METHODS OF SELECTION FOR THE EFFICIENT DEVELOPMENT OF ANIMALS BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION The present invention relates generally to methods of animal reproduction. More specifically, the invention relates to methods for selecting robustness between two or more animals based on the number of immune cell subtypes and the frequency of lymphocyte proliferative responses in animals. DESCRIPTION OF THE RELATED ART Animals have a complex array of molecular and cellular defenses, collectively referred to as the immune system, which recognizes and attacks foreign or endogenous but potentially harmful cells (respectively represented by, for example, pathogens such as bacteria or virus, and cancer cells or infected by pathogens), but that do not attack, but rather tolerate normal endogenous cells. When stimulated by foreign or abnormal biomolecules, the immune system undergoes a series of activities designed to neutralize and destroy pathogens, or cancer cells or cells infected with pathogens, with which strange or abnormal biomolecules are associated. These activities, collectively known as a x-immune response, may consist of a cell-mediated immune response, a humoral immune response (antibody-mediated) or an immune response "that includes elements of cell-mediated and humoral responses. The humoral antibodies are mediated by antibodies, glycoproteins that bind specific extraneous or abnormal biomolecules and attract other components of the immune system to them.Antibodies are immunoglobulin (Ig) molecules produced by B cells, lymphocytes that originate in the sac of the birds or in the bone marrow of mammals but migrate and mature in other organs, particularly the spleen (Robertson, "1983). Cell-mediated immune responses are the result of T-cell activities, lymphocytes that undergo maturation within the thymus of an animal (Tizard, 1988). The activities of T cells vary considerably between different subpopulations of T cells within an animal. Cytotoxic T cells recognize and destroy foreign cells (graft rejection) or endogenous but abnormal cells (eg, cancer cells or cells infected with intercellular parasites such as viruses and bacteria). T helper cells interact with, and produce biomolecules that influence the behavior of both B cells and cytotoxic T cells, in order to promote and direct antibody production and cytotoxic activities, respectively (Mossier, 1967). There are also other classes of T cells, including suppressor T cells- and memory T cells (Miedema and Melief, 1983; Tizard, supra, pp. 225-8). The classes of T cells are to some degree distinguished on the basis that different T cells exhibit different CD antigens on their surfaces. In order to function properly, the T and B cells of an animal's immune system must precisely and reliably identify a huge number of non-self compositions that are foreign or endogenous but abnormal compositions. Recognition and identification by the immune system occurs at the molecular level. An antigen, a molecular composition that has the potential to generate an immune response, is composed of one or more molecular size identification characteristics known as epitopes.A polypeptide antigen having an amino acid sequence comprising, example, one hundred amino acids could comprise dozens of epitopes, wherein each epitope is defined by a portion of polypeptide comprising from about 3 to about 15 amino acids.The number of epitopes derivable from polypeptides alone is estimated to be about ten million ( Tizard, supra, p.25). An antigen found by a T or B cell of an animal must be identified as being either associated with normal endogenous (ie, self) antigens, an immune response to which would be detrimental to the animal, or with foreign or abnormal antigens (that is, not own) to which an immune response must be mounted. The process can be explained by analogy to the identification of "friend or foe" in human combat. If the human system fails to identify associated antigens as invading pathogens or tumor cells as not their own, then these "enemies" can slip through the system's defenses. If the immune system erroneously identifies endogenous antigens of an animal as not own, in all parts of the body of the animal comprising the endogenous antigens they will face a "favorable fire" from the immune system. This last situation, in which the immune system of an animal erroneously engages in a cellular and molecular "war" against another, the normal part of an animal's body, is generally known as- "utoinmune disease". As part of the immune system's means of identifying antigens, the individual T and B cells produce antigen receptors that are displayed on the surface of the T or B cell and that bind specific antigens. Although each individual T or B cell exhibits identical antigen receptors, an animal's collection of different antigen receptors is very diverse. For T or B cells, the binding of the antigen to the antigen receptor of a cell activates the cell, that is, it stimulates the cell to undergo activity related to the generation of an immune response mediated by humoral cell. Although B cells can directly bind the antigen, T cells respond to the antigen only when it is exhibited in specific classes of other cells known generically as antigen presenting cells (APCs). APCs, for example, macrophages and dendritic cells, present polypeptide-derived antigens via glycoproteins, known as MHC proteins (major histocompatibility complex) that are displayed on the surface of APCs (Bevan et al., 1994). Without the MHC proteins, the T cells would be able to distinguish between foreign or endogenous antigens. During the last decade, most livestock industries have developed estimated breeding values to allow the identification of the best breeding animals. These estimated reproduction values are calculated based on the performance information of various attributes of the individual animal and its relatives, and represent the most accurate criteria for identifying animals of high generic merit. It is widely believed, however, that environmental stimuli similar to disease limit the expression of the "true" genetic potential of animals such as pigs. This may be due to the fact that selection occurs in units of high health status that differ significantly from the environments found in commercial production. This degradation of the state of health, and the concomitant reduction in the performance of the individual during multiplication is observed in many plant and animal systems. Similar to other systems of agriculture, today's livestock industry has a need for robust animals that perform well under different commercial facilities. Consequently, there is a need to develop tools that can identify and / or classify the most suitable animals for particular production systems. Due to the importance of the immune system of an animal in its global health, it is possible that there is a correlation between the robustness of an animal and the amount of specific immune cells that the animal possesses. Similarly, it is possible that there is a correlation between the robustness and functionality of particular immune cells. BRIEF DESCRIPTION OF THE INVENTION In accordance with an aspect of the invention, a method for selecting robustness between two or more animals is provided, which comprises providing two or more "animals of the same species; determine in each animal the number of cells expressing CD16 antigens, and select the animal with the lowest amount of cells expressing CD16 antigens. According to yet another aspect of the invention, there is provided a method for selecting robustness between two or more animals, comprising providing two or more animals of the same species; determine in each animal the number of cells expressing CD16 and CD2 double-positive antigen and select the animal with the lowest amount of cells expressing double-positive antigen * CD16 and CD2. According to yet another aspect of the invention, there is provided a method for selecting robustness between two or more animals, comprising providing two or more animals of the same species; determine in each animal the number of cells expressing CD8 antigen; and selecting the animal with the lowest amount of cells expressing CD8 antigens. According to yet another aspect of the invention, there is provided a method for selecting robustness between two or more animals comprising providing two or more animals of the same species; determine in each animal the number of cells that express MHC-DQ antigen; and selecting the animal with the highest amount of cells expressing MHC-DQ antigen. According to yet another aspect of the invention, there is provided a method for selecting robustness between two or more animals, comprising providing two or more animals of the same species; determining in each animal the amount of cells expressing an antigen that is directed by the MHC-DQ antibodies as "" MHC-DQB; and selecting the animal with the highest amount of cells expressing an antigen that is directed by the MHC-DQ antibodies as MHC-DQB. According to yet another aspect of the invention, there is provided a method for selecting robustness between two or more animals, comprising providing two or more animals of the same species; determine in each animal the number of cells that express an antigen that is directed by the MHC-DQ antibodies such as MHC-DQD, and select the animal. with the highest amount of cells expressing an antigen that is directed by the MHC-DQ antibodies as MHC-DBQ. According to yet another aspect of the invention, there is provided a method for selecting robustness between two or more animals, comprising providing two or more animals of the same species; determine in each animal the proliferation frequency of the cells expressing CD4 antibodies; and selecting the animal with the lowest proliferation frequency of cells expressing CD4 antigen.
