Use of sphingosine, kits, methods and devices for detecting the reproductive capacity of a male mammal and methods for selecting a male goat

Abstract

The present application relates to a kind of sphingosine purposes, kit, the method for detecting the reproductive capacity of male mammal and device and the method for screening ram, and sphingosine is used as the reproductive capacity marker of male mammal;The method for detecting the reproductive capacity of male mammal includes: the content of sphingosine in the sample to be measured is detected, and based on the content of the sphingosine, the reproductive capacity of the animal corresponding to the sample to be measured is determined.Sphingosine is used as marker, the reproductive capacity of male mammal can be directly judged, and it is helpful to screen out the male mammal of high reproductive capacity as paternal line to reproduce, and the reproduction rate of animal can be improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of animal reproduction technology, and particularly relates to a use of sphingosine, a kit, a method and device for detecting the reproductive capacity of a male mammal and a method for screening a ram. BACKGROUND

[0002] Livestock farming is a traditional and advantageous industry in Inner Mongolia Autonomous Region. With the strengthening of grassland resource protection in recent years, the traditional grazing mode has gradually been changed to an intensive breeding mode. In the intensive breeding mode, the addition of concentrate feed leads to an increase in the average body weight of livestock, but also increases the risk of metabolic disorder, thereby increasing the probability of spermatogenic impairment. Sheep are one of the most important sources of livestock products, and their reproductive performance is crucial to the scale of breeding and economic benefits. The selection of a ram directly determines the quality and economic benefits of the entire flock.

[0003] Therefore, there is an urgent need to provide a method for screening a ram with high reproductive performance. SUMMARY

[0004] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present application provides a use of sphingosine, a kit, a method and device for detecting the reproductive capacity of a male mammal and a method for screening a ram. The present application can quickly detect the reproductive capacity of a male mammal by detecting the content of sphingosine in the body of a male mammal.

[0005] The present application is based on the following findings of the inventors:

[0006] At present, most herdsmen choose rams with characteristics such as high spirits, liveliness, and robust physique, and then exclude rams with single testes or cryptorchidism by observing external reproductive organs, so as to select suitable rams. This method has the advantages of simple operation, etc., but its disadvantages are also obvious. The above-mentioned characteristics and external reproductive organs are not directly related to reproductive capacity, and the practicability of the selected rams is relatively low.

[0007] Sphingosine is a metabolite of sphingomyelin, and its chemical formula is C 18 H 37 NO2 (also known as 2-amino-4-octadecene-1, 3-diol). Sphingolipids are important components of cell membranes, and their metabolites include ceramide (Cer), sphingosine (SPH), and sphingosine-1-phosphate (S1P). The dynamic balance of ceramide, sphingosine, and sphingosine-1-phosphate is an important mechanism for controlling cell survival, apoptosis, and other life activities. Sphingosine is a negative regulator of cell proliferation, and has the effects of inhibiting cell growth and promoting cell apoptosis.

[0008] However, the inventors identified the fertility of the breeding rams, divided them into a high fertility group and a low fertility group, and then verified the difference between the two groups in the normal rate of testicular seminiferous tubule morphology and the normal rate of sperm morphology, proving that the normal rate of testicular seminiferous tubule morphology and the normal rate of sperm morphology of the low fertility group are significantly reduced. The inventors performed metabolomic analysis on the feces of the breeding rams, and unexpectedly found through correlation analysis that the metabolite sphingosine has a direct strong correlation with fertility; by comparing the content of sphingosine in the feces of the two groups of breeding rams, it was found that the content of sphingosine in the feces is closely related to the fertility of the breeding rams, specifically, the content of sphingosine in the low fertility group is significantly increased, and sphingosine has a strong correlation with the normal rate of seminiferous tubules and sperm quality of the breeding rams, and the inventors further found through experiments that sphingosine can enter the circulatory system through the intestinal tract and accumulate in the testis, causing the level of testicular cell apoptosis to increase, causing spermatogenic dysfunction. Therefore, the inventors believe that sphingosine can be selected as a potential indicator for screening the fertility of breeding rams, and the content of sphingosine in the feces and serum of breeding rams can be detected to screen high-fertility breeding rams.

