ES2941207B2 - METHOD FOR EARLY PREDICTION OF REPRODUCTIVE EFFICIENCY IN RUMINANTS - Google Patents

Description

DESCRIPTION

EARLY PREDICTION METHOD OF REPRODUCTIVE EFFICIENCY IN

RUMINANTS

The present invention relates to a method for early prediction of reproductive efficiency in ruminants, and more particularly, to an in vitro method based on the determination of the levels of a group of analytes that comprise, among others, HDL-cholesterol (HDL). , triglycerides (TGL) and oxocholanoic acid (OCO), and to uses of said levels for these purposes.

BACKGROUND OF THE INVENTION

On livestock farms, a percentage of animals are replaced annually for multiple reasons. This replacement rate is known as the replacement rate and from an economic point of view it is important that these replacement animals have high fertility, since the higher this is, the lower the number of reproductive animals that need to be raised annually. On the other hand, it is also important that these animals are precocial, that is, that they reach puberty and are fertile at the lowest possible age to advance the age at first birth and, thereby, reduce the unproductive period and the generation interval. as well as increasing productive life. In ruminants, it has also been observed that the most precocious animals have a higher fertility rate in the first mating than the less precocious ones, when they are inseminated at the same age.

In this productive context, therefore, it would be desirable to have a procedure to identify at the earliest possible age those animals that have the potential to be precocious and fertile, discarding as soon as possible those others that are less precocious and, therefore, more unproductive from the reproductive point of view.

There are some systems and methods to monitor different conditions in livestock, including fertility; for example, the system described in patent document US2010030036A1. There are also intelligent management methods for livestock reproduction that allow predicting reproductive efficiency, such as the one described in document US20200396962A1, in this case applied to the bovine subfamily.

Progesterone is a hormone involved in the estrous cycle of ruminant species. It is produced mainly in the ovaries, after the onset of puberty, presenting variations in plasma concentration associated with the ovarian cycle during the reproductive season. In this way, progesterone levels can be related to ovulation, since its plasma levels increase after ovulation. Thus, by periodically determining the concentration of progesterone in the blood, it can be determined whether an animal ovulates and is potentially fertile. This technique, however, can only be used after puberty and requires monitoring of plasma progesterone levels. This procedure, in addition to being costly in time and resources, both human and material, delays the identification of animals with different fertility potential, thus increasing the cost of production.

In order to identify animals with different precocity and fertility at the first mating, research has been carried out to find biomarkers related to puberty and ovarian activity in different livestock species. As a result of this effort, potential biomarkers have been identified, but none of them simultaneously meets the following two requirements: 1) provide a high predictive capacity, for which they must explain more than 75% of the variability in the parameter indicative of efficiency. reproductive used, and 2) can be determined in the early stages of life (before 40 days of age). Thus, for example, in the ovine species it has been observed that the plasma concentration of the anti-Mulerian hormone allows the identification of animals with different fertility at the first mating, although the studies are based on its determination from 3 months of age (Lahoz et al. 2012. BMC Ver. Res., 8, 118). There are also studies that relate the concentration of anti-Mulerian hormone with ovarian reserve, ovarian function and fertility in dairy cows (Mossa et al., 2019. Journal of Animal Science, 97 (4): 1446-1455). Different genomic association studies have allowed the identification of genes and polymorphisms related to precocity and fertility in ruminants (Haldar et al.

2014. Bio. Report., 90, 1-7. Juengel et al. 2014. J. Reprod. Fert. Develop., 28, 1318 1325. Meier et al. 2020. J. Dairy Sci., 104, 3707-3721), but these methods would not collect the genome-environment interaction that would be collected by the use of phenotypic markers, conditioned by both genetics and the environment, such as the one described in the present proposal. In any case, both methods - genomic and phenotypic - would be complementary.

The invention described here makes it possible to identify at an early age ruminants that will present different ovarian activity between weaning and 8 months of age, detecting animals with different degrees of precocity and fertility at first mating, allowing the reproductive efficiency of the herds to be improved. reducing the generation interval and production costs and increasing the productive life of the animals.

DESCRIPTION OF THE INVENTION

The present invention is aimed at solving the need to provide methods that allow animals to be identified at an early age that have a higher degree of precocity and fertility at the first mating, thus allowing their reproductive efficiency to be improved.

