Reprod Dom Anim 50, 522–525 (2015); doi: 10.1111/rda.12504 ISSN 0936–6768

Short Communication Aquagrams of Raw Milk for Oestrus Detection in Dairy Cows G Takemura1, G Baz ar1,2, K Ikuta3, E Yamaguchi3, S Ishikawa3, A Furukawa1, Y Kubota4, Z Kov acs1,5 and R Tsenkova1 1 Biomeasurement Technology Laboratory, Graduate School of Agricultural Science, Kobe University, Kobe, Japan; 2Institute of Food and Agricultural Product Qualification, Faculty of Agricultural and Environmental Sciences, Kaposv ar University, Kaposv ar, Hungary; 3Awaji Agricultural Institute, Hyogo Prefectural Technology Center for Agriculture, Forestry and Fisheries, Minamiawaji, Japan; 4Organization of Advanced Science and Technology, Kobe University, Kobe, Japan; 5Department of Physics and Control, Faculty of Food Science, Corvinus University of Budapest, Budapest, Hungary

Contents The purpose of this research was to develop rapid and costeffective method for oestrus detection in dairy cows by means of near infrared spectroscopy and aquaphotomics, using raw milk from individual cows. We found that aquaphotomics approach showed consistent specific water spectral pattern of milk at the oestrus periods of the investigated Holstein cows. Characteristic changes were detected especially in foremilk collected at morning milking. They were reflected in calculated aquagrams of milk spectra where distinctive spectral pattern of oestrus showed increased light absorbance of strongly hydrogen-bonded water. Results showed that monitoring of raw milk near infrared spectra provides an opportunity for analysing hormone levels indirectly, through the changes of water spectral pattern caused by complex physiological changes related to fertile periods.

for disease diagnosis in cows (Tsenkova et al. 2009). Although NIR spectroscopy has been applied for cow oestrus detection measuring in situ vulvar or vestibular spectra (Kunzler et al. 1992), it is difficult to measure under the same condition and measurements can stress cows. In case of NIR spectroscopy and aquaphotomics, there is a successful report of aquagrams based on NIR spectra of urine indicating the oestrus state in female giant pandas (Kinoshita et al. 2010). Further study on oestrus in giant panda using aquagrams (Kinoshita et al. 2012) suggested that the water spectral pattern of urine samples worked as a mirror on a molecular level and could be applied for fast and non-invasive detection of oestrus. The objective of this research was to develop a method to detect oestrus of dairy cows by means of NIR spectroscopy and aquaphotomics, when using raw milk of individual cows.

Introduction Sexual hormones cause complex physiological changes and monitoring of overall body fluid composition may provide a possibility to indicate fertile periods (Kinoshita et al. 2010). Cost-effective near infrared (NIR) spectroscopy has no harmful effect on the sample studied, making it ideal for online examination of living systems (Roberts et al. 2004). Water has very strong absorbance in the NIR region. As –OH bonds of water are altered easily by other molecules or physical conditions, there is a possibility for investigation of changes detected in water itself in order to get to wider conclusions regarding the entire system. Aquaphotomics is a new scientific discipline that uses rapid and comprehensive analysis of water–light interaction at various frequencies as a potential source of information for better understanding of the biological world (Tsenkova 2009). Different species and concentrations of solutes of aqueous systems structure the water solvent differently, thus, aquaphotomics investigates not the characteristic absorption bands of the solute in question, but the absorption at vibrational bands of water’s –OH bonds that have been altered by the solute. NIR spectroscopy and aquaphotomics have been successfully applied for measuring milk components (Tsenkova et al. 1999) and

Materials and Methods Animals Within four experiments (two in summer and two in winter), 18 Holstein cows (Cows-A~R) were monitored through 31-day milking period. Cows-A~N (n = 14) were hormonally treated according to the general practice in order to synchronize the sexual cycles and induce ovulation in the middle of the sampling period. Cows-O~R (n = 4) were diagnosed being off-cycle (persisting luteal phase or pregnancy), representing the non-oestrus control individuals for the experiment. Daily rectal examination confirmed ovulation of treated cows approximately 72 h after removal of progesterone releasing vaginal implants. During these experiments, the cows were managed according to the guideline for the care and use of experimental animals in Hyogo Prefectural Institute of Agriculture, Forestry and Fisheries. Blood serum and enzyme immunoassay test Blood samples (60 ml) were collected from jugular vein for several days before, during and after the planned © 2015 Blackwell Verlag GmbH