The species in which robustness can be selected include, but are not limited to, Bos taurus (cow), Sus scrofa (pig), Ovis arles (sheep), Bison bison (bison), Babalus babalus (buffalo), Gallus domesticus (Gallus domesticus). chicken), Meleagrus gallipavo (turkey), Anas rubripes (duck) and Branta canadensis (goose). It is contemplated that each of the methods of the invention described herein may be used alone or in combination with each other to select the most robust animals. DETAILED DESCRIPTION OF THE INVENTION The present invention provides methods for selecting robustness between two or more animals. Definitions As used herein, the terms
"Robust" and "robustness" are proposed to refer to the general condition in an animal characterized by greater than the average of (1) average daily gain (ADG) of the life time, (2) measurements of the hot channel, and (3) ) food conversion. As used herein, the term "CD" is intended to refer to the differentiation grouping. This designation is the international standard for leukocyte antigens for which monoclonal antibodies have been developed. As used herein, the term "antibody" is intended to refer to a protein molecule -synthesized by a cell: B on exposure to the antigen that is capable of combining specifically with that antigen; the term "monoclonal antibody" is intended to refer to an antibody molecule produced by a hybridoma that contains only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen. A "primary" antibody is one that binds the antigen directly. A "secondary" antibody is one that binds the primary antibody. As used herein, "the term" antigen "is intended to refer to a molecule or composition of matter that induces an immune response in an animal, and specifically interacts with components that recognize antigen of an animal's immune system. As used herein, the term "mitogen" is proposed to refer to a compound that stimulates lymphocytes to transit through the cell cycle. A suitable mitogen for T cells is Concanavalin? and phytohemagglutinin (PHA). Determination of the Percentage of Antigen Expressing Cells A determination of the percentage of antigen-expressing cells, including cells expressing the CD16, CD2, CD8 and MHC-DQ antigens, as well as cells expressing an antigen that is directed by the MHC-DQ antibodies as bright HC-DQ (B) or opaque MHC-DQ (D), in an animal having such cells is preferably performed by isolating peripheral blood mononuclear cells (PBMCs) from the animal; incubate the PBMCs with a primary monoclonal antibody specific for the antigen of interest; label the PBMCs with a conjugated secondary antibody - to a present dye; count the PBMCs that express the antigen of interest; and calculate the percentage of PBMCS expressing the antigen of interest. Determination of the Proliferation Frequency of Antigen Expressing Cells A determination of the proliferation frequency of antigen-expressing cells, including cells expressing the CD4 antigen, in an animal having such cells, is preferably done by isolating the cells peripheral blood mononuclear cells (PBMCS) of the animal; label the PBMCs with a suitable fluorescent dye; cultivate PBMCs with an adequate blastogenic medium; incubate the PBMCs with a mitogen; incubate the PBMCs with a monoclonal antibody specific for the cell expressing antigen of interest; and determining the frequency with which the cell expressing the antigen of interest has proliferated. Isolation of PBMCs - 'PBMCs are preferably isolated by gradient separation with a lymphocyte separation medium. A suitable lymphocyte separation means is LSM® (ICN Biomedicals). Counting Cells Expressing Antigen In the invention, the counting of cells expressing specific antigens, and the final determination of the percentage of PBMCs expressing that antigen, preferably by means of flow cytometry, more specifically fluorescence flow cytometry. Generally speaking, the flow cytometry consists of passing the cells one at a time through a detection zone of a flow cell. Since the cells are passed through the flow cell one at a time, it is typically necessary to dilute the cell sample before analysis so that individual cells can be isolated for detection. A fluorescence flow cytometer incorporates the principles of fluorescence cell analysis with light scattering. In general, this requires that the cells be stained with an appropriate color dye, or that a fluorochrome label be covalently bound to an antigen or antibody on the surface of the cells, thereby indicating the occurrence of a reaction of antigen-specific antibody. In fluorescence flow cytometry, a suspension of previously stained or fluorescently labeled particles, typically cells in a sample of blood or other biological fluid, is transported through a flow cell where the individual particles in the sample are removed with one or more focused light beams. One or more detectors detect the interaction between the beam (bundles) of light and the marked particles flowing through the flow cell. Commonly, some of the detectors are designed to measure fluorescent solutions, while other detectors measure the dispersion intensity or pulse duration. Thus, each particle that passes through the flow cell can be mapped in a characteristic space whose axes are the emission colors, light intensities, or other properties, that is, dispersion, measured by the detectors. Preferably, the different particles in the sample can be mapped in different and non-superimposed regions of the characteristic space, allowing each particle to be analyzed based on its cartography in the characteristic space. Determination of Proliferation Frequency In the invention, the determination of the frequency with which a cell expressing antigen has proliferated is preferably performed by flow cytometry, as described herein. The flow cytometric proliferation frequency data analysis is preferably performed with a software program containing the proliferation module such as ModFit LT (Verita Software House, Inc., Topsham, Maine). The determination of proliferation frequency is based on the principle that each generation of cells must have approximately half the dye of the cells of origin. Monoclonal Antibodies A composition of 'antibody. monoclonal typically exhibits a single binding affinity for a particular protein with which it immunoreacts. A monoclonal antibody to an epitope of an antigen (i.e., CD2) can be prepared by using a technique that provides for the production