Thus, in a first aspect, the present invention refers to an in vitro method, hereinafter "method of the invention", for the early prediction of reproductive efficiency in an animal, where said method comprises determining in an isolated sample of an animal levels of at least one analyte which is selected from the group consisting of: triglycerides (TGL); HDL cholesterol); and bile acids.

In the present invention, the term “in vitro” refers to the fact that the determination of the levels of the different analytes is carried out outside the animal's body. The term “animal” refers to any animal, preferably a mammal, and includes, but is not limited to, domestic and farm animals. In certain embodiments of the method of the invention, the animal is a ruminant. In certain embodiments of the method of the invention, the animal is a ruminant belonging to the caprine subfamily.

The expression "early prediction" or "predict early" in the present invention refers to the identification, before the animal's first two months of age, of those animals that have the potential to be precocial and fertile.

The term "reproductive efficiency" in the present invention refers to a production parameter in which it is expected to obtain a greater number of pregnant animals in a shorter time. For this, two factors are taken into account, sexual precocity and fertility.

The term “precocity” refers to the quality of some animals to reach a certain characteristic in a shorter time or age than another. Thus, in the present invention, the term "sexual precocity" refers to the fact that animals reach puberty and are fertile at the lowest possible age to advance the age at first birth and, thereby, reduce the unproductive period. and the generation interval.

In the present invention the term "fertility" or "fertile" refers to the ability of a living being to produce progeny.

In the present invention, “sample” is understood as any biological material of animal origin that is isolated from the animal to be subjected to study, analysis or experimentation. Examples of such samples include, but are not limited to, blood samples and other liquid samples of biological origin, solid tissue samples, such as biopsy samples or tissue cultures or cells derived from them and their progeny, such as cells in cell culture, supernatants cells, cell lysates, serum, plasma, biological fluids and tissue samples.

In certain embodiments of the invention, the isolated biological sample is serum or plasma.

The term “isolated” implies that the biological sample has been separated or extracted from the rest of the components that naturally accompany it. Techniques for obtaining biological samples from an animal are widely known in the state of the art, and any of them can be used in the implementation of the present invention.

In the present invention the term "analyte" refers to a compound present in a sample that has analytical interest. The analytes whose levels are determined in the method of the invention are widely known in the scientific literature, as are the techniques for measuring their levels.

As is known to one skilled in the art, an analyte can be determined in a sample by techniques and methods known in the state of the art, including, but not limited to, enzymatic methods, colorimetry, antibody tests, Western Blot, ELISA, PCR or Quantitative PCR, liquid chromatography-mass spectrometry (LCMS), SELDI-TOF (Surface-Enhanced Laser Desorption/lonization-Time of Flight), two-dimensional electrophoresis, use of antibodies, immunohistochemistry, flow cytometry , gas chromatography-mass spectrometry (GC-MS), gas chromatography-flame ionization detection (GC-FID), immunofluorescence, chromatography, spectrophotometric assays, ultraviolet (UV) spectroscopy, visible spectroscopy, or any combination thereof.

In the method of the invention, the levels of at least one analyte are determined, which is selected from the group consisting of: TGL; HDL; and bile acids.

A triglyceride (TGL) is an ester derived from glycerol and three fatty acids. The chain lengths of the fatty acids in natural triglycerides vary, but most contain 16, 18, or 20 carbon atoms. Triglycerides are the main constituents of body fat in animals. They are also present in the blood to allow the bidirectional transfer of adipose fat and blood glucose from the liver.

HDL-cholesterol (HDL) refers to high-density lipoproteins, which are those that transport cholesterol from the body's tissues to the liver. These are smaller and denser lipoproteins than LDL, they are composed of a high proportion of proteins. The liver synthesizes these lipoproteins as empty proteins and, after collecting cholesterol, they increase in size as they circulate through the bloodstream.

Bile acids are unfriendly molecules with a steroidal skeleton that are produced by liver cells from cholesterol. Bile acids are steroid acids found predominantly in the bile of mammals. The distinction between different bile acids is minimal, mostly depending on the presence or absence of hydroxyl groups at the 3,7,12 and 24 positions.

In certain embodiments of the invention, the bile acid is selected from the group consisting of: glycochenodeoxycholic acid (GCDO); hydroxycholenoic acid (HCO); and oxocholanoic acid (OCO).

Oxocholanoic acid has the molecular formula C24H36O3, hydroxycholenoic acid has the molecular formula C24H4003, and glycochenodeoxycholic acid has the molecular formula C26H43NO5.