[0009] Based on this, in one aspect of the present application, the present application provides a marker. According to an embodiment of the present application, the marker comprises sphingosine.

[0010] The inventors found through experiments that the content of sphingosine in the body of a male mammal (hereinafter referred to as a male animal) is closely related to the fertility of the male animal, that is, if the content of sphingosine in the body of the male animal is high, the fertility of the male animal is low; if the content of sphingosine in the body of the male animal is low, the fertility of the male animal is high. Therefore, the inventors found that sphingosine can be used as a marker, and by detecting the content of sphingosine in the body of a male animal, the fertility of the male animal can be directly determined, and male animals with high fertility can be screened for breeding, thereby providing a new technical means for screening male animals with high fertility.

[0011] In another aspect of the present application, the present application provides a use of sphingosine as a marker for the fertility of a male mammal. The inventors surprisingly found through a large number of experiments that sphingosine can be used as a marker for detecting the strength of the fertility of a male animal, which can directly determine the fertility of the male animal, and is helpful for screening male animals with high fertility for breeding as the paternal line, which can improve the reproduction rate of animals.

[0012] In yet another aspect of the present application, the present application provides a use of sphingosine in detecting the fertility of a male mammal. The inventors surprisingly found through a large number of experiments that by detecting the content of sphingosine in the body of a male animal, the fertility of the male animal can be directly determined, which is helpful for screening male animals with high fertility for breeding as the paternal line, which can improve the reproduction rate of animals.

[0013] In still another aspect of the present application, the present application provides a use of a sphingosine in detecting a testis seminiferous tubule morphology, a sperm morphology or a sperm quantity of a male mammal. The inventors surprisingly find that by detecting the content of the sphingosine in the male animal, the testis seminiferous tubule morphology, the sperm morphology or the sperm quantity of the male animal can be determined, and the male animal with a high normal rate of testis seminiferous tubule morphology, a high normal rate of sperm morphology and a high sperm quantity can be screened, which is helpful to obtain high-quality sperm and lay a foundation for subsequent research and analysis of animal reproductive performance.

[0014] In still another aspect of the present application, the present application provides a use of a reagent in preparing a kit for detecting a reproductive performance of a male mammal, the reagent being used for the above-mentioned marker. Thus, the kit can be used for directly detecting the reproductive performance of the male mammal, so as to screen the male animal with a high reproductive performance.

[0015] In still another aspect of the present application, the present application provides a kit. According to an embodiment of the present application, the kit is used for detecting a reproductive performance of a male mammal, and the kit comprises a reagent for detecting the above-mentioned marker. By using the above-mentioned kit, the reproductive performance of the male mammal can be detected, and the kit has the advantages of fast detection speed and high accuracy.

[0016] In still another aspect of the present application, the present application provides a use of the above-mentioned kit in detecting a reproductive performance of a male mammal. Thus, the kit can be used for detecting the reproductive performance of the male mammal, so as to screen the male animal with a high reproductive performance.

[0017] In still another aspect of the present application, the present application provides a use of the above-mentioned kit in detecting a testis seminiferous tubule morphology, a sperm morphology or a sperm quantity of a male mammal. Thus, the kit can determine the normal rate of testis seminiferous tubule morphology, the normal rate of sperm morphology or the sperm quantity of the male animal, and is helpful to obtain high-quality sperm.

[0018] In still another aspect of the present application, the present application provides a method for detecting a reproductive performance of a male mammal. According to an embodiment of the present application, the method comprises: detecting a content of a sphingosine in a to-be-detected sample; and determining a reproductive performance of an animal corresponding to the to-be-detected sample based on the content of the sphingosine. The method for detecting a reproductive performance of a male mammal according to the embodiment of the present application can detect the reproductive performance of the male mammal, so as to screen the male animal with a high reproductive performance, which is helpful to improve the reproductive rate of the animal.

[0019] In yet another aspect of the present application, the present application provides a method for screening a sire ram. According to an embodiment of the present application, the method comprises: detecting the fertility of a ram to be screened by using the method for detecting the fertility of a male mammal described above; and determining the sire ram based on the fertility of the ram to be screened. The method for screening a sire ram according to the embodiment of the present application can quickly screen a sire ram, and has the advantages of simple operation, fast screening speed, high accuracy, and the like.