of antibody molecules by continuous cell lines in culture. Esfos include but are not limited to the hybridoma technique originally described by Kohler and Milstein, and the most recent human B-cell hybridoma technique (Kozbor et al., 1983), EBV-hybridoma technique (Colé et al., (1985) ) and trioma techniques. Other methods that can effectively produce monoclonal antibodies useful in the present invention include phage display techniques (Arks et al., (1992)). Generally, the Kohler and Milstein hybridoma technique begins with the immunization of an animal with a protein or a fragment thereof. Immunization will typically be performed by administering the immunogen to an immunologically competent mammal in an immunologically effective amount. Preferably, the mammal is a rodent such as a rabbit, rat or mouse. The mammal is then maintained for a period of time sufficient for the mammal to produce cells that secrete antibody molecules that immunoreact with the immunogen. Such immunoreaction is detected by classifying the antibody molecules thus produced for immunoreactivity with a preparation of the immunogen protein. Optionally, it may be desired to classify the antibody molecules with a preparation of the protein in the form in which it will be detected by the antibody molecules in an assay, for example, a form of the membrane-associated antigen (i.e. CD2). These classification methods are well known to those skilled in the art. Then, a suspension of antibody-producing cells removed from each immunized mammal secreting the desired antibody is prepared. After a sufficient time, the mammal is sacrificed and the lymphocytes producing somatic antibodies are obtained. The cells that produce antibodies can be derived from the lymph nodes, spleens and peripheral blood of the primed animals. Spleen cells are preferred, and can be mechanically separated into individual cells in a physiologically sound medium using methods well known in the art. Mouse lymphocytes give a higher percentage of stable fusions with the mouse myelomas described later. Rat, rabbit and frog somatic cells can also be used. Chromosomes of the spleen cell encoding desired immunoglobulins are immortalized at? fusing the cells of the spleen with myeloma cells, generally in the presence of a fusion agent such as polyethylene glycol (PEG). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques. The resulting cells, which include the desired hybridomas, are then grown in a selective medium, such as the HAT medium, in which the unfused myeloma or lymphocyte cells of origin eventually die. Only the hybridoma cells survive and can be cultured under limiting dilution conditions to obtain isolated clones. The supernatants of the hybridomas are classified for the presence of antibody of the desired specificity, such as by immunoassay techniques in the antigen that has been used for immunization. Positive clones can then be subcloned under limiting dilution conditions and the monoclonal antibody produced can be isolated. There are several conventional methods for the isolation and purification of monoclonal antibodies to free them from other proteins and other contaminants. The methods commonly used to purify monoclonal antibodies include ammonium sulfate precipitation, ion exchange chromatography and affinity chromatography (see, for example, Zola et al., 1982). Hybridomas produced according to these methods can be propagated in vitxo or in vivo (ascites fluid) are two techniques known in the art. Generally, the line of individual cells can be propagated in vitro, for example in laboratory culture vessels, and the culture medium containing high concentrations of a single specific monoclonal antibody can be collected by decanting, filtration or centrifugation. Alternatively, the yield of the monoclonal antibody can be increased by injecting a sample of the hybridoma into a histocompatible animal of the type used to provide the somatic cells and myeloma for the original fusion. Tumors that secrete the specific monoclonal antibody produced by the fused cell hybrid develops in the injected animal.The body fluids of the animal, such as the ascites fluid or serum, provide monoclonal antibodies in high concentrations.
The media and animals useful for the preparation of these compositions are both well known in the art and commercially available and include synthetic culture medium, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM, Dulbecco et al., 1959) ^ supplemented with 4.5 gm / 1 glucose, 20 mM glutamine and 20% fetal calf serum. An exemplary inbred mouse breed is the Balb / c. Dyes / Fluorescent Markings Fluorescent labels that can be used in determining the percentage of an immune cell subtype present in a sample of PBMCs include, but are not limited to, phycoerythrin (PE), fluorescein isothiocyanate (FITC), allophycocyanin '(APC), Red' ~ from Texas (TR, Molecular Probes, Inc.), complex of peridinin chlorophyll (PerCo), Cy5 (Biological Detection System) and conjugates thereof coupled to PE (e.g. PE / CY5, PE / APC and PE / TR.) Fluorescent labels that can be used in determining the proliferation frequency of an immune cell subtype include, but are not limited to, PHK dyes such as PKH2, pKH26 and P H67. In the previous discussion, ^ a number of citations from professional journals and patents are included by reference.All of these citations are incorporated herein by reference in their entirety., "• EXAMPLE 1 Animals The productive performance of 199 pigs was inspected during a period of approximately seven months. The pigs were a product of three different internal genotypes: genotypes AxC, BXC and BXD. Eighteen sows in total were used in this experiment; eight sows had more than ten descendants and the other ten sows had less than ten. The male to female ratio in the population was 1-1.3. Flow of Animals The pigs were born in farm A, site I. Site I comprised the breeding, gestation and calving units. The piglets were kept with the sow during a lactation period of approximately 19 days. The sows were fed to meet or exceed the internal lactation curve guide lines,? The flow of pigs took place in batches of each week through the spawning, breeding and finishing growth spaces and the pigs moved to the next stage when they meet the specific target weights. The target weight for the breeding pigs was 4-6.5 kg, for the period "on the test" it was 31.7 kg and to be of "complete test" it was 122.31 kg.
