CN1717586A - Methods for selection for efficient animal growth - Google Patents

Abstract

The present invention relates generally to animal breeding methods. More specifically, the invention relates to methods for selecting for robustness among two or more animals based upon the quantity of immune cell subtypes and frequency of proliferative responses of lymphocytes in the animals.

Description

Be used to select the method for effective growth of animal

Background of invention

Technical field

Generality of the present invention relates to Perspective of Animal Breeding Methods.More particularly, the present invention relates to based on the quantity of the immunocyte hypotype (subtype) of animal and frequency that lymphocytic proliferative is replied and among two or more animal, select the method for robustness (robustness).

Background technology

Animal has a kind of molecule and cytophylaxis array of complexity, be referred to as immune system, its identification is also attacked potential harmful external source or endogenous but unusual cell (for example is respectively a pathogen, as bacterium or virus, and the cell of cancer cell or pathogen infection),, it tolerates endogenous normal cell but not attacking.When being subjected to external source or unusual biomolecule and stimulating, the immune system experience relevant with external source or unusual biomolecule, neutralize and a series of activity of the cell of destruction pathogen or cancer cell or pathogen infection.These activity that general designation is made immune response can or comprise that cell-mediated replying with a kind of immune response of humoral response element form by cell-mediated immune responses, body fluid (antibody-mediated) immune response.

Humoral immunoresponse(HI) by specificity in conjunction with external source or unusual biomolecule and attract the antibody of immune other composition, the glycoprotein mediation.Antibody is immunoglobulin (Ig) (Ig) molecule that is produced by the B cell, and the B cell is to originate from birds capsule (avian bursa) or mammal marrow but migrate to other organ especially spleen and ripe therein lymphocyte (Robertson, 1983).Cell-mediated immune responses is the result of T cytoactive, and the T cell is a lymphocyte (Tizard, 1988) ripe in the thymus gland of animal.Alter a great deal among the different in animal body T cell subsets (subpopulation) of T cytoactive.Cellular toxicity T cell recognition is also destroyed foreign cell (graft rejection) or endogenous but unusual cell (for example cancer cell or born of the same parents entozoa are as the cell of virus and bacterial infection).Helper cell and B cell and cellular toxicity T cell act on mutually and the exert an influence biomolecule of B cell and cellular toxicity T cell behavior is to promote respectively and to instruct antibody to produce and cellular toxicity activity (Mosier, 1967).The T cell that also has other classification comprises suppressor T lymphocyte and memory T cell (Miedema andMelief, 1983; Tizard is as preceding, pp.225-8).The classification of T cell is showed different T cell differentiation antigens in its surface based on different T cells to a certain extent and is distinguished.

Be correct functionating, the T cell of animal immune system and B cell must be correctly and are differentiated non-self composition or endogenous but unusual composition of a large amount of external sources reliably.Immune identification and discriminating occur in molecular level.Antigen has a kind of molecular composition that produces the immune response potentiality, is that epi-position is formed by the diagnostic characteristics of one or more molecular size.Have and for example comprise that a polypeptide antigen of 100 amino acid whose amino acid sequences can comprise many epi-positions, wherein each epi-position is all limited by the part of polypeptide, and it comprises about 15 amino acid of about 3-.(Tizard is as preceding, p.25) can to estimate at about 1,000 ten thousand derived from the number of the epi-position of polypeptide.

The antigen that is suffered from by T or the B cell of animal must be through differentiating to being with normally endogenous (being self) antigen is relevant, and its immune response is harmful to animal, and (non-self) antigen is relevant perhaps with external source or unusually, to it immune response should take place.Can the rest may be inferred " friendly or hostile " in the human body antagonism differentiate.If immune system can not differentiate and nonself invasion and attack pathogen or the relevant antigen of tumour cell that then these " enemies " can escape immune defence.If immune system is differentiated into right and wrong self mistakenly with the endogenous antigen of a kind of animal, then animal body comprises that part of of this endogenous antigen and will face from immune " the well-meaning attack ".The latter's situation is known usually to be " autoimmune disease ", and wherein the immune system mistake of animal starts to resist the cell and the molecule " war " of the other normal part of animal body.

Differentiate the part of the means of antigen as immune system, individual T cell and B cell produce antigen receptor, and this receptor is showed on T or the B cell surface and the specificity conjugated antigen.Although each individual T or B cell are all showed identical antigen receptor, the set of the different antigen receptors of animal is very various.With regard to T or B cell, antigen activates this cell with combining of cell antigen receptor, promptly stimulates this cell to carry out and generation activity cell-mediated or that humoral immunoresponse(HI) is relevant.Although the B cell is conjugated antigen directly, the T cell is only just replied it when antigen is being commonly referred to when showing on other special category cell of antigen presenting cell (APC).APC, for example macrophage and dendritic cell are presented by the antigen of glycoprotein derived from polypeptide, MHC (main histocompatibility complex) albumen (Bevan et al., 1994) of described glycoprotein as showing on the APC surface.Do not have MHC albumen, the T cell then can not be distinguished external source or endogenous antigen.

In the past ten years, most of domestic animals are already through having disclosed estimated breeding value (breedingvalue) can differentiate best breeding animal.These estimated breeding values are based on from some proterties information of each animal and correlative thereof and calculate, and the most accurate standard of representative has highly prepotent animal with discriminating.Yet, generally believe that environmental attack such as disease have limited the expression of " truly " genetic potential of animal such as pig.This perhaps be since with the significantly different high health status unit of the environment that in commodity production, meets with in select due to.The reduction of individual performance between the breeding period that in many plant and animals system, observes the degeneration of this health status and follow.The same with other agricultural system system, modern tame farm need be provided with the excellent healthy and strong animal of performance down in different commerce.

Therefore, need to disclose can differentiate and/or sorting at the instrument of the specific only animal of production system.Because the importance of animal immune system in its whole health status, it is possible therefore having correlativity between the quantity of the special immunocyte that good moral character and the animal of animal has.Similarly, it also is possible having correlativity between good moral character and special immunocyte functional.

Summary of the invention

According to an aspect of the present invention, the invention provides a kind of method of selecting robustness among two or more animal, described method comprises two or more animal that same species is provided; Determine the quantity of CD16 antigen presentation cell in each animal; And the minimum animal of selection CD16 antigen presentation cell quantity.

According to a further aspect in the invention, the invention provides a kind of method of selecting robustness among two or more animal, described method comprises two or more animal that same species is provided; Determine the quantity of the two positive antigen presentation cells of CD16 and CD2 in each animal; And selection CD16 and the two minimum animals of positive antigen presentation cell quantity of CD2.

In accordance with a further aspect of the present invention, the invention provides a kind of method of selecting robustness among two or more animal, described method comprises two or more animal that same species is provided; Determine the quantity of CD8 antigen presentation cell in each animal; And the minimum animal of selection CD8 antigen presentation cell quantity.

According to another aspect of the invention, the invention provides a kind of method of selecting robustness among two or more animal, described method comprises two or more animal that same species is provided; Determine the quantity of MHC-DQ antigen presentation cell in each animal; And the highest animal of selection MHC-DQ antigen presentation cell quantity.

According to a further aspect in the invention, the invention provides a kind of method of selecting robustness among two or more animal, described method comprises two or more animal that same species is provided; Determine in each animal to express by the MHC-DQ antibody target is the quantity of cell of the antigen of MHC-DQB; And the selection expression is the highest animal of cell quantity of the antigen of MHC-DQB by the MHC-DQ antibody target.

According to a further aspect in the invention, the invention provides a kind of method of selecting robustness among two or more animal, described method comprises two or more animal that same species is provided; Determine in each animal to express by the MHC-DQ antibody target is the quantity of cell of the antigen of MHC-DQD; And the selection expression is the highest animal of cell quantity of the antigen of MHC-DQD by the MHC-DQ antibody target.

According to another aspect of the invention, the invention provides a kind of method of selecting robustness among two or more animal, described method comprises two or more animal that same species is provided; Determine the propagation frequency of T4 antigen express cell in each animal; And select the T4 antigen express cell to breed the minimum animal of frequency.

