Method and system for the non-destructive in ovo determination of fowl gender

Abstract

The present invention relates to a method for non-destructively identifying a characteristic of a Gallus Gallus domesticus embryo in ovo, the method comprising:(a) obtaining a sample of material associated with an egg comprising the embryo, and(b) measuring a score value for the presence of, and concentration of at least a first biomarker in the sample indicative of the characteristic of the embryo, and(c) applying a threshold to the score value and concentration obtained in (b) to identify the characteristic for the embryo associated with the presence and concentration of the biomarker, wherein an at least first biomarker comprises an amino compound having a molecular weight in the range of from 140 to 190 g/mole, wherein step (c) further comprises:(i) correlating each relevant biomarker signal with a reference biomarker by matching the spectrum of each correlating signal with the expected spectrum of the correlating reference biomarker using a similarity measure, to define at least one positively correlating signal;(ii) measuring the intensity of each positively correlating signal and scoring its absolute and/or relative signal intensity; and(iii) applying a threshold to the score value obtained from a similarity function to determine the correlated embryo characteristic.

1 .- 74 . (canceled) 
     
     
         75 . A method for identifying a characteristic of an embryo of an oviparian species in ovo, the method comprising:
 (a) subjecting a sample of material associated with the egg comprising the embryo to a mass spectrometric analysis and recording retention time index and corresponding mass and mass for each signal detected;   (b) correlating the mass corresponding to each signal to a reference database of biomarker masses specific for the species to form a correlation between one or more signals and one or more reference biomarkers;   (c) confirming the correlation between each correlating signal and a reference biomarker by matching the mass spectrum of each correlating signal with the mass spectrum of the correlating reference biomarker using a similarity measure, to define at least one positively correlating signal;   (d) measuring the intensity of each positively correlating signal and scoring its absolute or relative signal intensity; and   (e) applying a threshold to the score value obtained from the discrimination function to determine the characteristic for the embryo associated with the presence and concentration of the correlated biomarker.   
     
     
         76 . The method according to  claim 75 , wherein the similarity measure involves correlating retention index and fragmentation pattern associated with the positively correlated signal, and/or wherein the test sample is processed prior to mass spectroscopic analysis. 
     
     
         77 . The method according to  claim 75 , wherein one or more internal standards of reference biomarkers labelled with an atomic tag are added to the sample prior to analysis by mass spectrometry; and/or wherein the absolute signal intensity is scored by measuring the biomarker signal intensity and comparing it to the signal intensity of one or more known internal standards. 
     
     
         78 . The method according to  claim 75 , wherein the characteristic comprises gender, age, developmental stage and/or viability of the embryo in the egg. 
     
     
         79 . The method according to  claim 78 , wherein the at least one biomarker is (3-[(2-aminoethyl)sulfanyl]buantoic acid for the determination of the gender of a domesticated chicken embryo in the egg. 
     
     
         80 . The method according to  claim 75 , comprising
 (a) obtaining a sample of an egg fluid or volatile compounds emanating from an egg;   (b) analyzing the sample for presence of at least one biomarker;   (c) determining the concentration of the at least one biomarker in the sample; and
 optionally, further comprising the steps of 
   (d) identifying at least one biomarker from the plurality of biomarkers from the sample; and   (e) comparing the concentration of the at least one biomarker with the values of the same biomarker in individuals with known characteristics, wherein the higher or lower concentration of the at least one biomarker is an indication of the embryo being male or female, viable or non-viable, and/or the developmental stage at which the embryo is.   
     
     
         81 . A method of determining suitable biomarkers to determine gender, age and/or viability of an oviparian species in ovo, the method comprising:
 (i) obtaining samples of egg fluids or egg volatiles from a representative group of eggs with known gender, age and/or viability;   (ii) subjecting the sample to a mass spectrometric analysis and recording retention time index and corresponding mass and mass for each signal detected;   (iii) separating, analyzing and identifying a signal pattern associated with at least one or more biomarkers from the sample showing a concentration relation associated with the gender, age and/or viability;   (iv) determining the concentration, and optionally at least one other characteristic of the at least one biomarker for a given condition;   (v) creating reference values characteristic of a given outcome associate with the concentration and optional additional characteristic; and   (vi) determining the prognosis of a selected outcome based on the evaluation; and preferably further comprising the steps of:
 A) correlating each relevant signal with a reference biomarker by matching the mass spectrum of each correlating signal with the mass spectrum of the correlating reference biomarker using a similarity measure, to define at least one positively correlating signal; and 
 B) measuring the intensity of each positively correlating signal and scoring its absolute or relative signal intensity; and applying a threshold to the score value obtained from the discrimination function to determine the characteristic for the embryo associated with the presence and concentration of the correlated biomarker. 
   
     
     
         82 . The method according to  claim 80 , further determining and storing pattern recognition information related with the measured biomarker pattern. 
     
     
         83 . The method according to  claim 82 , comprising identifying one or more biomarkers from the biomarker pattern recognition information. 
     
     
         84 . The method according to  claim 80 , wherein determining the prognosis of a selected outcome comprises determining that an embryo is likely to develop in a male or female adult; wherein the selected outcome is the prognosis of likely viability or non-viability of an embryo to achieve full growth to hatching; and/or wherein the selected outcome is the prognosis for the likely developmental stage and time required for the embryo to progress to hatching under incubation conditions; and/or wherein the similarity measure involves correlating retention index and fragmentation pattern associated with the positively correlated signal. 
     
     
         85 . The method according to  claim 75 , wherein the estimation of the likelihood of a gender for a sample comprises applying a supervised multivariate analysis, preferably Partial Least Squares Discriminate Analysis, PLS-DA or Orthogonal Partial Least Squares Discriminate Analysis to the data; and/or preferably, wherein outliers are removed from the population, optionally further comprising determining whether an embryo in an egg is viable and male, or viable and female, and separating a multitude of viable male eggs from a multitude of viable female eggs, and outliers, to form a predominantly male or predominantly female egg selection. 
     
     
         86 . A fully automated oviparian species embryo gender detection and analysis system, comprising:
 (i) a sample taking system for taking samples from individual eggs;   (ii) optionally, a means for processing the sample prior to analysis;   (iii) a mass spectrometer for collecting one or more spectra related to each sample;   (iv) a gender, developmental stage and/or viability identification facility programmatically identifying signals associated with one or more biomarkers from one or more samples analyzed by the mass spectrometer, the facility further performing an analysis comparing the signals with a stored library of control spectra collected on the sample data and/or with an internal standard, to identify the embryonic characteristic; preferably, wherein the identification facility is implemented in software on an electronic device interfaced with the mass spectrometer; and   (v) and an output means coupling the one or more embryonic characteristic information to a sample and/or an analyzed egg.   
     
     
         87 . The system according to  claim 86 , wherein the identification facility comprises a microprocessor implemented process of a multivariate statistical analysis program, preferably comprising a principal components analysis program, and/or a partial least-squares regression analysis program; such as wherein the comparison comprises an estimation of the likelihood of a gender for a sample using multivariate analysis of the measured spectra, and a determination of the association of an avian embryo with a certain population, preferably performed using partial least squares discriminate analysis. 
     
     
         88 . The system according to  claim 86 , wherein the similarity measure involves correlating retention index and fragmentation pattern associated with the positively correlated signal. 
     
     
         89 . The system according to  claim 86 , configured and operable of performing real time online analysis of eggs at remote locations, such as hatcheries or aquacultures, preferably for use in hatcheries.