All the pigs were processed 24 hours after birth. Each pig was weighed individually (Mortal scale model IQ-plus 390-DC) and identified with button ear tags. Each pig was given a mixture of 1 ce of iron dextran (Durvet, Inc.) and penicillin (Pfizerpen® G, Pfizer) and 1 ce of gentamicin (Garacin® Schering Plow) for the prevention of diarrhea. At the time of weaning, around 19 days of age, each pig was weighed individually and marked with color. The pigs were transported to a breeding space in site II where they were housed for at least seven weeks. Site II includes the spaces for nurturing and determining growth. There were 9 crianzas and the pigs were locked for sex and weight for seven weeks and were equally distributed with other non-experimental pigs of the same age and the genotype of the product. A total of 15 pigs / pen went. A four-stage breeding program was implemented as well as a rigorous economic feeding program. The pigs were vaccinated at 10 and 24 days of age with a bacterin Haemophilus parasuis. (Suvaxub® Respifend® Hps, Fort Dodge Laboratories). ^ The blood was obtained from each pig at 6-7 weeks of age by puncturing the anterior vena cava, and was collected in two tubes of 10 ml containing anticoagulant (EDTA). Blood samples are kept in coolers with ice packs at all times and sent for early delivery during the. night for laboratory processing. The time between blood collection and laboratory processing was 20 hours or less. The blood was sampled in eleven batches to obtain manageable numbers for laboratory processing and a maximum of 25 blood samples were directed per session with bleeding. In nine weeks of age, the pigs were weighed and the animals that reached the target weight of 31.7 kg were placed "on the test" by moving them out of the nursery spaces in the growth / termination spaces. Two to four parenting spaces were used to fill a growth / termination space. The pigs were housed with other non-selected pigs of the same age and genotype. The selected pigs were also represented in each pen. 60 castrated pigs and 60 young sows were distributed over eight pens within a space in the finishing facility. Six corral spaces were available to house a total of 720 pigs. The pigs were housed with a minimum variation in temperature in all the spaces (21.6 ° C high and 19.4 ° C low) and were fed a diet program based on four-phase soybean corn. Each pig was weighed individually * -in the test ^ scale of model 700 True-Test). In addition, measurements were taken of the living body, including the back fat in the first rib, the last rib (back fat), the last vertebra and the depth of the back with a real-time ultrasound (Moka model SSD-500 V). Each pig was weighed every two weeks until the pigs reached the weight of the complete objective test of 122.3 kg. At the end of the full test period, the pigs were transported and slaughtered in S ift (Louisville, Y) trail facilities. The attributes of the channel collected included the weight of the hot channel, the back fat with the fat meter, the depth of the back with the fat meter and the percentage of lean meat. Farm Health Status Veterinary visits monthly took place on Farm A for the duration of the experiment. A veterinarian estimated the presence of clinical disease and mortality in the nursery and growth / termination units. The farm was considered to have a low to medium level of exposure to Salmonella and that is positive for Mycroplasma hyopneumoniae by historical serological studies. The farm was constantly inspected for the detection of Salmonella antibodies using the Danish Mixed ELISA at Iowa State University. The results of the inspection of Salmonella (ten finishing pigs / ms) indicated that there was no active seroconversion during the present study, since seroconversion for Salmonella has occurred in previous years. No clinical signs of Salmonellosis were observed. The serology of Mycoplasma Hyopneumoniae was evaluated every year. During the last month of this study, 3/10 finishing pigs have antibodies against M. hyponeumoniae as detected by the "Tween-ELISA test at the University of Minnesota Diagnostic Laboratory." pre-aging and aging, 200 g / ton of neomycin sulfate and oxytetracycline (Neo-Terramycin®, Pfizer) were used as part of the routine feed additive program.Preparation of Cells, In the laboratory, blood mononuclear cells Peripheral (PBMC) of all 199 pig blood samples were isolated by gradient separation using the Lymphocyte separation medium (LSM, Cappel, ICN Biomedicals, Ohio) as described elsewhere (Solano-Aguilar et al. 2000) After the isolation of the PBMC, the cells were counted using Trypan blue dye exclusion and their live cell concentration was adjusted to 5 x 10 7 cells / ml. samples of >;
pig for all immune characterizations. Types of Monoclonal Cells and Antibodies Natural killer (NK) cells are cells that possess the ability to kill certain tumor cells and a wide variety of virally infected cells. Such terminations are part of the natural immunity before it specifies. In pigs, NK cells are identified as CD2 + CD16 + positive double cells using the monoclonal antibodies of pigs CD2 and CD16 (MAbs) MSA3 and G7, respectively. The CD2 MAb detects the T lymphocytes and the CD16 MAb detects the receptor, JFC? type III '.FcyRIII). The extermination of the target cells by NK requires that the target cell be pre-coated with specific immunoglobulins (IgG) and the lithic process is called antibody-dependent cell-mediated cytotoxicity. The recognition of the bound antibody occurs through a low affinity receptor for IgG Fe in the leukocyte, the FcyRIII, which is detected in the body with the CD16 MAb. The SLA molecules (Major Histocompatibility Complex of pig) play an important role in the recognition of the antigen. They direct the specific antigens and therefore the SLA belongs to the specific immune system. There are two classes of MHC class I and class II molecules. The CD8 + cells are mainly cytotoxic T lymphocytes (CTL) and recognize peptide fragments linked to the MHC class I molecules on cells that are the target of the lithic action of CTL. These peptides are generally derived from endogenously synthesized proteins such as viral antigens. The 'CD4 + cells' are mainly helper infocytes and recognize peptides linked to MHC class II molecules on the surface of other cells such as B cells, macrophages, and in pigs in some subsets of activated T cells. II are usually derived from extracellular microbes and soluble protein antigens.The subsets of CD4 + and CD8 + lymphocytes can be detected using MAbs 74-12-4 and 76-2-11, respectively.In MHC and SLA sites of human and pigs, there are distinctive chromosomal regions such as DP, DQ and DR In pigs, SLA-DQ antibodies target the SLA-II antigen as total SLA-DQ (T), bright or active (B), and opaque or less active (D) The SLA-DQ MAb recognizes a mono-anoric determinant in the SLA-DQ molecule that is thought to be a common component of the region and not a polymorphic determinant (the SLA-DQ gene expresses polymorphic products). of the ais 'of PBMC - from the whole blood, the lymphoid cells were tested under two conditions. First, aliquots of PBMC were directly immunostained and in the percentages of subsets of immune cells were determined by incubation of the PBMC with primary body mAb. Second, aliquots of PBMC were cultured with the mitogen with canabalin A (ConA, Sigma, St. Louis, MO) and the proliferative response of the cell subsets was determined using the flow cytometry and the combined spotting of MAb and PKH67 (Sigma, St. Louis, MO). The monoclonal antibodies and their target cells in the PBMC population are listed in Table 1. All the monoclonal antibodies used in this study were specific for pigs and the specificity to the target cell has been described elsewhere (Saalmueller et al., 1998). and Haverson et al., 2000). These MAbs have been chosen because they directed the surface markers that are expressed in monocytes and lymphocytes. A leukocyte marker in anti-pig trays (CD45) and multiple IgG isotype control MAbs were used as positive and negative controls, respectively. The percentage of cells immunostained with the specific MAbs was determined after labeling the cells with secondary monoclonal antibodies coupled with FITC or PE (Southern Biotechnology Associates, Birmingham, AL). Table 1. Monoclonal Antibodies of Puerco and Target Cells in PBMC Subset of Antibody Target Cell Cells (or Monoclonal? Combination) CD2 + / CD16 + positive double lymphocytes expressing both CD2 and CD16; Natural Exterminating Cells (NK) Cdl6 G7 low affinity receptor FcyRIII expressed mainly by multiple cell subsets: NK > B, macrophages