Can select the species of robustness to comprise but non-being limited to therein: Bos taurus (ox), Susscrofa (pig), Ovis aries (sheep), Bison bison (wild ox), Babalus babalus (buffalo), Gallus domesticus (chicken), Meleagrus gallopavo (turkey), Anas rubripes (duck) and Branta canadensis (goose).

Expect that every kind of method of the present invention can be used separately or combination with one another is used to select the most healthy and the strongest animal.

Detailed Description Of The Invention

The invention provides the method for among two or more animal, selecting robustness.

Definition

Term used herein " robustness (robust or robustness) " is meant the general state of animal, is characterised in that (1) average daily gain (ADG) in all one's life, (2) hot carcass measurement and (3) food conversion that is higher than average level.

Term used herein " CD " be meant the differentiation bunch.This title is to have developed the international standard title of the human leucocyte antigen of monoclonal antibody.

Term used herein " antibody " is meant a kind of protein molecule that is synthesized by the B cell when being exposed to antigen, it can make up with described antigentic specificity; Term " monoclonal antibody " is meant a kind of antibody molecule that is produced by hybridoma, its only contain can with a kind of an immunoreactive antigen binding site of special epi-position of antigen." elementary " antibody is the antibody of direct conjugated antigen." secondary " antibody is the antibody in conjunction with primary antibody.

Term used herein " antigen " is meant a kind of molecule or composition, immune response in its induced animal body, and interact with the immune antigen recognizing composition specificity of animal.

Term used herein " mitogen " is meant a kind of compound, and it stimulates the lymphocyte experience cell cycle.A kind of suitable mitogen of T cell is concanavalin A and phytolectin (PHA).

Determine the number percent of antigen presentation cell

Have to comprise the cell of expressing CD16, CD2, CD8 and MHC-DQ antigen and express to be to determine in the animal of cell of dark (D) antigen of MHC-DQ bright (B) or MHC-DQ that antigen presentation cell number percent preferably realizes as follows: separates blood monocyte (PBMC) on every side from animal body by the MHC-DQ antibody target; With PBMC and a kind of elementary monoclonal antibody incubation that is specific to interested antigen; PBMC is used a kind of secondary antibody mark of puting together with fluorescent dye; Counting is expressed the PBMC of interested antigen; And the PBMC number percent of the interested antigen of calculation expression.

Determine the propagation frequency of antigen presentation cell

Determine that in having the animal that comprises the cell of expressing T4 antigen antigen presentation cell proliferation frequency preferably realizes as follows: blood monocyte (PBMC) around separating from animal body; With PBMC with a kind of suitable fluorochrome label; PBMC is formed nutrient culture media (blastogenic medium) with a kind of suitable mother cell to be cultivated; With PBMC and a kind of mitogen incubation; With PBMC and a kind of monoclonal antibody incubation that is specific to interested antigen presentation cell; And the frequency of definite interested antigen presentation cell of having bred.

Separate PBMC

PBMC preferably uses a kind of LSM to separate by gradient separations.A kind of suitable LSM is LSM  (ICN Biomedicals).

Counting antigen presentation cell

In the present invention, the counting of specific antigen express cell and the final of PBMC number percent of expressing this antigen are determined preferably to realize by flow cytometry, more particularly use the fluorescence flow cytometry to realize.Generally speaking, flow cytometry is included in a time and cell is passed one at a time the induction zone of a chute (flow cell).Because cell is to pass chute one at a time, therefore typically need be before analyzing the diluting cells sample respond to so that can separate each cell.

The principle of fluorescence flow cytometry combined with fluorescent cell analysis and light scattering.Generally speaking, this need be with cell with a kind of suitable dyeing, and perhaps a kind of fluorochrome label covalently is attached to antigen or antibody on cell surface, therefore show the generation of specific antigen-antibody response.

In the fluorescence flow cytometry, suspending liquid with a kind of prestained or fluorescently-labeled particle, particularly the cell in blood or other biological fluid sample is transported through a chute, and each particle in the sample illuminates with one or more convergent pencil of rays herein.With one or more detector detect light beam with by the interaction between the mobile marking particle of chute.Usually, some detectors are designed to measure fluorescent emission, and other detector is measured scattering strength or duration of pulse.Therefore, each particle that passes chute all can be positioned a feature space, and its axle is the emission color, optical density or other character, the i.e. scattering of measuring by detector.Preferably, the variable grain in the sample can be positioned different and nonoverlapping zone of feature space, make each particle all based on it in the location of feature space and analyzed.

Determining of propagation frequency

Determining of the frequency of the antigen presentation cell of having bred in the present invention, is preferred by flow cytometry realization described herein.The analysis of fluidic cell propagation frequency data is preferably used and is contained the software program realization of breed module, as ModFit LT (Verity Software House, Inc., Topsham, Maine).The propagation frequency measurement is based on such principle, and promptly each of cell should have the only about half of dyestuff of parental cell from generation to generation.

Monoclonal antibody

Monoclonal antibody combination is typically showed the single binding affinity to a particular proteins immunoreactive with it.An a kind of monoclonal antibody of an epi-position (being CD2) of antigen can prepare by using the technology by the continuous cell line production antibody molecule of cultivating.These technology comprise but the non-hybridoma technology of being described by Kohler and Milstein at first that is limited to, and nearest human B cell hybridoma technology (Kozbor et al., 1983), EBV-hybridoma technology (Cole et al., 1985)), reach three knurls (trioma) technology.Other method that can be used for effective generation monoclonal antibody among the present invention comprises display technique of bacteriophage (Marks et al., 1992)).

Usually, the hybridoma technology of Kohler and Milstein is animal is carried out immunity and to begin with a kind of protein or its fragment.Immunity typically realizes by the immunogene that gives immune effective dose for the competent mammal of immunity.Preferably, described mammal is a kind of rodent, as rabbit, rat or mouse.Then this mammal is kept sufficiently long a period of time, make this mammal produce the cell of the antibody molecule of secretion and the reaction of described immunogen immune.This immune response by screening generation like this antibody molecule and a kind of immunoreactivity of immunogen protein goods detect.Randomly, can wish that described protein articles is the form that can be detected by described antibody molecule, for example antigen of film combining form (being CD2) in an analysis with a kind of protein articles screening antibody molecule.These screening techniques are well-known to those skilled in the art.Next, prepare a kind of suspending liquid that produces the cell of antibody, this cell takes out from the mammal of each inoculation of secreting desirable antibody.After time enough, put to death this mammal and therefrom obtain body cell antibody producing lymphocyte.The cell that produces antibody can derive from lymph node, spleen and the peripheral blood of the animal of contacted antigen.Preferred splenocyte can use method well known in the art that it mechanically is separated into single cell in the nutrient culture media of physiological tolerance.Mouse lymphocyte provides higher percent and stable fusion following mouse myeloma.Also can use the body cell of rat, rabbit and frog.The splenocyte chromosome of the immunoglobulin (Ig) that coding is wished is by with this splenocyte and myeloma cell's fusion and immortalization generally carries out under the situation that has fusion agent such as polyglycol (PEG).According to standard technique, the myeloma cell line of any number all can be used as a kind of fusion part.

The hybridoma that then the gained cell is comprised hope is grown in a kind of selection nutrient culture media such as HAT nutrient culture media, and wherein parental generation myeloma that does not merge or lymphocyte are dead at last.The clone who has only the hybridoma survival and can under the restriction diluting condition, grow and separate to obtain.In the supernatant of screening hybridoma desirable specific antibody have a situation, undertaken by immuno analytical method as using the antigen that has been used for immunity inoculation.Then with the positive colony monoclonal antibody that subclone also can separate generation under the restriction diluting condition.The separation of many monoclonal antibodies and purification process are arranged so that it dissociates from other protein or other pollutant.The normally used method of monoclonal antibody purification comprises ammonium sulfate precipitation, ion-exchange chromatography and affinity chromatography (seeing for example Zola et al., 1982).The hybridoma that produces according to these methods can use technology known in the art in external or proliferation in vivo (in ascites).