Aquagrams for Oestrus Detection


decrease of progesterone (P4) hormone, oestrus and ovulation. Blood serum was investigated for P4 level to certify the hormonal changes of cows at oestrus. Separated blood serum samples were stored frozen ( 20°C) until enzyme immunoassay (EIA) test on P4 was performed according to the modified method described by Kinoshita et al. (2011), without dilution. Milk samples and near infrared spectroscopy Individual foremilk samples of Cows-D~R (n = 15, 50 ml per sample) were collected by hand before milking during the entire sampling periods. Individual milk samples of all cows (n = 18, 50 ml per sample) were collected from the milk yield meter of the pneumatic milking system, during AM and PM milking. At the end of the sampling periods, frozen ( 20°C) foremilk and milk samples were warmed up in 40°C water bath and homogenized prior to NIR scanning. Transmittance spectra were recorded in the range of 400–2500 nm at 0.5 nm interval using a FOSS XDS spectrometer (FOSS NIRSystems, Inc., H€ ogan€ as, Sweden) equipped with Rapid Liquid Analyzer module. 1-mm cuvette was used with a temperature controlled cuvette holder (40°C). Acquisition of absorbance values (logT 1) was performed with the VISION 3.50 software (FOSS NIRSystems, Inc.). Three consecutive scans were recorded during random measurement of foremilk and milk samples (total nr. of spectra = 2910). NIR spectra were evaluated using Pirouette (ver. 4.0) spectral analytical program (Infometrics, Inc., Woodinville, WA, USA), MS Excel 2010 (Microsoft Co., Redmond, WA, USA) and R Project (ver. 3.0.2) statistical software package ( All data analyses were performed using the first overtone region of water, at 1300–1600 nm spectral interval. Savitzky–Golay smoothing with 2nd-order polynomials and 25 points, multiplicative scatter correction (MSC), and Savitzky–Golay 2nd derivative with 25 points were used as spectral treatments (Naes et al. 2002). The variation of the light absorbance at specific water matrix coordinates (WAMACs) (Tsenkova 2009) described the water spectral pattern (WASP), visualized in aquagrams (Tsenkova 2010; Kinoshita et al. 2012). Student’s t-test was applied to confirm the significance of the differences in the absorbance values used for aquagrams at each individual wavelength for the different stages (high P4 and low P4 period) and for all cows. The validity of t-test was verified by testing the equal variance of the groups using f-test.

Results EIA test of blood serum P4 level confirmed the hormonal changes of investigated cows, that is a drop of P4 level after removal of P4 releasing vaginal implants (Fig. 1) prepared for the treated cows for oestrus and ovulation. © 2015 Blackwell Verlag GmbH

Fig. 1. Normalized progesterone (P4) concentration changes in blood serum of 14 hormonally treated cows (solid lines) and 3 control cows (dashed lines) measured by enzyme immunoassay (EIA). To cancel individual differences among different cows, the concentration values were normalized within individual cows. One remaining control cow was pregnant (having high P4 concentration) and blood samples were rarely collected for safety. P4 concentration changes of 14 hormonally treated cows were well harmonized with the hormonal treatment and 4 control cows were different from them

Milk samples of each cow were sorted into two groups. High P4 group represented samples collected on the last 4 days of P4 treatment, 7–4 days prior to ovulation. Low P4 group contained samples collected after the hormone treatment, when P4 level decreased and the cows’ body started to prepare for oestrus, 3–0 days prior to ovulation (containing the day of ovulation). Mean  SD of serum P4 level in high P4 group = 4.21  3.63 ng/ml; in low P4 group = 1.11  1.25 ng/ml. Control cows showed comparatively constant high level of P4 along the entire sampling period (5.36  3.51 ng/ml). Spectra of milk samples from AM or PM milking were handled separately because of the considerably different baseline caused by the different fat content. Evaluations were performed on data of individual cows only. Based on the concept of aquaphotomics, water acts as a molecular mirror, and the complex changes of milk caused by tiny level of otherwise hardly detectable hormonal changes can be seen through the respective water spectrum. Systematic spectral treatments were applied to identify the specific spectral regions of water (WAMACs), representing the most information related to physiological changes caused by the hormonal changes in the oestrus period. Average milk spectrum of low P4 group was subtracted from that of high P4 group, in case of each animal, using foremilk and milk data, separately. The subtraction was done for treated and control cows, as well. The 2nd derivative-subtracted spectra of treated


G Takemura, G Bazar, K Ikuta, E Yamaguchi, S Ishikawa, A Furukawa, Y Kubota, Z Kov acs and R Tsenkova

and control cows were compared to select those spectral regions that showed variation only in case of treated cows. The absorbance bands showing the highest importance in the identification of spectral differences caused by oestrus were consistent and they defined the WAMACS related to oestrus, which were selected for further analysis. The selection of WAMACS was repeated iteratively, leaving out data of one cow at each iteration. The repeatability of the peak selection was over 85% in each sample types during the 18-times leave-one-cow-out evaluation. The average peak shift was

Aquagrams of raw milk for oestrus detection in dairy cows.

The purpose of this research was to develop rapid and cost-effective method for oestrus detection in dairy cows by means of near infrared spectroscopy...
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