CD2 MSA3 recipient of red blood cells of sheep; predominantly expressed in the T- and NK cells; opaque in CD4 + / CD8 + B cells. Double positive T lymphocytes that express both CD4 and CD8; normally CD8 opaque CD4 74-12-4 subset of T lymphocytes that reacts with the MHC type II antigen and the foreign antigen on the cell presenting antigen (APC) CD8 76-2-11 subset of T lymphocytes that reacts with the MHC antigen type I and the foreign antigen of APC
SLA-DQ T antigen of the pig lymphocyte class (SLA) II; equivalent to the MHC class II mouse I-E antigen; T = total PBMC (monocytes and lymphocytes)
SLA-DQ B TH-16 SLA-II B; detects only the bright SLA-DQ lymphocytes and / or macrophages SLA-DQ D SLA-II D; detects opaque or less reactive SLA-DQ lymphocytes, lymphocytes and / or macrophages Flow Cytometric Analysis Immunoblotted PBMCs were analyzed using flow cytometry as described elsewhere (Solano-Aguilar et al 2001). The percentage of stained cells was calculated based on the fluorescence intensity using the IgG isotype background as a control. For certain markers it is important to distinguish between opaque (D) and bright (B) immunofluorescence. For SLA-DQ, total populations (T), D and B are reported. The flow cytometric analysis for each sample included 16,000 to 20,000 events Staining of PKH67 For the proliferation test, PBMC were washed once in RPMI 1640 medium, followed by filtration through a nylon mesh in a 17 x 100 ml polypropylene conical tube. The cell pellet was taken in Diluent C (Sigma, St. Louis, MO) to obtain a final suspension of 5 x 106 cells / ml). This cell suspension was then added to an equal volume of the PKH67 dye material (1.5 L dye / 1 x 107 cells) and incubated for three minutes at room temperature. Cell concentrations were previously established to stain cells homogeneously and brightly. A volume of warm inactivated fetal bovine serum equal to the total volume of cells + dye was added to the suspension. The cells were then centrifuged in a pellet three times, washed the first time with the RPMI medium with serum and the final time in blastogenic medium (RPMI 1640 supplemented with 5% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine). , non-essential amino acids, 5.5 x 10-5 M 2-Mercaptoethanol, buffer solution Hepes 25 mM), and were counted with a hemacytometer. The homogeneity of the immunoblotting procedure was verified by flow cytometry on day 0 for an aliquot of these labeled cells. Proliferation A total of 8 x 106 cells labeled with PKH67 were placed in individual cavities of a 24-well tissue culture plate. Concanavalin A at a final dose of 5 μg / ml was added to the cavities at the beginning of the culture period. The control cavities in the medium without mitogen. The final volume was 2 mi / cavity. Plates were incubated at 37 ° C in 5% CO2 for three days. The top part of 1 ml of the medium was exchanged with 1 ml of fresh blastogenic medium in the cavities after 48 hours of culture. Cell Collection for Proliferation Analysis On day three, cells were harvested with two washes in the RPMI medium and counted. A concentration of 1 x 10 7 cells / ml was used for differential immunoblotting as described elsewhere (Solano-Aguilar et al., 2001). Then the culture cells were stained with control MAbs or CD.4 or CD8 MAbs. The data collected were used to calculate lymphocyte proliferation after exposure to ConA as a measure of lymphocyte function. The proliferation of CD4 positive (CD4 +), CD8 positive (CD8 +), and incorporation of P H67 (proliferation of total cells) was compared between cells cultured with Con A or with the medium to determine if any subunit of cells was preferentially implemented after the stimulation. Data Analysis of the Cell-Immune Proliferation The flow cytometry data was analyzed with the Proliferation Wizard module in the ModFit LT software (Verita Software House, Inc., Topsham, Maine). The cells were confined according to the scattered forward and lateral signals of the lymphocyte population to exclude debris. The intensity of the non-proliferating cells (of origin) was determined by analyzing the sample that has been grown without mitogen. Each generation of cells must have approximately half of the PKH67 dye of the cells of origin. Working from the intensity of the source generation, the ModFit software executes the fluorescence intensity histogram with Gaussian distributions centered on the maximum at different channel intervals (Giban et al., 1999). Using the data returned by the software for the percentage of cells in each daughter generation at the time of analysis, the individual frequency of each subset of | cell (CD4 +, CD8 +, PKH67 +) was calculated in the original population that has proliferated (frequency of precursor or FP). The proliferation index (PI) was calculated as the sum of the cells in all generations divided by the number of original origin cells theoretically present in the stimulated population. The PI is a measure of the increase in the number of cells in the culture during the course of the experiment (three days). Precursor frequencies and proliferation indices were used as measurements of lymphocyte proliferation and functional assays are considered in this study. ^ Test of Immune Parameters as Predictors of Growth The productive parameters evaluated included the average daily gain (ADG) in 'the following stages of production: a) on the test, b) from birth to weaning, c) from birth to the complete test or the daily gain of time of life, d) weaning to the complete test and e) weaning to over the test. Additional production parameters such as live body and channel measurements, feed intake and feed conversion were also analyzed. The effects of proliferation assays (FP and
PI) and the immune phenotypes (percentages of cells expressing CD16, CD2, etc.) in production parameters were estimated by including them as regresators in a mixed model analysis. A mixed model (SAS Proc. MIXED) was used to determine which parameters should be used when estimating the effects of the growth attributes. A complete model was used that included the product, progenitor (spliced into the product, breeding space, space, finishing, sex, and sow feeding, where the progenitor is a random effect and all others are fixed. spliced into the product due to partial confusion The product was not found significant when tested against the parent within the product and therefore was excluded from the final model.The breeding space and the termination space are strongly confused, so only the breeding space is included in the final model since it makes the most significant contribution.The predictive effect of markers of subtype t of immune cell in the proliferation assays in the productive attributes were estimated as well as the predictive effect of the Proliferation tests on the productive attributes The final models for each of these are shown in Table 2. Table 2. M Parameter odels