In certain specific embodiments, the method of the invention comprises determining OCO levels.

The method of the invention allows early prediction of reproductive efficiency in an animal by determining the levels of at least one analyte that is selected from the group consisting of: TGL, HDL and bile acids. As demonstrated in the experimental section, the discrimination capacity in said prediction is significantly superior to that obtained by determining the levels of other analytes that are selected from the group consisting of: aspartate aminotransferase (AST); alanine aminotransferase (ALT); ceruloplasmin (CRL); bilirubin (BIL); total cholesterol (COL-T); LDL-cholesterol (LDL); p-hydroxybutyrate (PHB); non-esterified fatty acids (NEFA); glucose (GLU); creatine kinase (CK); creatinine (CRT); albumin (ALB); total proteins (TP); urea; calcium; zinc; and magnesium.

The method of the invention allows animals to be identified at an early age that have a higher degree of precocity and fertility, thus allowing their reproductive efficiency to be improved.

In the context of the present invention, “early age” is understood to mean an age between three and eight weeks, or between 21 and 56 days, inclusive.

In certain embodiments of the invention, the biological sample is isolated from the animal between 30 and 50 days of age of the animal, inclusive.

In certain specific embodiments of the invention, the biological sample is isolated from the animal between 35 and 40 days of age of the animal, inclusive.

As explained at the beginning of the present description, the determination of the levels of at least one analyte that is selected from the group consisting of: TGL; HDL; and bile acids, is useful for early prediction of reproductive efficiency in an animal. Therefore, in the present invention, the uses of said analytes are also contemplated as inventive aspects.

Thus, a second aspect of the present invention relates to the in vitro use, hereinafter "use of the invention", of the levels in an isolated sample of an animal of at least one analyte that is selected from the group consisting of : TGL; HDL; and bile acids to early predict reproductive efficiency in an animal.

In certain embodiments of the invention, the bile acid is selected from the group consisting of: GCDO; HCO; I co.

In certain embodiments of the use of the invention, the bile acid is OCO.

The terms and expressions, “in vitro”, “early prediction” or “predict early”, “reproductive efficiency”, “animal”, “sample”, “isolate” and “analyte” have been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect.

The implementation of the use and methods of the invention is based on the determination of the levels of different analytes in biological samples isolated from an animal. The media used for this determination of the levels of these analytes can be part of a kit.

Thus, a third aspect of the present invention refers to a kit, hereinafter "kit of the invention", which comprises means for determining in vitro, in an isolated sample of an animal, the levels of at least one analyte that is select from the group consisting of: TGL; HDL; and bile acids.

In certain embodiments, the bile acid is selected from the group consisting of: GCDO; HCO; I co.

In certain embodiments, the kit of the invention comprises means for determining OCO levels.

The terms and expressions, “in vitro”, “animal”, “sample”, “isolate” and “analyte” have been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect.

The kit may comprise other components useful in the implementation of the present invention, such as buffers, material supports, positive and/or negative control components, etc. In addition to the mentioned components, the kits may also include instructions for practicing the object of the invention. These instructions may be present in the aforementioned kits in a variety of forms, one or more of which may be present in the kit. One way in which these instructions may be present is as printed information on a suitable medium or substrate, e.g. e.g., a sheet or sheets of paper on which the information is printed, on the kit packaging, on a package insert, etc. Another medium would be a computer-readable medium, for example, a CD, USB, etc., on which the information has been recorded. Another medium that may be present is a website address that can be used over the Internet to access information at a remote site. Any convenient media may be present in the kits.

The inventors of the present invention have developed an intelligent management method that allows early in vitro prediction of reproductive efficiency in an animal. Therefore, the present invention also refers to an intelligent management method for early in vitro prediction of reproductive efficiency in an animal where the method is based on the parametric method of estimating the linear discriminant function, and where the plasma HDL concentrations; TGL; I co.

Linear discriminant analysis is a generalization of Fisher's linear discriminant, a method used in statistics to find a linear combination of features that characterize or separate two or more classes of objects or events.

Discriminant analysis develops a discriminant criterion to classify a set of observations (calibration set) containing one or more quantitative variables and a classification variable that defines groups of observations. In the present invention, these are fertility categories.

When the distribution within each group is multivariate normal, a parametric method can be used to develop a discriminant function. The discriminant function, also known as the classification criterion, is determined by a generalized squared distance measure. The classification criterion can be based on the individual covariance matrices within the group (which produce a quadratic function) or the grouped covariance matrix (which produces a linear function); It also takes into account the groups' prior probabilities.