Normally, can for example breed in the laboratory cultures container with single clone at in-vitro multiplication, the nutrient culture media that contains the single specificity monoclonal antibody of high concentration can be by decant, filtration or centrifugal the results.Perhaps, the output of monoclonal antibody can be used to provide body cell and myeloma cell to carry out initial fusion by strengthening in the animal that a kind of hybridoma sample is injected into tissue compatible.Secretion is being grown in the animal of injection by the tumour of the monoclonal antibody specific that the hybrid cell that merges produces.The body fluid of animal provides the monoclonal antibody of high concentration as ascites or serum.

The nutrient culture media and the animal that are used to prepare these compositions are known in the art and normally can be purchased, and comprise synthetic nutrient culture media, inbreeding mouse etc.A kind of synthetic nutrient culture media for example is the Dulbecco ' s minimum essential medium (DMEM that has added 4.5gm/l glucose, 20mM glutamine and 20% hyclone; Dulbecco et al., 1959).A kind of inbreeding mouse for example is Balb/c.

Fluorescent dye/mark

The fluorescence labeling that can be used for determining the immunocyte hypotype number percent that exists in the PBMC sample comprises but non-being limited to: phycoerythrin (PE), fluorescein isothiocynate (FITC), allophycocyanin (APC), the red (TR of Texas, Molecular Probes, Inc.), peridinin chlorophyll compound (PerCp), CY5 (Biological Detection System) and with the conjugate (for example PE/CY5, PE/APC and PE/TR) of PE coupling.The fluorescence labeling that can be used for the propagation frequency of definite immunocyte hypotype comprises but the non-PKH of being limited to dyestuff, as PKH2, PKH26 and PKH67.

In the discussion in front, comprise from quoting of many technical magazines and patent and be used for reference.All this quoting at this are all incorporated reference in full with it.

Embodiment 1

Animal

To the production behavior monitoring of 199 pigs about 7 months.Described pig is the product of 3 kinds of different interior genotype (internal genotype): AxC, BXC and BXD genotype.In this test, use 18 boars altogether; 8 boars have the filial generation more than 10, and other 10 chieftain is lower than 10 in generation.Male and female sex rate is 1-1.3 in the colony.

The animal flow process

Pig is at farm A, and place I is born.Place I comprises raising, gestation and production unit.Piggy was fed about 19 days with sow milk.The feeding of sow meets or surpasses domestic lactation curve standard (internal lactation curve guidelines).

Carry out the pig flow process one time by producing (farrowing), child care (nursery) and grow-finish chamber weekly, when this pig meets the specificity target weight, pig moved to the next one stage then.The target weight of child care pig is 4-6.5kg, and the weight of testing period is 31.7kg, and weight is 122.31kg during end of test (EOT).

All pigs were all begun to handle in birth in back 24 hours.Every pig is weighed (Mosdalscale model IQ-plus 390-DC) separately also with the identification of button-type eartag.For every pig give ferrodextranum (Durvet, Inc) and penicillin (Pfizerpen  G, 1cc potpourri Pfizer) and 1cc gentamicin (Garacin , Schering Plough) are with prevention diarrhoea.

At weaning period, promptly about 19 day age, every pig is weighed separately and use color mark.Pig is transported to the nursery of place II, in these at least 7 weeks of stable breeding.Place II comprises nursery and grow-finish chamber.9 nurseries are arranged, pig by 7 weeks in sex and the weight pound, is distributed stable breeding equally with the non-experiment pig of other same age and product gene type.Experimental subjects is totally 15 pig/hurdles.Carry out one 4 stage child care program and a strict feed budget process.Pig was inoculated with haemophilus parasuis (Haemophilus parasuis) vaccine (Suvaxyn  Respifend  Hps, Fort Dodge Laboratories) in the 10th day age and the 24th day age.

From 6-7 every pig in age in week, obtain blood, and be collected in two 10ml test tubes that contain anti-coagulants (EDTA) by the vena cava anterior puncture.Blood sample is all being remained in the refrigeratory with ice bag and deliver to the laboratory by the delivery service of delivering goods all through the night and handle period.Time between handle in blood sample collection and laboratory is 20 hours or still less.So that handle in the laboratory, 25 blood samples are got in each drainage at most with 11 batches of blood samplings.

In 9 weeks during ages, pig weighed and the animal that will reach the 31.7kg target weight moves to growth/fattening and experimentizes in the chamber from the nursery.With growth/fattening chamber of 2-4 nursery's filling.With pig with same age and genotypic other unselected pig stable breeding.The pig of selecting in each hurdle is similarly indicated.60 boars of castrating and 60 sows of in a room of fattening chamber, distributing 8 hurdles.Utilize stable breeding chamber, 6 hurdle 720 pigs of stable breeding altogether.Stable breeding under the condition of pig temperature minimum change (21.6 ℃-19.4 ℃) in all rooms is also carried out feeding with 4 stages (four-phase) based on the dietary programmes of corn-soybean.

At when test every pig is weighed separately (True-Test model 700scale).In addition, use real-time ultrasound (Aloka model SSD-500V) to carry out living body measurement, comprise the back fat that is determined at first rib, aft rib (back fat), last vertebra and the loin degree of depth (loin depth).Will every pig weigh in per two weeks and reach target weight 122.3kg when finishing to test until this pig.

When end of test (EOT), pig is transported to the Swift killing machinery, and (Louisville KY) butchers.The carcass trait of collecting comprises hot genetic ability for carcass weight, the back fat that the fat-o-meter instrument records, the loin degree of depth that the fat-o-meter instrument records and lean meat number percent.

Farm health status

Inspected a farm A in every month the experimental session animal doctor.The animal doctor assesses the clinical disease in nursery and the growth/fattening chamber and the situation that exists of mortality ratio.Think that farm A has Salmonella (Salmonella) exposure level that is low to moderate medium level, thinks mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) positive by historical serological research.

The Danish MixedELISA that the farm is continued to monitor to use Iowa State University detects Salmonella antibody.Salmonella monitoring result (10 fattening pig/moons) shows non-activity seroconversion during this research, and the seroconversion of Salmonella is several years ago occurring.Do not observe the clinical indication of Salmonella.Mycoplasma hyopneumoniae serology of annual assessment.During last month of this research, detecting by Tween-ELISA test in University of Minnesota diagnostic test chamber has 3 antibody with anti-mycoplasma hyopneumoniae in 10 fattening pigs.

Medicine/feed adds program

Before child care and in the child care diet, (Neo-Terramycin  Pfizer) is used as the part that conventional feed adds program for 200g/ ton neomycinsulphate and terramycin.

Cell preparation

In the laboratory, use the LSM (LSM, Cappel, the ICN Biomedicals that describe elsewhere, Ohio) from all 199 pig blood samples, separate blood monocyte (PBMC) (Solano-Aguilar et al., 2000) on every side by gradient separations.After PBMC separates, use trypan blue dye excretion counting cells and its viable cell concentrations is adjusted into 5 * 10 ⁷Individual cell/ml.Not every pig sample all is used for all immunity to be identified.

Cell type and monoclonal antibody

Natural killer cell (NK) is the cell with the cell ability that kills some tumour cell and numerous virus infectionses.This killing and wounding is natural rather than specific immunity.In pig, the NK cell uses CD2 and CD16 pig monoclonal antibody (MAb) MSA3 and G7 respectively and is differentiated two positive cells as CD2+/CD16+.CD2MAb detects the T lymphocyte, and CD16MAb detects III type Fc γ acceptor (Fc γ RIII).

NK needs target cell to be wrapped quilt in advance by specific immune globulin (IgG) to killing and wounding of target cell, and cracking process is called the cellular toxicity of antibody dependent cellular mediation.In conjunction with the low affinity receptor Fc γ RIII of identification by IgG Fc on the leucocyte of antibody take place, described acceptor detects with CD16MAb in pig.

SLA (the main histocompatibility complex of pig) molecule plays an important role in antigen recognizing.Their targets are in special antigen, so SLA belongs to the specific immunity system.Two class MHC molecules are arranged, i.e. I class and II class.The CD8+ cell is the strongest T lymphocyte (CTL) of cellular toxicity, the fragments of peptides that combines with I class MHC molecule on its recognizing cells, and this is the target position of the splitting action of CTL.These peptides are usually derived from endogenous synthetic protein, as viral antigen.The CD4+ cell is the strongest auxiliary lymphocyte, and it discerns other cell, reaches the peptide that combines with II class MHC molecule on some subgroups (subset) surface of activated T cells in pig as B cell, macrophage.The peptide that the II class is correlated with is usually derived from outer microorganism of born of the same parents and soluble proteantigen.CD4+ and CD8+ lymphocyte subgroup can use 74-12-4 and 76-2-11MAb to detect respectively.