[0020] In yet another aspect of the present application, the present application provides an apparatus for detecting the fertility of a male mammal. According to an embodiment of the present application, the apparatus comprises: a determination device configured to determine the content of the marker in a sample to be detected; and an analysis device connected to the determination device and configured to compare and analyze the content of the marker obtained by the determination device, so as to determine the fertility of the animal corresponding to the sample to be detected. The apparatus according to the embodiment of the present application can quickly detect the fertility of a male mammal, and has the advantages of convenient use, fast detection speed, high accuracy, and the like.

[0021] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0022] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0023] Figure 1 is a tissue map of testis and epididymal tail and a sperm morphology picture detected by H&E according to Example 1 of the present application;

[0024] Figure 2 is a statistic of normal ratio of testicular seminiferous tubules according to Example 1 of the present application;

[0025] Figure 3 is a statistic of sperm count according to Example 1 of the present application;

[0026] Figure 4 is a correlation analysis between metabolic markers and fertility according to Example 2 of the present application;

[0027] Figure 5 is a relative content of fecal sphingosine between two groups of metabolomics according to Example 2 of the present application;

[0028] Figure 6 is a content of serum sphingosine of two groups of sire rams detected by competitive ELISA according to Example 3 of the present application;

[0029] Figure 7The two groups of male goat fecal sphingosine content is detected according to the competitive ELISA method in the embodiment 3 of the present application;

[0030] Figure 8 The testicular seminiferous tubule normal ratio statistics is according to the embodiment 4 of the present application;

[0031] Figure 9 The schematic diagram of the device for detecting the reproductive ability of the male mammal is according to an embodiment of the present application;

[0032] Figure 10 The schematic diagram of the device for detecting the reproductive ability of the male mammal is according to an embodiment of the present application. DETAILED DESCRIPTION

[0033] The embodiments of the present application are described in detail below. The embodiments described below are exemplary only, and are not intended to limit the present application.

[0034] It should be noted that the terms "first", "second" are used only for descriptive purpose, and should not be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features with "first", "second" can explicitly or implicitly include one or more features. Further, in the description of the present application, unless otherwise specified and limited, the meaning of "a plurality of" is two or more.

[0035] In this document, unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connecting", "fixing" and the like should be understood in a broad sense, for example, can be fixed connection, can also be detachable connection, or integral; can be mechanical connection, can also be electrical connection or communication with each other; can be directly connected, can also be indirectly connected through intermediate medium, can be the internal communication of two elements or the interaction relationship between two elements, unless otherwise explicitly limited. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0036] In the present document, the term "about" refers to an acceptable error for a particular value depending on how that value is being measured or determined, or can be adjusted according to factors such as the animal's living environment, breed, etc., as determined by those of ordinary skill in the art. In certain embodiments, the term "about" refers to within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" refers to within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range. Illustratively, "about 60 ng / ml" refers to 50 ng / ml, 51 ng / ml, 52 ng / ml, 53 ng / ml, 54 ng / ml, 55 ng / ml, 56 ng / ml, 57 ng / ml, 58 ng / ml, 59 ng / ml, 61 ng / ml, 62 ng / ml, 63 ng / ml, 64 ng / ml, 65 ng / ml, 66 ng / ml, 67 ng / ml, 68 ng / ml, 69 ng / ml, 70 ng / ml; illustratively, "about 2000 ng / ml" refers to 1990 ng / ml, 1991 ng / ml, 1992 ng / ml, 1993 ng / ml, 1994 ng / ml, 1995 ng / ml, 1996 ng / ml, 1997 ng / ml, 1998 ng / ml, 1999 ng / ml, 2001 ng / ml, 2002 ng / ml, 2003 ng / ml, 2004 ng / ml, 2005 ng / ml, 2006 ng / ml, 2007 ng / ml, 2008 ng / ml, 2009 ng / ml, 2010 ng / ml.

[0037] In the present document, the term "ruminant" refers to an animal having a ruminant digestive system, a suborder of the order Artiodactyla, including but not limited to camels, deer, cattle, sheep, etc. "Ruminant digestive system" refers to the process of returning semi-digested food in the stomach to the mouth for re-chewing after a period of time.