Discriminant functions are a linear combination of the independent variables that provide the greatest possible discrimination between categories. In the present invention the independent variables are the plasma concentrations of TGL, HDL and OCO.

In certain embodiments of the intelligent management method of the invention, the greatest discriminating capacity is achieved by transforming the plasma concentration of OCO to a natural logarithm and raising the plasma concentration of triglycerides (TGL) to a power of two. Thus, in the present invention, the two discriminant functions (D1 and D2) that provide the greatest discrimination capacity between fertility categories are:

•D1 = -19.048 1.6964 x ln (OCO) 0.00103 x (TGL)2+ 0.55367 x HDL

•D2 = -37.452 -10.966 x ln (OCO) -0.0061 x (TGL)2+ 0.0198 x HDL

The means for executing the method of the invention for early in vitro prediction of reproductive efficiency in an animal may be comprised in kit form.

Therefore, a fourth aspect of the invention refers to a kit for early in vitro prediction of reproductive efficiency in an animal, where said kit comprises at least one electronic device with at least one processor and a memory, and where the memory stores instructions that, when executed by the at least one processor, cause the electronic device to execute the method of the invention.

Likewise, the invention relates to a computer program with instructions configured to be executed by at least one processor, where said instructions cause the execution of the method of the invention by the electronic device.

DESCRIPTION OF THE FIGURES

Fig. 1.Plasma concentration of progesterone in animals classified into different categories.

Fig. 2. Chromatograms with identification of bile acids in the sample corresponding to an animal from category 0 and category 1.

Fig.3. Fertility rate of the group of animals classified within the different reproductive categories.

Fig. 4. Spatial distribution of individuals from the two reproductive categories based on the values obtained with the discriminant functions (data generated in the calibration subset).

Fig.5. Spatial distribution of individuals from the two reproductive categories based on the values obtained with the discriminant functions (data generated in the validation subset).

EXAMPLES

Next, the invention will be illustrated through tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.

1. Materials and methods

1.1. Animals, feeding and management

Data and samples from 25 Assaf breed lambs were used, from the flock of the Mountain Livestock Institute (IGM/CSIC-ULE). Part of these animals (n=18) were mated at 9 months of age, after synchronization of heat with sponges with progestins (Chronogest® 20 mg, MSD) and administration of equine serum gonadotropin (Foligon, MSD). This subset of animals (calibration subset) was used to obtain the reproductive behavior prediction model. The remaining animals (n=7) were not covered and were used to validate the prediction model obtained (validation subset).

The animals used, both for obtaining and validating the prediction model, were separated from their mothers 24 hours after birth, to ensure colostrum consumption. Subsequently, they were raised by artificial lactation, using an automatic wet nurse (JR, Spain) with milk replacer (Cordevit, Leches Maternizada S.A., León, Spain). The animals had free access to the nurse 24 hours a day.

At 40 days of age, the animals were progressively weaned over 7 days, allowing them free access to a starter feed (Babymix, Inatega, Spain), barley straw and alfalfa hay, distributed in different feeders. During this period, they were allowed access, on days 41, 42, 43, 45 and 47, for one hour a day to an automatic wet nurse. After weaning, the animals were provided with cereal straw, alfalfa hay and the same starter feed used in the previous phase, all administered ad libitum, for 6 weeks. Subsequently, they were fed ad libitum with a complete pelleted diet, containing 54.5% forage and 45.5% concentrate (6.5% wheat bran, 12% cereal straw, 36% alfalfa,14, 5% barley, 14% corn, 7% wheat, 5% soybean cake, 2% molasses, 1% butter and 2% vitamin-mineral corrector) up to 8 months of age.

1.2. Obtaining blood samples

To study the biochemical and metabolomic profile, a blood sample is taken from all animals between 35 and 40 days of age, both included. After weaning, samples were collected weekly for 7 months to determine progesterone concentration. Samples were always collected in the morning, at 8:30 a.m., by venipuncture in the jugular vein.

Blood samples for biochemical and metabolomic profiling were collected using a lithium-heparin vacutainer and immediately transferred to the laboratory in ice water, where they were centrifuged at 3520 g for 16 min at 50C.

Plasma samples were then collected and stored at -80 0C until analysis.