Results The data analyzed by flow cytometry with the ModFit program indicated that 99.37% of the cells were stained with PKH67 demonstrating the high efficiency of the procedure. The results obtained with the MAb CD45 panleukocyte, indicated that an average of 92.04% of the isolated cells were recognized by the monoclonal antibody CD45 as leukocytes, which indicated an appropriate lymphoid gate. The background staining was controlled by the use of the IgG isotype controls. All the MAb used showed higher values than the antecedent controls indicating that the detection system was effective. The proportion of the immune marker detected in the blood is given to 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%). The percentages for the controls were: IgGa / Ig2b (04 /%), Ig2A / IgGl (0.22 / .06, CD45 (98.38).) As expected, some subsets of immune cells similar to CD8, CD16, CD2 / CD16 were relatively The relative percentages of the other markers such as SWC3 and CD21 were less common in the circulating blood but still several times above the antecedent level, the mean weaning weight was 6.4 kg + 0.16, and the age of average weaning it was 19.4 days ± 0.2 In weaning, 76% of the pigs fall within the 9.0-14.5 pounds of the weight range, the rest 1% of the pigs fall below a weight of <8.9 pounds and 23% were> 14.6 lb. The initial average weight of the test was 33.3 kg + 0.5 and the average age in the test was 70.8 days ± 0.6 The average weight of the complete test was 123.1 kg ± 0.7 and the average age of the "complete" test was 166.8 days ± 0.9. they observed under a flow of pigs that start the production stages with similar weights before similar ages. The standardization of the flow of pigs for the weight was considered important to compare the performance in pigs where the evaluation of ADG and the channel are the measured attributes because the weight of the pig will influence the daily gain. Similarly, the association analysis for the channel attributes takes into account the weight of the animal at the time of measurement. The results of the analysis of the linear model indicated that globally, the proliferation assays were influenced by the parent and sex; the attributes of gain were influenced by the parent, the parenting and termination spaces, sex and the birth of the sow. The measurements in the final stage and the channel were influenced by the product, the parent, the sex and the sowing of the sow. After taking into account the various sources of variation, significant associations were found between immune markers and productive performance. These associations were found in the specific production stage that covered the period when the blood sample was collected (breeding) but they were also associated with other stages of production suggesting that these markers can predict productive performance through the full productive life of a pig. Proliferation of lymphocytes Markers of immune csubtypes were found to be good predictors of lymphocyte proliferation. The frequency of total cproliferation (pkH67 PF) was influenced by the percentage of CD4 + (p = 0.09) and the SLA-DQT + c (p = 0.009). The total cproliferation index (PKH67 PI) was significantly influenced by the percentage of SLA-DQB + c. Finally, the proliferation frequency of CD8 + c was influenced by the percentage of CD4 + / CD8 + c (p = 0.07), CD8 + (p = 0.03), and SLA-DQD + (p = 0.06). Daily Gain Immunoclar subtype markers predicted ADG. Three phenotypes were significantly associated with ADG during the lifetime of the pig including CD16 + c (p = 0.02), CD2 + / CD16 + c (p = 0.0053, CD8 + c (p = 0.04) Table 3). A functional assay was associated with ADG during the lifetime of the pig: CD4 PF (p = 0.08). These four phenotypes were negatively correlated with growth, higher percentages of marker were associated with lower ADG. Another way to look at the influence of markers on growth is to compare the tails or extremes. It should be mentioned, however, that the return model as used in the above is a better indicator than the observation at the extremes since this includes all the data. However, to estimate the impact of the frequency of CD2 + / CD16 + c on ADG lifetime, the weight gain and the average number of days for the market was compared among the 30 pigs showing the highest percentages of high percentages of c and 30 pigs that show lower percentage. The weight gain was calculated by multiplying ADG by the average number of days needed to reach the test weight (average APRA all pigs was 166.8 + 0.9 days). The average number of days to the market was calculated by dividing the objective test weight of 116 kg between the ADG of the upper part against the bottom of 30 pigs. The results indicated that a 1% increase in CD2 + / CD16 + c is predicted to create a .0018 kg reduction in ADG lifetime. Thus, the 30 respondents of the fund had an average of 5.4% for the CD2 + / CD16 + c and the 30 from the top had an average of 33.1%. This means the difference predicted in the life time gain is (33.1-5.4) x .0018 = .0498 kg / day. For 167 days the difference between the high and low% of CD2 + / CD16 + c was approximately 8.32 kg. The pigs that have a higher proportion of double positive c had UH ADG of 0.7556 kg, to reach the weight of the objective test of 116 kg that they required on average 156 days. Pigs expressing a high proportion of CD2 + / CD16 + required 166 days to reach the same target weight making a difference of ten days between the groups. Some phenotypic markers and proliferative responses were significantly associated with ADG in specific production stages but not in the complete productive life of the pig. Those included ": 'CD4 + c were associated with ADG from birth to weaning (p = 0.04), SLA-DQB + c were associated with ADG from weaning to on the test (p = 0.08), and S C3 + cells were associated with ADG on the test (p = 0.07) and from weaning to on the test (p = 0.09). All the associations had a positive correlation, the higher the percentage of cells the ADG is better (Tables 3 and 4). Channel Measurements Significant associations were found between live body measurements and cell subtype markers and a proliferation assay. These associations included CD4 + / CD8 + cells and the last vertebrae on the test (p = 0.015), CD8 PF and the first rib in the complete test (p = 0.08 =, and SLA-DQD and fat back to on the test ( p = 0.08) (Table 4) These associations do not remain significant when the channel attributes of the same group of animals were evaluated in the slaughterhouse The proportion of cells expressing pig leukocyte antigen (SLA) class II, or SLA -DQ, the markers significantly influenced the attributes of the important channel.The proportion of SLA-DQB + and SLA-DQT + cells were associated with the hot channel weight (p = 0.04) .The correlation was positive, the highest proportion of SLA-DBQ cells and positive T cells were associated with the weight of the largest warm channel (Table 4) Food Ingestion and Efficiency Food intake was significantly associated with the proportion of SLA-DQQ + cells (p = 0.04), and SLA-DQT + cells (p = 0 .06) The SLA-DQ markers were also associated with feed conversion. SLA-DQB + (p = 0.05), SLA-DQD + (p = 0.06), and SLA-DQT (p = 0.015) were significantly associated with feed conversion. Pigs with a higher percentage of SLA-DQ positive cells showed better feed conversion. The high CD8 PF was associated with the conversion of food (p = 0.09). No associations were found between production parameters and CD2 +, CD21 +, CD4 PI, CD8 PI, PKH67 PI, or PKH67 PF. Overall, the most relevant results from the economic point of view are the significant associations between immune phenotypes and ADG. during the life of the pig, the attributes of the channel and the conversion of food. Consequently, the biological aspects of the immune cells or subsets of cells targeted with the antibodies involved in those key associations will be discussed. Significant associations were found between the CD2 + / CD16 + cells and the gain attributes during the complete productive of the pigs. These double positive cells are most likely NK cells. NK cells have the ability to kill certain tumor cells and a wide variety of virally infected cells. Such terminations are not induced by the specific antigen and thus are part of the natural rather than specific immunity. NK cells are capable of lysing several target cells. The level of NK-mediated cytotoxicity is typically not regulated by the antigen but by cytokines and hormones similar to interleukin-2, interferons (IFN), prolactin and growth hormone. NK cells also secrete IFN-gamma that activates monocytes to develop into macrophages.