[0038] In the present document, the term "normal rate of seminiferous tubule morphology" refers to the proportion of seminiferous tubules of the testis in which the spermatogenic epithelium is full and the arrangement of the spermatogenic cells at all levels is normal and orderly.

[0039] In the present document, the term "normal rate of sperm morphology" refers to the proportion of sperm observed in a sperm smear that have normal morphology, i.e., no abnormalities such as double heads, double tails, fragmentation, etc.

[0040] In the present text, the term "high rate of normality of testicular seminiferous tubule morphology" refers to the high fertility of the animal, and the critical value of the high rate of normality can be adjusted according to the experience of the person skilled in the animal breeding art, and the critical value is exemplarily 60%, 62%, 64%, 66%, 68%, 70%, 75%, 80%, 85%, 90% of the normal rate, which is not specifically limited.

[0041] In the present text, the term "high rate of normality of sperm morphology" refers to the high fertility of the animal, and the critical value of the high rate of normality can be adjusted according to the experience of the person skilled in the animal breeding art, and the critical value is exemplarily 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 75%, 80%, 85%, 90% of the normal rate, which is not specifically limited.

[0042] In the present text, the term "high sperm quantity" refers to the high fertility of the animal, and the critical value of the high rate of normality can be adjusted according to the experience of the person skilled in the animal breeding art, and the critical value is not specifically limited.

[0043] The present application provides a use of sphingosine, a kit, a method and a device for detecting the fertility of a male mammal, and a method for screening a male goat, which will be described in detail below.

[0044] Use

[0045] In some embodiments, the present application provides a use of sphingosine as a marker for the fertility of a male mammal. The inventors have found through experiments that the content of sphingosine in a male animal is closely related to the fertility of the male animal, and if the content of sphingosine in the male animal is high, the fertility of the male animal is low; if the content of sphingosine in the male animal is low, the fertility of the male animal is high. Therefore, by taking sphingosine as a marker and detecting the content of sphingosine in the male animal, the fertility of the male animal can be directly judged, and a male animal with high fertility can be screened for breeding, thereby providing a new technical means for screening a male animal with high fertility.

[0046] In some preferred embodiments, the content of the sphingosine in the serum is not higher than about 60 ng / ml or the content of the sphingosine in the feces is not higher than about 2000 ng / ml, which is an indication of high fertility of the male mammal. The inventors have obtained the critical value of the content of the sphingosine through a large number of experiments, and if the content of the sphingosine in the male animal to be detected is not higher than the critical value, the fertility of the male animal is high.

[0047] In some embodiments, the present application provides a use of sphingosine in detecting the fertility of a male mammal. The inventors have found through a large number of experiments that the fertility of a male mammal can be determined by detecting the content of sphingosine in the male mammal, and the male mammal with high fertility can be screened as the father for reproduction, thereby providing a new technical means for screening the male mammal with high fertility.

[0048] In some preferred embodiments, the content of the sphingosine in the serum is not higher than about 60 ng / ml or the content of the sphingosine in the feces is not higher than about 2000 ng / ml, which is an indication of high fertility of the male mammal. The inventors have obtained the critical value of the content of the sphingosine through a large number of experiments, and if the content of the sphingosine in the male mammal to be detected is not higher than the critical value, the male mammal has high fertility.

[0049] In some embodiments, the present application provides a use of sphingosine in detecting the testis seminiferous tubule morphology, sperm morphology or sperm quantity of a male mammal. The inventors have surprisingly found that the sphingosine has a strong correlation with the seminiferous tubule abnormality and sperm quality, and the testis seminiferous tubule morphology, sperm morphology or sperm quantity of a male mammal can be determined by detecting the content of the sphingosine in the male mammal, and the male mammal with high normal rate of testis seminiferous tubule morphology, high normal rate of sperm morphology and high sperm quantity can be screened, which is helpful to obtain high-quality sperm and lay a foundation for the subsequent research and analysis of animal reproductive performance.

[0050] In some preferred embodiments, the content of the sphingosine in the serum is not higher than about 60 ng / ml or the content of the sphingosine in the feces is not higher than about 2000 ng / ml, which is an indication of high normal rate of testis seminiferous tubule morphology or high normal rate of sperm morphology or high sperm quantity of the male mammal. The inventors have obtained the critical value of the content of the sphingosine through a large number of experiments, and if the content of the sphingosine in the male mammal to be detected is not higher than the critical value, the male mammal has high normal rate of testis seminiferous tubule morphology, high normal rate of sperm morphology and high sperm quantity, and can be used as the male mammal with high fertility.