To measure progesterone, samples collected after weaning were allowed to clot in a bath at 37°C for 30 minutes and then centrifuged at 3520 g for 16 min at 4°C. The serum samples thus obtained were stored at -80oC until the progesterone determination was performed.

1.3. Reproductive categories

The animals, both from the calibration and validation subset, were distributed into two categories based on the number of progesterone peaks detected in the period of time between weaning (at 40 days of age) and 8 months of age. . Thus, within each subset, the animals were classified into two categories:

•Category 0: lambs that presented less than 2 peaks of progesterone with a concentration >0.5 ng/mL before 8 months of age (indicating less than 2 ovulations in the period). This subgroup consisted of 11 animals and their fertility rate was 45.5%.

•Category 1: lambs that presented at least 2 peaks of progesterone with a concentration >0.5 ng/mL before 8 months of age (indicating a minimum of 2 ovulations). This subgroup consisted of 7 animals and their fertility rate was 85.7%.

Fig. 1 shows the changes in the concentration of progesterone in the blood of some of the animals included in categories 0 and 1. As can be seen, the animals in category 0 presented a single peak during the study period. of progesterone, while those in category 1 presented at least 2 peaks of progesterone. A valid progesterone peak was considered when the plasma concentration exceeded 0.5 ng/mL, with a decrease in this concentration being observed between two sequential peaks.

1.4. Analytical determinations

Determination of progesterone in serum samples was performed by competitive sequential immunoassay (lmmulite®/lmmulite® 1000 Progesterone).

In the plasma samples collected at 35 days of age, the following parameters were determined: aspartate aminotransferase (AST), alanine aminotransferase (ALT), ceruloplasmin (CRL), bilirubin (BIL), triglycerides (TGL), total cholesterol (COL- T), HDL-cholesterol (HDL), LDL-cholesterol (LDL), p-hydroxybutyrate (PHB), non-esterified fatty acids (NEFA), glucose (GLU), creatine kinase (CK), creatinine (CRT), albumin ( ALB), total proteins (TP), urea, calcium, zinc and magnesium. Plasma samples were thawed overnight at 4°C and analyzed using an ILAB.650 instrument (Instrumentation Laboratory, Lexington, MA, USA).

In an aliquot of these same plasma samples, the concentration of bile acids was determined by liquid chromatography and detection by mass spectrometry, specifically glycochenodeoxycholic (GCDO), hydroxycholenoic (HCO) and oxocholanoic (OCO) acids, which could be detected. such as the pseudomolecular ion ([M+H]+) and the dehydrated pseudomolecular ion ([M-H2Ü+H]+) using positive ionization. For this, an Acquity Ultraperformance LC chromatographic equipment (UPLC®; WATERS, Barcelona, Spain) was used, equipped with an Acquity UPLC HSS T3 column (1.8 μm particle size, and dimensions 2.1 * 100 mm) and a guard column of the same filler (VanGuard 2.1 mm * 5 mm, 1.8 μm particle size). Samples were analyzed with two different elution methods, injecting 7.5 μL of each sample into both methods. In method 1, a binary allusion gradient was used, with solvent A of methanol: water (2:8; v/v) and 0.1% formic acid; Solvent B was a mixture of 100% acetonitrile with 0.1% formic acid. The flow was 0.35 mL/min. The elution gradient was as follows: 99.9% A; 1 min, Socratic; 3.5 min, 20% A; 5 min, Socratic; 9.5 min, 0.1% A; 11 min, Socratic; 14 min, 99.9% A (the total time of each chromatographic run was 15 min). In method 2, a binary gradient was also used, but solvent A (methanol: water: formic acid, 50:50:0.5; v/v/v), and solvent B (methanol: acetonitrile: formic acid, 59:40:0.5; The flow was 0.30 mL/min and the elution gradient: 99.9% A; 1 min, Socratic; 2.5 min, 20% A; 4 min, Socratic; 5.5 min, 0.1% A; 8 min, Socratic; 10 min, 99.9% A (the total time of each chromatographic run was 12 min). The samples were distributed randomly to distribute the propagation errors and a joint sample was used as a control sample (QC), which was injected at the beginning, in the middle and at the end of the set of analyzed samples. The values of the areas of each peak were subsequently adjusted according to the values obtained with this control sample. Reserpine was also added to each sample as a reference compound and after integration, the areas were normalized using this reference. The detection of the analytes was performed on a SYNAPT HDMS G2 mass spectrometer (WATERS, Barcelona, Spain), equipped with an elestrospray ionization source (ESI, Z-spray®) and a time-of-flight analyzer (ESl-QToF -MS). Samples were analyzed in positive and negative ionization modes (this mode only in method 1). The mass spectrometer was operated with the MSE method, which includes low energy (equivalent to full-scan) and high energy functions, the latter allowing the fragments of the base peak to be determined continuously and at each instant. The MassLynx™ program was used to control the analysis and data acquisition.