In people and pig MHC and SLA locus, distinguished chromosomal region is arranged, as DP, DQ and DR.In pig, SLA-DQ antibody target SLA-II antigen is respectively SLA-DQ overall (T), bright or active (B) and dark or low activity (D).On the SLA-DQ MAb identification SLA-DQ molecule one be considered to described zone a common component the singlet determinant but not discern a polymorphic determinant (SLA-DQ gene expression polymorphism product).

After from whole blood, separating PBMC, under two kinds of conditions, test lymphocyte.At first, with PBMC equal portions direct immunization dyeing and by PBMC is measured the number percent of immunocyte subgroup with elementary pig MAb incubation.Secondly, (St.Louis MO) cultivates together for ConA, Sigma, and (MO) proliferative of mensuration cell subsets is replied for Sigma, St.Louis to use flow cytometry and combination MAb and PKH67 dyeing with the mitogen concanavalin A with the PBMC equal portions.Monoclonal antibody and target cell thereof are listed in table 1 among the PBMC group.All monoclonal antibodies of using in this research all are that pig is specific, and its target cell specificity is described (Saalmueller et al., 1998 and Haverson et al., 2000) elsewhere.Select these MAb, because the surface indicia of expressing on its target monocyte and the lymphocyte.A kind of anti-pig whiting cell (panleukocyte) mark (CD45) and multiple IgG isotype contrast MAb are used separately as the positive and negative control.

(Southern Biotechnology Associates, Birmingham AL) after the secondary labeling of monoclonal antibody cell of coupling, determine the number percent with the cell of specificity MAb immunostaining with FITC or PE using.

Table 1: the target cell among pig monoclonal antibody and the PBMC

Cell subsets (or combination)	Monoclonal antibody	Target cell
			CD2+/CD16+		Express two positive lymphocytes of CD2 and CD16; NK cell (NK) cell
CD16	G7	The main low affinity receptor Fc γ RIII that expresses by a plurality of cell subsets: NK, B, macrophage
			CD2	MSA3	Sheep erythrocyte receptor; Mainly on T cell and NK cell, express, on the B cell dim (dull)
CD4+/CD8+		Express two positive t lymphocytes of CD4 and CD8; An Dan CD8 just often
			CD4	74-12-4	T lymphocyte subset group with MHC II type antigen on the antigen presenting cell (APC) and exotic antigen reaction
CD8	76-2-11	T lymphocyte subset group with MHC I type antigen on the APC and exotic antigen reaction

SLA-DQ T	TH-16	Pig II quasi-lymphocyte antigen (SLA); Be equivalent to mouse MHC II class I-E antigen; The total PBMC of T=(monocyte and lymphocyte)
			SLA-DQ B		SLA-II B; Only detect bright lymphocyte of SLA-DQ and/or macrophage
SLA-DQ D		SLA-II D; Detect SLA-DQ dim or active lower lymphocyte and/or macrophage

Flow cytometry is analyzed

Use the flow cytometry of describing elsewhere to analyze the PBMC (Solano-Aguilar et al.2001) of immunostaining.The number percent of the cell that calculating is dyeed based on fluorescence intensity uses IgG isotype background in contrast.With regard to some mark, importantly distinguish the immunofluorescence of dark (D) and bright (B), therefore reported total (T), D and the B group of SLA-DQ.Flow cytometry analysis at each sample comprises 16,000-20,000 result.

PKH67 dyeing

For breeding test, PBMC is washed once in RPMI 1640 nutrient culture media, subsequently by the nylon net filter in the tapered test tube of the polypropylene of 17 * 100ml.With cell precipitation place dilution C (Sigma, St.Louis, MO) in, reach 5 * 10 ⁶The final suspending liquid of individual cell/ml.Then this cell suspending liquid is added (1.5L dyestuff/1 * 10 in isopyknic PKH67 stock staining solution ⁷Individual cell), room temperature incubation 3 minutes.Pre-determine cell concentration with even matter and bright ground staining cell.The long-pending heat-inactivated hyclone of monoploid that will equal cell+dyestuff cumulative volume adds in this suspending liquid.Then cell centrifugation is precipitated 3 times, with RPMI nutrient culture media and serum washing, form nutrient culture media at mother cell for the last time and (add 5%FBS, 1mM Sodium Pyruvate, 2mM L-glutaminate, nonessential amino acid, 5.5 * 10 for the first time ^-5The M2-mercaptoethanol, the RPMI 1640 of 25mM Hepes damping fluid) middle washing, count with hemacytometer.The even matter of dyeing procedure detects by flow cytometry at the 0th day cell equal portions at these marks.

Propagation

Will be altogether 8 * 10 ⁶The cell of individual PKH67-mark is plated in each hole of 24 hole tissue culturing plates.Adding final dose when cultivating beginning in this hole is the concanavalin A of 5 μ g/ml.There is not mitogen in the nutrient culture media control wells.Final volume is the 2ml/ hole.Should flat board at 37 ℃ in 5%CO ₂Middle incubation 3 days.After cultivating 48 hours, the nutrient culture media of middle and upper part, hole 1ml is changed with the fresh mother cell formation of 1ml nutrient culture media.

Harvesting carries out proliferation assay

At the 3rd day, collecting cell, washing 2 times and counting in the RPMI nutrient culture media.With 1 * 10 ⁷The concentration of individual cell/ml is carried out as the described differential immunostainings in other places (differentialimmunostaining) (Solano-Aguilar et al., 2001).After the cultivation, cell is dyeed with contrasting MAb or CD4 or CD8MAb.The data of collecting are used for calculating after being exposed to ConA lymphocytic propagation to measure lymphocytic function.With ConA or and the nutrient culture media cultured cells between the contrast CD4 positive (CD4+), the CD8 positive (CD8+) and PKH67 mix the propagation situation of (total cell proliferation), to determine whether any cell subsets all increases potentially after stimulation.

The data analysis of immune cell propagation

Proliferation Wizard module analysis fluidic cell metering data in the use ModFit LT software (Verity Software House, Inc., Topsham, Maine).Cell is carried out gate to remove cell fragment according to the forward scattering and the lateral scattering signal of lymphocyte populations.The intensity of non-proliferative (parental generation) cell is determined by analyzing the sample of not cultivating with mitogen.The generation of each cell should have the PKH67 dyestuff of only about half of parental cell.From the intensity research of parental generation, ModFit software is the Gaussian distribution at center (Deconvolute) fluorescence intensity histogram (Givan et al., 1999) that deconvolutes with different channel spacings in order to the peak.Use the number percent data of cell in each female filial generation when analyzing of software repayment, calculate each cell subsets (CD4+, CD8+, each frequency PKH67+) in (preceding body frequency or the PF) initial population of having bred.The proliferation index (PI) that calculates is that all cell summations in from generation to generation are divided by the original parental cell number that exists in theory in the colony that does not stimulate.PI is a kind of assay method that experimental session (3 days) cultured cells number increases.The preceding body frequency and the proliferation index that use are lymphopoietic mensuration, and think functional selection in this research.

Test as the immune parameter of growth indexes

The manufacturing parameter of assessment is included in the average daily gain (ADG) of following growth phase: a) testing period, b) from being born to weaning period, c) from be born to the end of test (EOT) phase or all one's life daily gain, d) wean, and e to the end of test (EOT) phase) wean to the testing period.Extra manufacturing parameter such as live body and carcass measurement, feed intake and food conversion are also analyzed.

Proliferation assay (PF and PI) and immunophenotype (expressing the number percent of the cell of CD16, CD2 etc.) to the effect of manufacturing parameter by it is assessed as regressor in a mixed model analysis.