[0051] Kit

[0052] In some embodiments, the present application provides a use of a reagent in the preparation of a kit. According to the embodiments of the present application, the kit is used for detecting the fertility of a male mammal, and the reagent is used for detecting sphingosine. The inventors have found through a large number of experiments that the fertility of a male mammal can be determined by detecting the content of sphingosine in the male mammal through the reagent in the kit, and the male mammal with high fertility can be screened.

[0053] In some preferred embodiments, the reagent comprises an ELISA reagent or a mass spectrometry reagent, preferably an ELISA reagent. Thus, the above-mentioned reagents can be used to detect the content of the sphingosine in the male animal; and the inventors have found through experiments that when the content of the sphingosine in the male animal is detected by using the ELISA reagent, the content of the sphingosine can be quickly detected, and the method has the advantages of fast detection speed and high accuracy.

[0054] In some embodiments, the present application provides a kit. According to an embodiment of the present application, the kit is used for detecting the fertility of a male mammal, and the kit comprises a reagent for detecting sphingosine. The above-mentioned kit can be used to detect the fertility of the male mammal, and has the advantages of fast detection speed and high accuracy.

[0055] In some preferred embodiments, the reagent comprises an ELISA reagent or a mass spectrometry reagent, preferably an ELISA reagent. Thus, the above-mentioned reagents can be used to detect the content of the sphingosine in the male animal; and the inventors have found through experiments that when the content of the sphingosine in the male animal is detected by using the ELISA reagent, the content of the sphingosine can be quickly detected, and the method has the advantages of fast detection speed and high accuracy.

[0056] In some preferred embodiments, the detection object of the kit is selected from the serum of the male mammal or the feces of the male mammal. The inventors have found through a large number of experiments that the serum and the feces can accurately reflect the content of the sphingosine in the male animal.

[0057] In some preferred embodiments, the detection object of the kit is the feces of the male mammal. The inventors have found through experiments that by detecting the feces of the male mammal, the fertility of the male animal can be non-invasively detected, and the feces sample is easy to obtain.

[0058] In some preferred embodiments, the mammal is a ruminant, preferably a sheep.

[0059] In some embodiments, the present application provides a use of the above-mentioned kit in detecting the fertility of the male mammal. Thus, the kit can be used to detect the fertility of the male mammal, so as to screen the male animals with high fertility.

[0060] In some embodiments, the present application provides a use of the above-mentioned kit in detecting the morphology of the testicular seminiferous tubule, the morphology of the sperm, or the number of the sperm of the male mammal. Thus, the kit can be used to detect the abnormality of the seminiferous tubule and the quality of the sperm of the male mammal, so as to screen the male animals with high normal rate of the morphology of the testicular seminiferous tubule, high normal rate of the morphology of the sperm, and high number of the sperm, which is helpful to obtain high-quality sperm and lay a foundation for the subsequent research and analysis of the animal reproductive performance.

[0061] Method

[0062] In some embodiments, the present application provides a method for detecting the fertility of a male mammal. According to an embodiment of the present application, the method comprises: detecting the content of sphingosine in a sample to be tested; and determining the fertility of the animal corresponding to the sample to be tested based on the content of sphingosine. The method for detecting the fertility of a male mammal according to an embodiment of the present application can detect the fertility of a male mammal, and can screen a male animal with high fertility, which can be used as a sire for subsequent breeding, thereby helping to improve the breeding rate of animals.

[0063] In some preferred embodiments, the further comprising: the content of sphingosine in serum higher than about 60 ng / ml or the content of sphingosine in feces higher than about 2000 ng / ml is an indication of low fertility of the animal; and the content of sphingosine in serum not higher than about 60 ng / ml or the content of sphingosine in feces not higher than about 2000 ng / ml is an indication of high fertility of the animal. The inventors have obtained the critical values of the content of sphingosine through a large number of experiments, and the fertility of a male animal corresponding to the sample to be tested can be determined according to the detected content of sphingosine.