Table 1 shows the chromatographic data of the three bile acids indicated above. In Fig.2. A typical chromatogram of one of the samples analyzed is presented for both method 1 (with ionization in positive and negative modes) and for method 2 (ionization only the positive).

Table 1. Chromatographic and mass spectrum data for the different bile acids determined.

1.5. Statistic analysis

All procedures performed were carried out using the SAS statistical package (SAS Inst. Inc., Cary, NC, USA).

In the calibration set, a discriminant analysis was performed to evaluate the ability to classify the animals into their corresponding reproductive categories (Category 0vs1) using the blood biochemical profile determined in samples obtained before weaning (35-40 days of age). In addition to the raw parameters, new variables were included, a combination of the original ones or simply transformed, using powers or the natural logarithm. Predictor variables were selected using the STEPDISC procedure. Once the variables were selected, the DISCRIM procedure evaluated the correct classification capacity through resubstitution and cross-validation, as well as in the external validation subset.

The goodness of fit of models obtained using different procedures for estimating the classification criterion was compared: 1) parametric method using the joint covariance matrix to obtain the classification criterion (linear discriminant function); 2) parametric method using the individual covariance matrices to obtain the classification criterion (quadratic discriminant function) and 3) non-parametric method based on the K nearest neighbors, using the joint covariance matrix to estimate the Mahalanobis distance. The 3 methods compared allowed 100% of the individuals to be correctly classified in both the calibration and validation sets, but the parametric method with the estimation of the linear discriminant function was the one that generated higher classification probabilities.

As mentioned previously in the description, the discriminant functions are a linear combination of the independent variables, in the present invention, the plasma concentrations of the selected markers, which provide the greatest possible discrimination between the categories. In the present invention, 2 discriminant functions (D1 and D2) were obtained:

• D1 =-19.048+ 1.6964xln(OCO) 0.00103x(TGL)2+ 0.55367xHDL

• D2 = -37.452 -10.966 x ln (OCO) -0.0061 x (TGL)2+ 0.0198 x HDL

It should be noted that the ordinate and the coefficients in the discriminant functions may vary depending on whether different calculation methods are used (linear or quadratic parametric function or non-parametric methods), whether or not the prior probabilities are taken into consideration or even if they are used. data from other laboratories, which present some type of bias. The discriminant capacity of the identified markers would be the same, as long as the bias is constant.

The discriminant functions of the present invention allow animals to be classified into reproductive categories according to the following criteria:

•If the value obtained in function D1 is greater than in function D2, the animal is classified in category 0.

•If the value obtained in function D1 is lower than in function D2, the animal is classified in category 1.

2. Results

2.1. Number of progesterone peaks and fertility

The lambs of the calibration subset were inseminated at 9 months of age and the fertility rate was determined, defined as the n0 of lambs lambed/n0 of lambs inseminated. The fertility rate, as can be seen in Fig. 3, was numerically higher in category 1 (animals that presented at least 2 progesterone peaks before 8 months of age). The association between fertility (fertile vs. non-fertile) and reproductive categories (category 0 vs. 1) showed a trend towards significance{X2=2.92; p=0.0876).

2.2. Biochemical profile of animals

Table 2 shows the mean values for the different parameters indicative of the biochemical profile corresponding to the two reproductive categories, in the calibration subset.

1 chromatographic peak area values of the compound normalized to the chromatographic peak area of the reference compound (reserpine)

Table 2. Mean values ± standard error of the mean of the different parameters indicative of the blood biochemical profile at 35 days of age of the animals corresponding to the two reproductive categories studied in the calibration subset (Category 0 vs 1).

23. Prediction of reproductive behavior

The combination that allowed the greatest discriminating capacity was that constituted by the plasma concentration of oxocholanoic acid (OCO, transformed as a natural logarithm), triglycerides (TGL, raised to a power of 2) and high-density cholesterol-binding lipoproteins (HDL). .