Use a mixed model (SAS Proc.MIXED) to determine to use which parameter when the time spent of doing of assessment growth traits.A complete model that uses comprises the parity (parity of sow) of product, sire (being nested in the product), nursery, fattening chamber, sex, sow, and wherein male animal is a chance mechanism, and all other factorses are all fixed.Sire is owing to partial hybrid is nested in the product.Do not find that when testing product is significant, therefore gets rid of from final model at the sire in the product.Nursery and fattening chamber mix very much, so include only the nursery in the final mask, because it produces the most significant effect.

Assessment immunocyte subunit phenotypic marker is to the predictive role of the proliferation assay and the production traits and the proliferation assay predictive role to the production traits.The final mask of these effects is shown in table 2.

Table 2: parameter model

Proliferation assay	Sire (product)+nursery+sex
		Immune labeled	Sire (product)+nursery+sex+parity
Live body/carcass trait	Body weight during sire (product)+nursery+sex+parity+measurement
		Raise parameter	Sire (product)+nursery+parity

The result:

Show that by the data of using the flow cytometry analysis that the ModFit program carries out 99.37% cell is dyeed by PKH67, has proved the efficient of this program.The result who obtains with CD45 whiting cell MAb shows that average 92.04% isolated cells is identified as leucocyte by the CD45 monoclonal antibody, shows a kind of suitable lymph gate.Background dyeing contrasts by using IgG isotype tester.The MAb of all uses all illustrates than the higher value of background contrast, shows that this detection system is effective.

The immune labeled ratio that detects in blood is given at a typical pig (pig 1867): CD4 (14.92%), CD8 (48.24%), CD4/CD8 (8.32%) SWC3 (4.12%), SLADQ T (46.4%) SLADQB (16.34%), SLADQ D (30.06%), CD16 (20.16%) CD2 (63.32%), CD2/CD16 (18.98%), CD21 (7.26%).Tester number percent is: IgGa/Ig2b (0.4/0%), Ig2a/IgG1 (0.22/.06, CD45 (98.38).Just as expected, some immunocyte subgroups are relative enrichments as CD8, CD16, CD2/CD16.The relative percentage of other mark such as SWC3 and CD21 is lower in circulation blood, but still is higher than background level sometimes.

Average weaning period weight is 6.4kg ± 0.16, and be 19.4 days ± 0.2 the age of on average weaning.When wean, 76% piggy weight is in the weight range of 9.0-14.5 pound; Remaining, 1% pig weight＜8.9 pound, 23% pig weight＞14.6 pound.Weight is 33.3kg ± 0.5 when on average beginning to test, and be 70.8 days ± 0.6 age when on average beginning to test.Weight was 123.1kg ± 0.7 when average test finished, and be 166.8 days ± 0.9 age when average test finished.

These discoveries are to observe under the similar rather than similar pig flow process of carrying out of age in pig initial production weight.Think to the weight in the pig flow process in addition standardization be important to contrast properties in pig, wherein the assessment of ADG and trunk is the proterties of measuring, because the weight of pig will influence daily gain.Similarly, the animal weight when measuring is considered in the association analysis of carrying out at carcass trait.

The result of linear model analysis shows that whole proliferation assay are subjected to the influence of sire and sex; The weightening finish proterties is subjected to the influence of sire, nursery and fattening chamber, sex and sow parity.Live body and carcass measurement are subjected to the influence of product, sire, sex and sow parity.

After the difference of having considered various sources, between immune labeled and prouctiveness, find significantly related.These associations are to find in the special producing stage that includes blood sampling period (nursery), but they are also related with other production phase, point out these marks to predict prouctiveness in the whole production lifetime of pig.

Lymphopoiesis

Find that the immunocyte subunit phenotypic marker is lymphopoietic good indication.Total cell proliferation frequency (PKH67PF) is subjected to the influence of the number percent of CD4+ (p=0.09) and SLA-DQT+ cell (p=0.009).Total cell proliferation index (PKH67PI) significantly is subjected to number percent (p=0.009) influence of SLA-DQB+ cell.At last, the propagation frequency of CD8+ cell is subjected to the influence of the number percent of CD4+/CD8+ cell (p=0.07), CD8+ (p=0.03) and SLA-DQD+ (p=0.06).

Daily gain

Immunocyte subunit phenotypic marker indication ADG.The lifelong ADG significant correlation of three kinds of phenotypes and pig comprises CD16+ cell (p=0.02), CD2+/CD16+ cell (p=0.0053), CD8+ cell (p=0.04) (table 3).A kind of functional selection is relevant with the lifelong ADG of pig: CD4PF (p=0.08).These four kinds of phenotypes and growth negative correlation, the higher percent of mark is relevant with lower ADG.

The research mark is contrast tails or extremes to the another kind of mode of the influence of growth.Yet it must be noted that the regression model as above-mentioned use is than checking that extremes better indicates, because it comprises all data.Yet, be the influence of the frequency of assessment CD2+/CD16+ cell, the average fate that contrast is increased weight and delivered for sale between 30 pigs of the highest number percent that these cell subsets are shown and 30 pigs that lowest percentage is shown to lifelong ADG.Calculate weightening finish and be ADG be multiply by the necessary average fate of weight (all pig average out to 166.8 ± 0.9 days) when reaching end of test (EOT).It is that target weight 116kg during with end of test (EOT) is divided by the highest ADG with respect to 30 pigs of described lowest percentage that average fate is delivered in calculating for sale.

The result shows that increasing by 1% expection in the CD2+/CD16+ cell makes lifelong ADG reduce 0.0018kg.Minimum 30 respondents' CD2+/CD16+ cell average out to 5.4%, 30 the highest average out to 33.1%.The forecasted variances that this means lifelong weightening finish is (33.1-5.4) * 0.0018=0.0498kg/ days.With regard to 167 days, the difference between the high and low % of CD2+/CD16+ cell is about 8.32kg.

ADG with pig of the two positive cells of higher proportion is 0.7556kg, and the target weight of 116kg needs average 156 days when therefore reaching end of test (EOT).The pig of expressing CD2+/CD16+ cell at high proportion needed 166 days reach same target weight, between group 10 days difference was arranged.

Some phenotypic markers and proliferative reply with in the specific production phase but not the lifelong ADG significant correlation of the whole production of pig.These comprise: CD4+ cell and the ADG relevant (p=0.04) that extremely weans from birth, the SLA-DQB+ cell with from the ADG relevant (p=0.08) of wean when testing, and the SWC3+ cell during with test ADG (p=0.07) reach from the ADG (p=0.09) that weans when testing relevant.All these associations all have positive correlation, and higher cell number percent correspondence is ADG (table 3 and 4) preferably.

Carcass measurement

Find remarkable related between living body measurement and cell subsets mark and the proliferation assay.These associations be included in when test the CD4+/CD8+ cell with last vertebra (p=0.015), CD8PF and first rib (p=0.08) reach related (table 4) between the SLA-DQD and back fat (p=0.08) when testing when end of test (EOT).When the carcass trait of animal of assessing same group in the slaughterhouse, these associations are not obvious.

Express the important carcass trait of ratio appreciable impact of the cell of swine leukocyte antigen (SLA) II type or SLA-DQ mark.The ratio of SLA-DQB+ and SLA-DQT+ cell related with hot genetic ability for carcass weight (p=0.04).This correlativity is positive, the SLA-DQB of higher proportion and T positive cell and higher hot genetic ability for carcass weight relevant (table 4).

Feed picked-up and effectiveness

The feed picked-up is significantly related with the ratio of SLA-DQD+ cell (p=0.04) and SLA-DQT+ cell (p=0.06).The SLA-DQ mark is also related with food conversion.SLA-DQB+ (p=0.05), SLA-DQD+ (p=0.06) is significantly related with food conversion with SLA-DQT (p=0.015).Pig with higher SLA-DQ positive cell percentage illustrates food conversion preferably.High-caliber CD8PF and relatively poor food conversion relevant (p=0.09).

Between manufacturing parameter and CD2+, CD21+, CD4PI, CD8PI, PKH67PI or PKH67PF, find onrelevant.

Generally speaking, the more heterogeneous pass result who derives from the economics viewpoint is the significant correlation between lifelong ADG, carcass trait and the food conversion of immunophenotype and pig.Therefore, with the content of setting forth by the biology aspect of immunocyte that participates in the related antibody target of these keys or cell subsets.