[0064] In some preferred embodiments, the content of sphingosine in the sample to be tested is detected by using the kit.

[0065] In some preferred embodiments, the sample to be tested is selected from the feces of a male mammal or the serum of a male mammal. The inventors have found through a large number of experiments that the content of sphingosine in the serum and the content of sphingosine in the feces can accurately reflect the content of sphingosine in a male animal.

[0066] In some preferred embodiments, the sample to be tested is selected from the feces of a male mammal. The inventors have found through experiments that the fertility of a male animal can be non-invasively detected by detecting the feces of a male mammal, and the feces sample is easy to obtain.

[0067] In some preferred embodiments, the sample to be tested is derived from a ruminant, preferably a sheep. In this way, the fertility of a ruminant can be detected.

[0068] In some embodiments, the present application provides a method for screening a sire ram. According to an embodiment of the present application, the method comprises: detecting the fertility of a ram to be screened by using the method for detecting the fertility of a male mammal; and determining a sire ram based on the fertility of the ram to be screened. The method for screening a sire ram according to an embodiment of the present application can quickly screen a sire ram, and has the advantages of simple operation, fast screening speed, high accuracy, and the like.

[0069] In some preferred embodiments, the high fertility of the male goat to be screened is an indication of a breeding male goat. The inventors have found through experiments that selecting a male goat with high fertility as a breeding male goat can improve the breeding rate.

[0070] Apparatus

[0071] In some embodiments, the present application provides an apparatus for detecting the fertility of a male mammal. According to an embodiment of the present application, referring to Figure 9 , the apparatus comprises: a determination device 100 for determining the content of sphingosine in a sample to be tested; and an analysis device 200 connected to the determination device for comparing and analyzing the content of the marker obtained by the determination device to determine the fertility of the animal corresponding to the sample to be tested. The apparatus according to the embodiment of the present application can quickly detect the fertility of a male mammal, and has the advantages of convenient use, fast detection, high accuracy, etc.

[0072] In some preferred embodiments, referring to Figure 10 , the apparatus further comprises: a determination device 300 connected to the analysis device for screening animals with high fertility based on the fertility determined by the analysis device. Thus, animals with high fertility can be directly screened according to the fertility.

[0073] In some preferred embodiments, the content of sphingosine in serum higher than about 60 ng / ml or the content of sphingosine in feces higher than about 2000 ng / ml is an indication of low fertility of the animal; and the content of sphingosine in serum not higher than about 60 ng / ml or the content of sphingosine in feces not higher than about 2000 ng / ml is an indication of high fertility of the animal. The inventors have obtained the critical values of the content of sphingosine through a large number of experiments, and according to the content of sphingosine detected, the fertility of the male animal corresponding to the sample to be tested can be determined.

[0074] In some preferred embodiments, the sample to be tested is derived from a ruminant, preferably a sheep. Thus, the fertility of a ruminant can be detected or animals with high fertility can be screened.

[0075] In some preferred embodiments, the sample to be tested is selected from the feces of a male mammal or the serum of a male mammal.

[0076] The schemes of the present application will be explained below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If no manufacturer of the reagent or instrument is specified, it is a conventional product that can be obtained from the market.

[0077] Example 1: Fertility of a ram was identified

[0078] Eighteen rams were selected, and small pieces of 0.5x0.5x0.5cm3 were cut from the testes and the tails of the epididymides of the rams, respectively. The small pieces from the testes and the epididymides were placed in 5ml centrifuge tubes, 4ml Bouin's tissue fixative was added, and the tubes were fixed at 4°C overnight. The fixed tissues were washed 3 times with 1xPBS, dehydrated with ethanol gradient, transparentized with xylene, embedded with wax, and sliced. The slices of different rams were mounted on one glass slide, deparaffinated, rehydrated, washed 3 times with PBS for 5 minutes each time, stained with hematoxylin and eosin, and mounted. The normal rate of the seminiferous tubules of the testes was calculated according to the indexes of spermatogenic cell disorder, multinucleation, and vacuolization, see Figure 2 At the same time, the sperm morphology and the sperm count of the tails of the epididymides were detected, see Figure 1 and 3 According to the normal rate of the seminiferous tubules, the sperm morphology, and the sperm count, the above rams were divided into a high-fertility group (10 rams) and a low-fertility group (8 rams). Compared with the high-fertility group, the normal rate of the seminiferous tubules, the sperm count, and the normal rate of the sperm morphology of the low-fertility group decreased significantly. The tissue maps of the testes and the tails of the epididymides are shown in Figure 1 .