Significant associations found that CD16 + cells and gain attributes during the complete productive life of pigs. In many cases, the extermination of target cells by NK requires that the target cell be pre-coated with specific IgG, and the lithic process is called antibody-mediated cell-mediated cytotoxicity. The recognition of the bound antibody occurs through a low affinity receptor for the IgG Fe on the leukocyte, called Fe? receptor III (FcyRIII) that is detected by CD16 MAb. Although there is no absolute marker to identify NK cells in pigs, the authors believe that from the monoclonal antibodies available today, the combination of CD2 / CD16 antibodies is found to be the most appropriate. The expression of CD2 in humans occurs in thymic cells, peripheral T cells and natural killer cells. 50% of thymic B cells are also expressed and expression in mature B cells is controversial. CD16 detects FcyRIII. For the pig a subset of NK cells are considered to be CD2 * CD16 +. The subset of CD16 cells is mainly expressed by T and NK cells but B cells can also express it. B and NK cells are negative CD3. The cells ? NK in the pig are divided between opaque cells "CD8- and CD8.The function of B cells as a source subset of CD16 + cells in this study can not be significant since there were no associations between the B cell antigen (CD21) and ADG CD8 + cells are mainly cytotoxic T lymphocytes (CTL) and recognize peptide fragments linked to MHC class I molecules on cells that are targets for the lithic action of CTL These peptides are generally derived from endogenously synthesized proteins such as Viral antigens: Class I MHC antigens were not targeted in this study.CD4 + lymphocytes are mainly helper cells and recognize peptides bound to MHC class II molecules are from the surface of other cells such as B cells, macrophages and in pigs in some subsets of activated T cells The associated peptides of class II are usually derived from extracellular microbes and soluble protein antigens. The proportion of SLA-DQ + cells showed significant associations with the attributes of the channel and the conversion of the food. SLA or MHC pig molecules play an important role in the recognition of the antigen. The pig MAb is SLA-DQ directs the class II SLA antigen, which is thought to be equivalent to the murine class II MHC site, I-E and human HLA-DQ. At the level of the gene, the DQ region comprises the sites for the alpha and beta chains;
SLA-DQ is usually a heterodimer of an alpha chain and a beta chain which is the product expressed on the surface detected by the antibody. The monoclonal antibody SLA-DQ recognizes a monomorphic determinant on the SLA-DQ molecule that is thought to be a conserved sequence of one of the genes in the SLA-DQ genes. MAb SWC3 was associated with ADG at limited production stages. SWC3 targets mononuclear cells. Mononuclear cells represent a population of cells that is critical in natural immunity and also plays a central role in specific acquired immunity. Some functions of the monocyte. they are phagocytosis of foreign particles, production of mediators to kill microbes and control the spread of infections, production of cytokines and growth factors, function of the cell that presents antigen and promotion of the activation of the T cell. The high proliferation of lymphocytes has a damaging effect on production parameters such as ADG and feed conversion. It has been reported that under a stimulation of acute Salmonella, the proliferation of lymphocytes is a good indicator of Salmonella resistance and growth. In that study, the highest proliferative responses of blood lymphocytes before stimulation were associated with decreased disease resistance and growth under experimental conditions (Van Diemen et al., submitted). In the present study, high proliferative responses were associated with detrimental effects on production parameters including growth that could possibly be associated with resistance to subclinical diseases. Much more importantly, this deleterious effect was observed in a commercial set in a herd without particular acute disease and could be an indicator of robustness. Microbial exposure occurs in any environment that is not completely sterile. Microbial exposure certainly occurs in cleaner commercial operations. Exposure of the pig to specific pathogens was not estimated in this study but the association results (ADG) point to the involvement of key immune cells that act in response to viral infections (NK cells, CD8 + cells) and bacterial infections (CD4 + , SLA-DQ +, monocytes). This is observed to be relevant to an environment where there are no obvious clinical signs of viral infections such as in the case of farm A. Several factors could influence the percentages of immune markers present in the blood, one of these being vaccination. Vaccination with a Haemophilus paraseis bacterin was administered on days 10 and 24 days of age in the pre-breeding and rearing stages. The second vaccination occurred at least three weeks before the blood was collected for cell characterization. Due to the time lag after the second vaccination that was at least three weeks before the blood samples were collected for immunoblotting, it is speculated that the vaccination did not have a significant impact on immune cell populations. Veterinary reports indicated that in farm A, site II some pigs showed clinical disease in the breeding during the study period. These signs included pigs that are seen as occasional sick, that do not develop well, and that cough. The mortalities of global breeding and growth / termination were 1-2% and 2% respectively. These percentages are within the normal mortality ratios in commercial operations. Therefore, 98% of the pigs whose immune cells were tested, reached the end of the experiment. Only 4/199 of the experimental pigs did not survive until the end of the experiment. Management practices on farm A including enclosure by sex, feeding protocols, vaccination, pig density, are similar to many commercial operations. In the same way, the degree of exposure to Salmonella and mycoplasmas is common in many pig operations. Therefore, the conclusions drawn from this study could be extrapolated to similar operations. The immune cells targeted in this study have conserved functions in other animal species and these subcellular type markers can potentially be used in a wider spectrum of animal hosts. Table 3. Association between the Subtypes of Immune Cells (CD16, CD2 / CD16, CD4, CD8) and the Proliferative Responses, Average Daily Gain, Channel Attributes and Feeding Parameters. Attribute Estimated