As mentioned above, if the value obtained in function D1 is greater than in function D2, the animal is classified in category 0 (presents less than two ovulations before eight months of age). Therefore, these animals will be less precocious and fertile. If the value obtained in function D1 is lower than in function D2, the animal is classified in category 1 (it will present at least two ovulations before eight months of age). Therefore, these animals will be more precocious and fertile. Table 3 shows the mean values ± standard error of the mean of the markers in each of the categories.

Table 3. Mean values ± standard error of the mean of the markers in each of the categories (mean values including the animals of the calibration and validation subsets).

Fig. 4 shows the values obtained with each discriminant function, corresponding to each of the individuals in the calibration subset. Table 4 presents the results of the assignment process, both the resubstitution process and the cross-validation process.

Table 4. Error rates in the classification of animals in the different reproductive categories based on the biochemical profile determined before weaning (data generated in the calibration subset through resubstitution or cross-validation).

In order to confirm the goodness of the prediction model, the classification capacity was evaluated in a subset of animals (n=7) that were not used to obtain the model. Fig. 5 shows the values obtained with each discriminant function, corresponding to each of the individuals in the validation subset. It should be noted that the classification error rate was 0% and the assignment probabilities were very high, as can be seen in Table 5.

Table 5. Probability of a posteriori assignment of the animals in the reproductive categories studied (Category 0vs1)

Claims (18)

1. In vitro method for the early prediction of reproductive efficiency in a ruminant animal, where said method comprises determining triglyceride levels (TGL) in an isolated serum or plasma sample from said animal at an early age; HDL cholesterol); and bile acids, where the early age is before the first two months of age of the animal and where the prediction of reproductive efficiency is made by determining two discriminant functions (D1 and D2): •D1 = -19.048 1.6964 x In (bile acids) 0.00103 x (TGL)2+ 0.55367 x HDL, and •D2 = -37.452 -10.966 x In (bile acids) -0.0061 x (TGL)2+ 0.0198 x HDL, where if the value obtained in function D1 is greater than in function D2, the animal is classified in category 0 (it has less than two ovulations before eight months of age and therefore will be less precocious and fertile) and if the value obtained in function D1 is lower than in function D2, the animal is classified in category 1 (it presents at least two ovulations before eight months of age and therefore will be earlier and more fertile). 2. Method according to claim 1, wherein the bile acid is selected from the group consisting of: glycochenodeoxycholic acid (GCDO); hydroxycholenoic acid (HCO); and oxocholanoic acid (OCO). 3. Method according to any of claims 1 or 2, wherein the method comprises determining OCO levels. 4. Method according to any of claims 1 to 3, where the animal belongs to the goat subfamily. 5. Method according to any of claims 1 to 4, wherein the biological sample is isolated from the animal between 30 and 50 days of age of the animal, both included. 6. Method according to any of claims 1 to 5, wherein the biological sample is isolated from the animal between 35 and 40 days of age of the animal, both included. 7. In vitro use of the levels, in an isolated serum or plasma sample of a ruminant animal at an early age, of TGL; HDL; and bile acids to early predict reproductive efficiency in an animal. 8. Use according to claim 7, wherein the bile acid is selected from the group consisting of: GCDO; HCO; I co. 9. Use according to any of claims 7u8, where the bile acid is OCO. 10. Use according to any of claims 7 to 9, where the animal belongs to the goat subfamily. 11. A kit that comprises means to determine in vitro, in an isolated sample of serum or plasma from a ruminant animal at an early age, the levels of TGL; HDL; and bile acids. 12. Kit according to claim 11, wherein the bile acid is selected from the group consisting of: GCDO; HCO; I co. 13. Kit according to any of claims 11 or 12 wherein the kit comprises means for determining OCO levels. 14. Kit according to any of claims 11 to 13, where the animal belongs to the goat subfamily. 15. Kit according to any of claims 11 to 14, wherein the biological sample is isolated from the animal between 30 and 50 days of age of the animal, both included. 16. Kit according to any of claims 11 to 15, wherein the biological sample is isolated from the animal between 35 and 40 days of age of the animal, both included. 17. A kit for the early in vitro prediction of reproductive efficiency in a ruminant animal at an early age, where said kit comprises, at least, an electronic device with at least one processor and a memory, and where the memory stores instructions that, when they are executed by the at least one processor, cause the electronic device to execute the method of any of claims 1 to 6. 18. A computer program with instructions configured to be executed by at least one processor, wherein said instructions cause execution of the method of any of claims 1 to 6 by the electronic device defined in claim 17.