[0079] Example 2: Detection of the fecal metabolites of a ram

[0080] The fecal samples of the rams in Example 1 were collected and mixed with 10% DMSO physiological saline extract. The mixture was homogenized, ultrasonicated in an ice water bath for 5 minutes, incubated at -40°C for 1 hour, centrifuged at 12,000 rpm at 4°C for 15 minutes, and the supernatant was collected. After drying by a vacuum concentrator at 37°C, 50% acetonitrile was added, the mixture was resuspended by ultrasonication on ice for 10 minutes, and the mixture was centrifuged at 13,000 rpm at 4°C for 15 minutes. The supernatant was collected, and the metabolomics was detected by UHPLC-QTOF-MS. Specifically, the UHPLC system 1290 Infinity was used to separate the metabolites on a UPLC BEH Amide column with 25mm ammonium acetate and 25mm ammonium hydroxide as the mobile phase, and then the TripleTOF 6600 mass spectrometry system was used for LC / MS experiments (the experimental method is referred to R Michael Sgambelluri, et al., 2014). The quantified metabolites were analyzed for correlation with fertility, and the metabolite marker sphingosine with significant correlation was finally screened out, see Figure 4 Subsequently, by comparing the sphingosine content of the metabolomics of the high-fertility group and the low-fertility group, it was found that the sphingosine in the low-fertility group was significantly increased, see Figure 5Therefore, the present example shows that the sphingosine expression is highly correlated with spermatogenic disorders and can be used as an indicator for identifying the breeding ability of a ram.

[0081] Example 3: Detection of sphingosine in the feces of a ram by competitive ELISA

[0082] Fresh serum and feces of the 18 rams in Example 1 were collected and resuspended in physiological saline at 20 mg / 100 μl, except for the blank wells. 50 μl of the standard or sample to be tested and 50 μl of the biotinylated sphingosine antigen were added to each well, mixed thoroughly, and incubated at 37°C for 60 minutes. After washing the plate manually three times, 50 μl of enzyme-labeled avidin was added to each well, mixed, and incubated at 37°C for 30 minutes. 50 μl of color developing reagent A and B were added to each well, and incubated at 37°C for 15 minutes. 50 μl of the stop solution was added to each well, and the OD value at 450 nm was detected by an enzyme-labeled instrument. The blank was zeroed, the OD value of the standard was fitted with the concentration by using a power function to obtain a standard curve, and the absolute concentration of sphingosine in the sample was obtained. See Figure 6 and Figure 7 The results show that the sphingosine in the low fertility group is significantly higher than that in the high fertility group, which is consistent with the metabolic group detection. Therefore, the results show that the sphingosine ELISA detection value of the serum and feces of a ram can be used as a breeding ability screening indicator.

[0083] In subsequent experiments, the inventors detected the sphingosine in the serum and feces of a large number of high-fertility rams, and finally determined that the sphingosine content in the serum of a high-fertility ram is not higher than about 60 ng / ml, and the sphingosine content in the feces of a high-fertility ram is not higher than about 2000 ng / ml.

[0084] Example 4:

[0085] 1. Collection of fresh feces of a ram

[0086] Randomly select 18 rams (completely different from the rams in Example 1) and mark them with ear tags. After 1 hour of feeding, disinfect the tail with 75% ethanol, and fix a high-pressure treated clean and sterile bag to the tail of the ram so that the feces can fall into the bag. Check every 15 minutes, and use sterile tweezers to collect fresh feces into a collection tube. (Note: The feces should be normal in appearance, uniform in texture and humidity, and free from infection, diarrhea, constipation and other interference factors.)

[0087] 2. Treatment of feces

[0088] The first collected feces were stored at -30°C. After all the samples were collected, the feces were cut open in a sterile environment, and 20-50 mg of feces at different positions were weighed with an analytical balance, resuspended in physiological saline at a concentration of 20 mg / 100 μl, and centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was collected for use.