number of p dependent values observations regression (se) Effect of CD16 ADG on the 136 -0.00.4 (0.0023) 0.0951 ADG test from 139 -0.00.2 (0.0009) 0.031 weaning birth ADG of 136 - C.0.0.3 (0.0014) 0.021 weaning ADG life at 136 -0.00.3 (0.0014) 0.031 ADG complete weaning test at 139 -0.00.6 (0.0019) 0.00131 on the effect test of CD2 / CD16 ADG on the 136 -0.005 (0.0023) 0.031 ADG test from 139 -0.002 (0.0009) 0.0111 weaning birth ADG time of 136 -0.004 (0.0014) 0.00531 weaning ADG life at 136 -0.004 (0.0014) 0.011 complete weaning ADG test at 139 -0.006 (0.0019) 0.0031 on the test effect of CD8 ADG on the 137,., - • 9.002 (0.0015) 0.021 ADG test from 140 -0.001 (0.0006) 0.2 weaning birth ADG of 137 -0.002 (0.009) 0.041 weaning ADG life to 137 -0.002 (0.009) 0.051 complete ADG weaning test at 140 -0.004 (0.0012) 0.0051 on the test effect of CD4 PF ADG on the 118 -0.099 (0.0932) 0.029 ADG test from 120 -0.013 (0.0494) 0.81! weaning birth ADG of time of 118 -0.109 (0.0624) 0.081 weaning ADG life 120 -0.105 (0.0605) 0.091 to the complete weaning test ADG 120 -0.0141 (0.0818) 0.091 a on the significant test at the p <level; 0.01 Table 4. Effect of the SLA-DQ Subtypes on Channel Attributes and Power Parameters. Attribute Estimated Number of Value p? dependent observations regression (se) Effect of SLA-DQB first rib 139 -0.0213 (0.0164) 0.2 a on the first rib test 136 -0.001 (0.0462) 0.98 to the full test back fat at 139 -0.005 (0.0122) 0.69 on the test back fat to 136 - .019 (0.03-33) 0.57 full test depth of 139 -0.071 (0.3346) 0.83 spine to the test depth of 136 0.93-0.417 0l45 spine to test (00.52.055) complete last vertebra 139 -0.0004 0.98 a on the 0.76 (0.0195) test last vertebra 136 -0.0050.279 0.82 a test (0.0258) 0.05 complete weight of the channel 131 -0.3587 (0.1753) 0.041 hot fat meter 131 -6.003 (0.03-47) 0.93 of the back fat 'fat meter 131 -0.0509 (0.0792) 0.52 of the loin depth percentage of 131 -0.0078 (0.0249) 0.76 lean meat ingestion of 105 0.0048 (0.0043) 0.27 food conversion of 105 Ck0Q91 (0.0046) 0.051 food effect of SLA- DQD first rib 139 0.0034 (0.0192) 0.86 a on the first rib test 136 0.036 (0.0536) 0.51 to the full test back fat to 139 -0.025 (0.014) 0.081 on the back fat test to 136 0.0130 (0.0388) 0.74 the full depth test of 139 0.3441 (0.3877) 0.38 spine a on the test depth of 136 0.0615 (0.0638) 0.34 lomo to the complete test. last vertebra 139 0.0091 (0.0226) 0.69 a on the test last vertebra 136 0.007 (0.0301) 0.82 a full test weight of the channel 131 0.1956 (0.2101) 0.35 hot fat gauge 131 0.0164 (0.0402) 0.68 of the fat back fat gauge 131 0.0510 (0.0919) 0.58 of loin depth percentage of 131 0.004 (0.0288) 0.9 lean meat ingestion of 105 0.0109 (0.0053) 0.041 food conversion of 105 0.0111 (0.0058) 0.061 food SLA-DQT effect first rib 139 0.0122 (0.0118) ) 0.3 a on the first rib test 136. -0.014 (0.0329) 0.67 to the test s full back fat to 139 -0.012 (0.0087) 0.17 on the back fat test to 136 -0.005 (0.0238) 0.85 the full test depth of 139 0.0942 (0.2394) 0.69 back to on the test depth from 136 0.0442 (0.039) 0.26 back to full test last vertebra 139 0.0037 (0.0139) 0.79 a on the test last vertebra 136 0.0005 (0.0184) 0.98 a test? full channel weight 131 0.2616 (0.1269) 0.041 hot fat gauge 131 0.0048 (0.0251) 0.85 fat back fat gauge 131 0.0466 (0.0573) 0.42 'of the loin depth percentage of 131 0.0026 (0.018) 0.89 lean meat intake of 105 0.0057 (0.0029) 0.061 food conversion of 0.00 0.0078 (0.0031) 0.0151 gnificant food at the p < 0.01 Example 2 The size of the population of pigs studied increased to 286 additional pigs. The objective was to collect more data to confirm associations between the immune phenotypes (CD16, CD2 / CD16, CD8) and productive performance (growth). The pigs included in this study originated from 2 different farms, one of the farms was the same as in Example 1. The management of the animals, the recording of the productive performance and the measurement of the immunological attributes was similar to aguel described in the Example 1. Similarly, the statistical analysis was performed in an identical manner to that described in Example 1. The association analysis reported for the 1st year included 139 animals. The results of the 1st and 2nd year are presented with a total of 425 years. See labia 5 right away. Table 5. Effect of immune phenotypes on average daily gain (ADG). The statistical model was used: progenitor (product) + breeding space (year) + sex + calving. For a data set of this size the results with a value of p > 0.1 are considered significant. In bold, years 1 and 2 are combined together. Results not in bold are from year 1 only. CD16 Parameter of Estimated Number of Value P
Growth observations regression (se) ADG on the test 425 -.829 (.5170) 0.11 ADG from 139 -.002 (.0009) .003 birth at weaning ADG time 425 -.580 (.3451) 0.09 life ADG weaning at 225 -.002 (.0013) 0.13 complete ADG weaning test at 139.006 (.0019) .0012 on the CD2 / CD16 test Parameter of Estimated Number of P Value Growth regression observations (se) ADG about the test 425 -.572 (.5387) 0.29
ADG from 139 -.002 (.0009) 0.11 birth at weaning $ DG of time from 425 -.339 (.3598) 0.35 life ADG of weaning to 225 -.002 (.0014) 0.096 complete ADG test of weaning to 139 -.006 (.0019) 0.003 on the CD8 test Parameter Number of Estimated Value P Growth observations regression (se) ADG on the test 425 -.134 (.3288) 0.68
ADG from 139 -.001 (.0006) 0.19 birth at weaning ADG of time from 425 -.068 (.2212) 0.76 life ADG of weaning to 225 -.002 (.0008) 0.002 complete ADG test of weaning to 139 -.004 (.0012) 0.0045 on the test from Table 5 above 'it is important to indicate this: These additional results provide evidence that the percentage of CD16 positive cells is associated with growth during the full productive life of the pigs (p> 0.09). Additionally, these results provide evidence that the percentage of CD2 + / CD16 + cells is associated with growth. In this case the associations were significant for ADG from weaning to the full test (p> 0.096) but not necessarily during the ADG of time of life. In addition, these results provide evidence that the percentage of CD8 positive cells is associated with growth in pigs. In this case the associations were significant for ADG from weaning to the full test (p> 0.002) but not necessarily during the ADG of full life time. deferences Bevan et al., Science 264: 796-7 (1994). Colé et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96 (1985) Dulbecco et al., Virology 8: 396 (1959) Givan et al., J. Immunol. Meth. 230: 99-112
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