At the whole production life period of pig, between CD2+/CD16+ cell and weightening finish proterties, find significantly related.These pairs positive cell is the NK cell most likely.The NK cell has some tumour cell and the ability of the cell of virus infections widely of killing.This killing and wounding is not by special antigen induction, is the part of autarcesis rather than specific immunity therefore.The NK cell can the multiple target cell of cracking.The level of the cellular toxicity of NK mediation is not typically regulated by antigen, but by cell factor and hormone, regulates as interleukin 2, interferon (IFN), prolactin and growth hormone.The NK cell is secretion of gamma-IFN also, and its activated mononuclear cell development is a macrophage.

At the whole production life period of pig, between CD16+ cell and weightening finish proterties, find significantly related.In many cases, NK needs this target cell in advance with special IgG bag quilt to killing and wounding of target cell, and this cracking process is called the cellular toxicity of antibody dependent cellular mediation.In conjunction with a kind of low affinity receptor of identification by IgG Fc on the leucocyte of antibody take place, this receptor is called Fc γ receptor II I (Fc γ RIII), it is detected by CD16MAb.

Although there is not the absolute mark of differentiating the NK cell in the pig, in the monoclonal antibody that author's phase believer in a certain religion can obtain now, the combination of CD2/CD16 antibody is seemingly only.CD2 gives expression on present thymocyte, periphery T cell and the natural killer cell in human body.It is also expressed on 50% thymus gland B cell, and the expression on mature B cell is disputable.CD16 detects Fc γ RIII.For pig, a NK cell subsets it is believed that it is CD2+/CD16+.The CD16 cell subsets is mainly by T and NK cellular expression, but the B cell also can be expressed it.B and NK cell are the CD3 feminine genders.NK cell in the pig is divided into CD8-and the dim cell (dull cell) of CD8.The B cell is not remarkable as the effect in CD16+ cell subsets source in this research because between B cell antigen (CD21) and ADG onrelevant.

The CD8+ cell is the T lymphocyte (CTL) of cellular toxicity, its identification be the fragments of peptides that I type MHC molecule combines on the cell of splitting action target of CTL.These peptides are usually derived from endogenous synthetic protein, as viral antigen.In this research, do not study MHC I type antigen.

The CD4+ lymphocyte is topmost auxiliary cell, and identification reaches the peptide that the lip-deep II type MHC molecule of some activated T cells subgroups combines in pig with other cell such as B cell, macrophage.The peptide that the II type is correlated with is usually derived from outer microorganism of born of the same parents and soluble proteantigen.

The ratio of SLA-DQ+ cell illustrates remarkable related with carcass trait and food conversion.SLA or pig MHC molecule play an important role in antigen recognizing.SLA-DQ pig MAb target SLA II type antigen is thought itself and mouse MHC II type locus, and I-E is suitable with people HLA-DQ.At gene level, the DQ zone comprises α and β chain gene seat; SLA-DQ is the heterodimer of a α chain and a β chain normally, and it is the product by the surface expression of antibody test.A singlet determinant on the SLA-DQ monoclonal antibody identification SLA-DQ molecule thinks that it is gene conservative sequence in the SLA-DQ gene.

SWC3 MAb is with relevant at the ADG of limit production in the stage.SWC3 target monocyte.Monocyte is such cell mass, and it is critical in autarcesis, and also plays a crucial role in special acquired immunity.More monocytic functions are the phagocytosiss to the external source particle, produce the medium of kill microorganisms and control infection diffusion, produce cell factor and growth factor, antigen presenting cell function and promotion t cell activation.

Lymphocyte height propagation has illeffects to manufacturing parameter such as ADG and food conversion.Reported that under the attack of acute salmonella lymphopoiesis is a good indication of salmonella resistance and growth.In the research, before attacking blood lymphocyte reply than higher proliferation relevant with disease resistance under experiment condition with the growth reduction (VanDiemen et al., submitted).In this research, higher proliferation is replied relevant with illeffects to the manufacturing parameter that comprises growth, and it may be relevant with the resistance of subclinical disease.Most important ground, this illeffects observes in the drove of the no special acute illness of business contexts, and this may be the indication of robustness.

Microbial exposure occurs in not exclusively aseptic any environment.Microbial exposure also exists in the commercial operation that cleans most really.In this research, do not assess pig and be exposed to special pathogen, but association results (ADG) is pointed out crucial immunocyte and participated in replying virus (NK cell, CD8+ cell) and bacterial infection (CD4+, SLA-DQ+, monocyte).Even in a kind of as the environment that does not wherein have obvious virus infections clinical indication in the situation of farm A as if this be correlated with.

Some factors can influence the immune labeled number percent that exists in the blood, and wherein a kind of factor is inoculation.Before child care and the child care stage inoculate Haemophilus parasuis bacterin in 10 day age and 24 day age.Before carrying out the cell evaluation, collection blood carries out the inoculation second time at least 3 weeks.Because be at least 3 weeks, infer that therefore inoculation does not make significant difference to immune cell population in the time before the collection blood sample carries out immunostaining after the inoculation for the second time.

Animal doctor's report shows at farm A, clinical disease shown in the child care of some pigs during studying among the II of place.These signs comprise the accidental pig that seems morbid state, and its growth is bad, and cough.Whole child care and growth/fattening mortality ratio is respectively 1-2% and 2%.These number percents are in commercial operation in normal mortality ratio scope.When therefore, 98% of its test immunocyte pig survives to end of test (EOT).When having only 4 not survive in the pig that 199 experimentize to end of test (EOT).

The processing of carrying out at farm A comprises according to the sex subfield, the raising scheme, and vaccine inoculation, pig density, similar to many commercial operations.Equally, the degree that is exposed to Salmonella and mycoplasma is total in many pig operation schemes.Therefore, the conclusion that derives from this research can be inferred similar operation.Immunocyte in this research has conservative function in other animal species, these cell subsets marks can be used for animal reservoir's pedigree widely potentially.

Table 3: (CD4 CD8) replys with proliferative, average daily gain, carcass trait and raise related between the parameter immunocyte hypotype for CD16, CD2/CD16

The dependence proterties	Observed number	Return valuation (se)	The P value
				The effect of CD16
ADG when beginning to test	136	-0.004(0.0023)	0.095 1
				ADG from birth to wean	139	-0.002(0.0009)	0.03 1
Lifelong ADG	136	-0.003(0.0014)	0.02 1
				ADG when weaning to end of test (EOT)	136	-0.003(0.0014)	0.03 1
The ADG of wean when beginning to test	139	-0.006(0.0019)	0.0013 1
				The effect of CD2/CD16
ADG when beginning to test	136	-0.005(0.0023)	0.03 1
				ADG from birth to wean	139	-0.002(0.0009)	0.011 1
Lifelong ADG	136	-0.004(0.0014)	0.0053 1
				ADG when weaning to end of test (EOT)	136	-0.004(0.0014)	0.01 1
The ADG of wean when beginning to test	139	-0.006(0.0019)	0.003 1
				The effect of CD8
ADG when beginning to test	137	-0.002(0.0015)	0.021
				ADG from birth to wean	140	-0.001(0.0006)	0.2
Lifelong ADG	137	-0.002(0.009)	0.04 1
				ADG when weaning to end of test (EOT)	137	-0.002(0.009)	0.05 1
The ADG of wean when beginning to test	140	-0.004(0.0012)	0.005 1
				The effect of CD4PF
ADG when beginning to test	118	-0.099(0.0932)	0.29
				ADG from birth to wean	120	-0.013(0.0494)	0.8
Lifelong ADG	118	-0.109(0.0624)	0.08 1
				ADG when weaning to end of test (EOT)	120	-0.105(0.0605)	0.09 1
The ADG of wean when beginning to test	120	-0.0141(0.0818)	0.09 1