[0089] 3. Serum collection, jugular vein blood was collected by vacuum tube, 4°C for 1h, 3000rpm centrifugation for 5min at 4°C, and serum was collected.

[0090] 4. Detection of serum or fecal sphingosine of rams by competitive ELISA

[0091] The competitive sphingosine ELISA kit (BYabscience, BY-99863) was removed from 4°C and equilibrated at room temperature for 30min. Except for the blank wells, 50μl of standard or supernatant or serum collected in the previous step and 50μl of biotinylated sphingosine antigen were added to each well, mixed well, and incubated at 37°C for 60min. After washing the plate 3 times to remove unbound antigen, 50μl of enzyme-labeled avidin was added to each well, mixed well, and incubated at 37°C for 30min. Then, 50μl of color developing reagent A and B were added to each well, and incubated at 37°C for 15min. Finally, 50μl of stop solution was added to each well, and the OD value at 450nm was detected using a microplate reader. The blank was set to zero, and the standard curve was fitted using a power function. The absolute concentration of sphingosine in the serum and fecal samples was obtained. Rams with sphingosine content in serum below 60ng / ml were selected as high-breeding rams, and vice versa. Finally, 10 rams with lower sphingosine content were selected as high-breeding rams, and the remaining 8 rams were low-breeding rams, and their ear tags were recorded. At the same time, rams with sphingosine content in feces below 2000ng / ml were selected as high-breeding rams, and vice versa. Finally, 10 rams with lower sphingosine content were selected as high-breeding rams, and the remaining 8 rams were low-breeding rams, and their ear tags were recorded. It was found that the detection results of fecal samples and serum samples were the same.

[0092] 5. Verification of the breeding ability of rams

[0093] The testis and epididymal tail of 18 rams in step 1 were cut into small pieces of 0.5x0.5x0.5cm3, and the small pieces from the testis and epididymis were respectively placed in 5ml centrifuge tubes, 4ml Bouin's tissue fixative was added, and the tubes were fixed at 4°C overnight. The fixed tissues were washed with 1xPBS for 3 times, dehydrated with ethanol gradient, transparentized with xylene, embedded with paraffin, and the sections of different rams were spread on a glass slide. After dewaxing and rehydration, the sections were washed with PBS for 3 times, 5 minutes each time, stained with hematoxylin and eosin, and mounted. According to the indexes of spermatogenic cell disorder, multinucleation, and vacuoles, the normal rate of testicular seminiferous tubules of each group was counted, and the number and morphology of sperm in the epididymal tail and testis were detected. According to the results of the normal rate of testicular seminiferous tubules, the number and morphology of sperm, the rams were grouped according to their fertility, and finally 10 high-fertility rams (i.e. the selected group) and 8 low-fertility rams (i.e. the eliminated group) were obtained. The ear tag marks of the two groups of rams were recorded, and it was found that the ear tag marks of the rams in the selected group were the same as those of the high-fertility rams in step 3.

[0094] In addition, the normal rate of testicular seminiferous tubules of the 10 high-fertility rams was greater than 60%, and the normal rate of testicular seminiferous tubules of the 8 low-fertility rams was less than 20%. The normal rate of testicular seminiferous tubules of the selected group and the eliminated group was calculated, and the results are shown in Table 1. Figure 8

[0095] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and modified by those skilled in the art without contradiction.

[0096] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.​

Claims

1. Use of a reagent for detecting sphingosine in the manufacture of a kit for detecting the fertility of a male mammal.

2. Use according to claim 1, characterized in that, The reagent comprises an ELISA reagent or a mass spectrometry reagent.

3. Use according to claim 1, characterized in that, The kit is for detecting a sample selected from the group consisting of serum of a male mammal or faeces of a male mammal.

4. Use according to claim 1, characterized in that, The mammal is a ruminant.

5. Use according to claim 1, characterized in that, A level of sphingosine in the serum of not more than 60 ng / ml or a level of sphingosine in the faeces of not more than 2000 ng / ml is an indication of high fertility of the male mammal; A level of sphingosine in the serum of more than 60 ng / ml or a level of sphingosine in the faeces of more than 2000 ng / ml is an indication of low fertility of the animal.

6. Use according to claim 3, characterized in that, The mammal is a sheep.

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