ovum pick up-derived oocytes have different kinetics and requirements regarding maturation media. Theriogenology. 2014a;81(8):1021-31. PMid:24582267.

Souza-Fabjan JMG, Panneau B, Duffard N, Locatell Y, Figueiredo JR, Freitas VJF, Mermillod P. In vitro production of small ruminant embryos: late improvements and further research. Theriogenology. 2014b;81(9):1149-62. PMid:24650929.

Souza-Fabjan JMG, Pereira AF, Melo CH, Sanchez DJ, Oba E, Mermillod P, Melo LM, Teixeira DIA, Freitas VJ. Assessment of the reproductive parameters, laparoscopic oocyte recovery and the first embryos produced in vitro from endangered Canindé goats (Capra hircus). Reprod Biol. 2013;13(4):325-32. PMid:24287041.

Szymańska E, Saccenti E, Smilde AK, Westerhuis JA. Double-check: validation of diagnostic statistics for PLS-DA models in metabolomics studies. Metabolomics. 2012;8(Suppl 1):3-16. PMid:22593721.

Vergouw CG, Botros LL, Roos P, Lens JW, Schats R, Hompes PGA, Burns DH, Lambalk CB. Metabolomic profiling by near-infrared spectroscopy as a tool to assess embryo viability: a novel, non-invasive method for embryo selection. Hum Reprod. 2008;23(7):1499-504. PMid:18424459.

Wiweko B, Zakia Z, Tedjo A, Widyahening IS, Pratama G, Hestiantoro A, Natadisastra M, Sumapraja K, Harzif AK, Zakirah SC. Prediction of good quality blastocyst formation by metabolomic profile of spent embryo culture media using FTIR Spectroscopy in Women undergoing IVF Cycle: a Cohort prospective study. Res Sq. 2020:1-19.

Xiaobo Z, Jiewen Z, Povey MJ, Holmes M, Hanpin M. Variables selection methods in near-infrared spectroscop. Anal Chim Acta. 2010;667(1-2):14-32. PMid:20441862.

Zhang YL, Zhang GM, Jia RX, Wan YJ, Yang H, Sun LW, Han L, Wang F. Non‐invasive assessment of culture media from goat cloned embryos associated with subjective morphology by gas chromatography–mass spectroscopy‐based metabolomic analysis. Anim Sci J. 2018;89(1):31-41. PMid:28833899.

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