¹The conspicuousness of p＜0.01 level

Table 4:SLA-DQ hypotype is to the effect of carcass trait and raising parameter

The dependence proterties	Observed number	Return valuation (se)	The P value
				The effect of SLA-DQB
First rib when beginning to test	139	-0.0213(0.0164)	0.2
				First rib during end of test (EOT)	136	-0.001(0.0462)	0.98
Back fat when beginning to test	139	-0.005(0.0122)	0.69
				Back fat during end of test (EOT)	136	-0.019(0.0333)	0.57
The loin degree of depth when beginning to test	139	-0.071(0.3346)	0.83
				Loin degree of depth during end of test (EOT)	136	-0.0417(0.055)	0.45
Last vertebra when beginning to test	139	-0.0004(0.0195)	0.98
				Last vertebra during end of test (EOT)	136	-0.0059(0.0258)	0.82
Hot genetic ability for carcass weight	131	-0.3587(0.1753)	0.04 1
				The fat-o-meter back fat	131	-0.003(0.0347)	0.93
The fat-o-meter loin degree of depth	131	-0.0509(0.0792)	0.52
				Lean meat number percent	131	-0.0078(0.0249)	0.76
Feed is taken in	105	0.0048(0.0043)	0.27
				Food conversion	105	0.0091(0.0046)	0.05 1
The effect of SLA-DQD
				First rib when beginning to test	139	0.0034(0.0192)	0.86
First rib during end of test (EOT)	136	0.036(0.0536)	0.51
				Back fat when beginning to test	139	-0.025(0.014)	0.08 1
Back fat during end of test (EOT)	136	0.0130(0.0388)	0.74
				The loin degree of depth when beginning to test	139	0.3441(0.3877)	0.38
Loin degree of depth during end of test (EOT)	136	0.0615(0.0638)	0.34
				Last vertebra when beginning to test	139	0.0091(0.0226)	0.69
Last vertebra during end of test (EOT)	136	0.007(0.0301)	0.82
				Hot genetic ability for carcass weight	131	0.1956(0.2101)	0.35
The fat-o-meter back fat	131	0.0164(0.0402)	0.68
				The fat-o-meter loin degree of depth	131	0.0510(0.0919)	0.58
Lean meat number percent	131	0.004(0.0288)	0.9
				Feed is taken in	105	0.0109(0.0053)	0.04 1
Food conversion	105	0.0111(0.0058)	0.06 1
				The effect of SLA-DQT

First rib when beginning to test	139	0.0122(0.0118)	0.3
				First rib during end of test (EOT)	136	-0.014(0.0329)	0.67
Back fat when beginning to test	139	-0.012(0.0087)	0.17
				Back fat during end of test (EOT)	136	-0.005(0.0238)	0.85
The loin degree of depth when beginning to test	139	0.0942(0.2394)	0.69
				Loin degree of depth during end of test (EOT)	136	0.0442(0.039)	0.26
Last vertebra when beginning to test	139	0.0037(0.0139)	0.79
				Last vertebra during end of test (EOT)	136	0.0005(0.0184)	0.98
Hot genetic ability for carcass weight	131	0.2616(0.1269)	0.04 1
				The fat-o-meter back fat	131	0.0048(0.0251)	0.85
The fat-o-meter loin degree of depth	131	0.0466(0.0573)	0.42
				Lean meat number percent	131	0.0026(0.018)	0.89
Feed is taken in	105	0.0057(0.0029)	0.06 1
				Food conversion	105	0.0078(0.0031)	0.015 1

¹The conspicuousness of p＜0.01 level

Embodiment 2

The size of swinery body of research is increased to extra 286 pigs.Purpose be collect more data with confirm immunophenotype (CD16, CD2/CD16, CD8) with production performance (growth) between related.Pig in this research is derived from two different farms, and farm is identical with embodiment's 1.The mensuration of the management of animal, the record of production performance and immunology proterties is described similar to embodiment 1.Similarly, to carry out statistical analysis with embodiment 1 described identical mode.Association analysis at the 1st year report comprises 139 animals.The result of the 1st year and the 2nd year is totally 425 animals.See the following form 5.

Table 5: immunophenotype is to the effect of average daily gain (ADG).The statistical model that uses is: sire (product)+nursery's (year)+sex+parity.Da Xiao data set hereto, the result of p value＞0.1 thinks significantly.The result that runic is represented represents the 1st year and combined in the 2nd year.The result that non-runic is represented is only from the 1st year.

CD16

Growth parameter(s)	Observed number	Return valuation (se)	The P value
				ADG when beginning to test	425	-.829(.5170)	0.11
ADG from birth to wean	139	-.002(.0009)	.003
				Lifelong ADG	425	-.580(.3451)	0.09
ADG when weaning to end of test (EOT)	22.5	-.002(.0013)	0.13
				The ADG of wean when beginning to test	139	-.006(.0019)	.0012

CD2/CD16

Growth parameter(s)	Observed number	Return valuation (se)	The P value
				ADG when beginning to test	425	-.572(.5387)	0.29
ADG from birth to wean	139	-.002(.0009)	0.011
				Lifelong ADG	425	-.339(.3598)	0.35
ADG when weaning to end of test (EOT)	225	-.002(.0014)	0.096
				The ADG of wean when beginning to test	139	-.006(.0019)	0.003

CD8

Growth parameter(s)	Observed number	Return valuation (se)	The P value
				ADG when beginning to test	425	-.134(.3288)	0.68
ADG from birth to wean	139	-.001(.0006)	0.19
				Lifelong ADG	425	-.068(.2212)	0.76
ADG when weaning to end of test (EOT)	225	-.002(.0008)	0.002
				The ADG of wean when beginning to test	139	-.004(.0012)	0.0045

Be important to note that following content in the last table 5:

These extra results provide such evidence, i.e. the number percent of CD16 positive cell relevant with the growth of the whole production life period of pig (p＞0.09).

In addition, these results provide such evidence, and promptly the number percent of CD2+/CD16+ cell is relevant with growth.In this case, this relevant be significant (p＞0.096) for the ADG when weaning to end of test (EOT), but optional to lifelong ADG.

Moreover these results provide such evidence, and promptly the number percent of CD8 positive cell is relevant with the growth of pig.In this case, this relevant be significant (p＞0.002) for the ADG when weaning to end of test (EOT), but optional to lifelong ADG.

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Those skilled in the art are by means of announcement of the present invention, can carry out multiple change, replace or carry out other variation with equivalent, to implement the present invention method and composition as herein described.Therefore, except the described content of following claims, protection scope of the present invention is not limited by other content.

Claims (58)

1. method of among two or more animal, selecting robustness, described method comprises the steps: to provide two or more animal of same species; Determine the quantity of CD16 antigen presentation cell in each animal; And select the minimum animal of CD16 antigen presentation cell quantity, thereby among two or more animal, select robustness.

2. method of among two or more animal, selecting robustness, described method comprises the steps: to provide two or more animal of same species; Determine the quantity of the two positive antigen presentation cells of CD16 and CD2 in each animal; And select CD16 and the two minimum animals of positive antigen presentation cell quantity of CD2, thereby among two or more animal, select robustness.

3. method of among two or more animal, selecting robustness, described method comprises the steps: to provide two or more animal of same species; Determine the quantity of CD8 antigen presentation cell in each animal; And select the minimum animal of CD8 antigen presentation cell quantity, thereby among two or more animal, select robustness.

4. method of among two or more animal, selecting robustness, described method comprises the steps: to provide two or more animal of same species; Determine the quantity of MHC-DQ antigen presentation cell in each animal; And select the highest animal of MHC-DQ antigen presentation cell quantity, thereby among two or more animal, select robustness.

5. method of among two or more animal, selecting robustness, described method comprises the steps: to provide two or more animal of same species; Determining to express by the MHC-DQ antibody target in each animal is the quantity of the cell of MHC-DQB antigen; Reaching and selecting expression is the highest animal of cell quantity of MHC-DQB antigen by the MHC-DQ antibody target, thereby selects robustness among two or more animal.

6. method of among two or more animal, selecting robustness, described method comprises the steps: to provide two or more animal of same species; Determining to express by the MHC-DQ antibody target in each animal is the quantity of the cell of MHC-DQD antigen; Reaching and selecting expression is the highest animal of cell quantity of MHC-DQD antigen by the MHC-DQ antibody target, thereby selects robustness among two or more animal.

7. method of among two or more animal, selecting robustness, described method comprises the steps: to provide two or more animal of same species; Determine the propagation frequency of T4 antigen express cell in each animal; And select the minimum animal of T4 antigen express cell propagation frequency, thereby among two or more animal, select robustness.

8. the process of claim 1 wherein that described species are selected from: Bos taurus, Sus scrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrusgallopavo, Anas rubripes and Branta canadensis.

9. the method for claim 2, wherein said species are selected from: Bos taurus, Sus scrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrusgallopavo, Anas rubripes and Branta canadensis.

10. the method for claim 3, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

11. the method for claim 4, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

12. the method for claim 5, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

13. the method for claim 6, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

14. the method for claim 7, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

15. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Determine the two positive antigen presentation cells of CD16 antigen presentation cell, CD16 and CD2 in each animal, CD8 antigen presentation cell, MHC-DQ antigen presentation cell, express by the MHC-DQ antibody target and be the cell of MHC-DQB antigen and be the quantity of the cell of MHC-DQD antigen by the MHC-DQ antibody target; Determine the propagation frequency of T4 antigen express cell in each animal; And select that CD16 antigen presentation cell quantity is minimum, CD16 and the two positive antigen presentation cell quantities of CD2 are minimum, CD8 antigen presentation cell quantity is minimum, MHC-DQ antigen presentation cell quantity is the highest, express by the MHC-DQ antibody target be the cell quantity of MHC-DQB antigen the highest, be the minimum animal of propagation frequency of the highest and T4 antigen express cell of the cell quantity of MHC-DQD antigen by the MHC-DQ antibody target, thereby among two or more animal, select robustness.

16. the method for claim 15, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

17. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Determine the quantity of CD16 antigen presentation cell in each animal; The quantity of CD16 antigen presentation cell of determining animal is significantly related with the statistics between the robustness; Reach based on described related selection animal with the improvement robustness.

18. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Determine the quantity of the two positive antigen presentation cells of CD16 and CD2 in each animal; The quantity of determining the two positive antigen presentation cells of the CD16 of animal and CD2 is significantly related with the statistics between the robustness; Reach based on described related selection animal with the improvement robustness.

19. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Determine the quantity of CD8 antigen presentation cell in each animal; The quantity of CD8 antigen presentation cell of determining animal is significantly related with the statistics between the robustness; Reach based on described related selection animal with the improvement robustness.

20. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Determine the quantity of MHC-DQ antigen presentation cell in each animal; The quantity of MHC-DQ antigen presentation cell of determining animal is significantly related with the statistics between the robustness; Reach based on described related selection animal with the improvement robustness.

21. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Determining to express by the MHC-DQ antibody target in each animal is the quantity of the cell of MHC-DQB antigen; The expression of determining animal is that the cell quantity of MHC-DQB antigen is significantly related with the statistics between the robustness by the MHC-DQ antibody target; Reach based on described related selection animal with the improvement robustness.

22. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Determining to express by the MHC-DQ antibody target in each animal is the cell quantity of MHC-DQD antigen; The expression of determining animal is that the cell quantity of MHC-DQD antigen is significantly related with the statistics between the robustness by the MHC-DQ antibody target; Reach based on described related selection animal with the improvement robustness.

23. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Determine the propagation frequency of T4 antigen express cell in each animal; The propagation frequency of T4 antigen express cell of determining animal is significantly related with the statistics between the robustness; Reach based on described related selection animal with the improvement robustness.

24. the method for claim 17, wherein said species are selected from Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

25. the method for claim 18, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

26. the method for claim 19, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

27. the method for claim 20, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

28. the method for claim 21, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

29. the method for claim 22, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

30. the method for claim 23, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

31. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Obtain biological sample from animal, wherein said sample comprises whole blood; Determine the quantity of CD16 antigen presentation cell in each animal; And select the minimum animal of CD16 antigen presentation cell quantity, thereby among two or more animal, select robustness.

32. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Obtain biological sample from animal, wherein said sample comprises whole blood; Determine the quantity of the two positive antigen presentation cells of CD16 and CD2 in each animal; And select CD16 and the two minimum animals of positive antigen presentation cell quantity of CD2, thereby among two or more animal, select robustness.

33. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Obtain biological sample from animal, wherein said sample comprises whole blood; Determine the quantity of CD8 antigen presentation cell in each animal; And select the minimum animal of CD8 antigen presentation cell quantity, thereby among two or more animal, select robustness.

34. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Obtain biological sample from animal, wherein said sample comprises whole blood; Determine the quantity of MHC-DQ antigen presentation cell in each animal; And select the highest animal of MHC-DQ antigen presentation cell quantity, thereby among two or more animal, select robustness.

35. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Obtain biological sample from animal, wherein said sample comprises whole blood; Determining to express by the MHC-DQ antibody target in each animal is the quantity of the cell of MHC-DQB antigen; Reaching and selecting expression is the highest animal of cell quantity of MHC-DQB antigen by the MHC-DQ antibody target, thereby selects robustness among two or more animal.

36. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Obtain biological sample from animal, wherein said sample comprises whole blood; Determining to express by the MHC-DQ antibody target in each animal is the quantity of the cell of MHC-DQD antigen; Reaching and selecting expression is the highest animal of cell quantity of MHC-DQD antigen by the MHC-DQ antibody target, thereby selects robustness among two or more animal.

37. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Obtain biological sample from animal, wherein said sample comprises whole blood; Determine the propagation frequency of T4 antigen express cell in each animal; And select the minimum animal of T4 antigen express cell propagation frequency, thereby among two or more animal, select robustness.

38. the method for claim 31, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

39. the method for claim 32, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

40. the method for claim 33, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

41. the method for claim 34, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

42. the method for claim 35, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

43. the method for claim 36, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

44. the method for claim 37, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

45. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Obtain biological sample from animal, wherein said sample comprises whole blood; Determine the quantity of CD16 antigen presentation cell in each animal; The quantity of CD16 antigen presentation cell of determining animal is significantly related with the statistics between the robustness; Reach based on described related selection animal with the improvement robustness.

46. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Obtain biological sample from animal, wherein said sample comprises whole blood; Determine the quantity of the two positive antigen presentation cells of CD16 and CD2 in each animal; The quantity of determining the two positive antigen presentation cells of the CD16 of animal and CD2 is significantly related with the statistics between the robustness; Reach based on described related selection animal with the improvement robustness.

47. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Obtain biological sample from animal, wherein said sample comprises whole blood; Determine the quantity of CD8 antigen presentation cell in each animal; The quantity of CD8 antigen presentation cell of determining animal is significantly related with the statistics between the robustness; Reach based on described related selection animal with the improvement robustness.

48. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Obtain biological sample from animal, wherein said sample comprises whole blood; Determine the quantity of MHC-DQ antigen presentation cell in each animal; The quantity of MHC-DQ antigen presentation cell of determining animal is significantly related with the statistics between the robustness; Reach based on described related selection animal with the improvement robustness.

49. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Obtain biological sample from animal, wherein said sample comprises whole blood; Determining to express by the MHC-DQ antibody target in each animal is the quantity of the cell of MHC-DQB antigen; The expression of determining animal is that the quantity of cell of MHC-DQB antigen is significantly related with the statistics between the robustness by the MHC-DQ antibody target; Reach based on described related selection animal with the improvement robustness.

50. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Obtain biological sample from animal, wherein said sample comprises whole blood; Determining to express by the MHC-DQ antibody target in each animal is the quantity of the cell of MHC-DQD antigen; The expression of determining animal is that the quantity of cell of MHC-DQD antigen is significantly related with the statistics between the robustness by the MHC-DQ antibody target; Reach based on described related selection animal with the improvement robustness.

51. a method of selecting robustness among two or more animal, described method comprises the steps: to provide two or more animal of same species; Obtain biological sample from animal, wherein said sample comprises whole blood; Determine the propagation frequency of T4 antigen express cell in each animal; The propagation frequency of T4 antigen express cell of determining animal is significantly related with the statistics between the robustness; Reach based on described related selection animal with the improvement robustness.

52. the method for claim 45, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

53. the method for claim 46, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

54. the method for claim 47, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

55. the method for claim 48, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

56. the method for claim 49, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

57. the method for claim 50, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.

58. the method for claim 51, wherein said species are selected from: Bos taurus, Susscrofa, Ovis aries, Bison bison, Babalus babalus, Gallus domesticus, Meleagrus gallopavo, Anas rubripes and Branta canadensis.