Methods in molecular biology.

Sex-sorting sperm using flow cytometry/cell sorting.

Methods in molecular biology (Clifton, N.J.) September 21, 2012 Volume 927 279-295 doi: 10.1007/978-1-62703-038-0_26

Garner DL, Evans KM, Seidel GE.The sex of mammalian offspring can be predetermined by flow sorting relatively pure living populations of X- and Y-chromosome-bearing sperm. This method is based on precise staining of the DNA of sperm with the nucleic acid-specific fluorophore, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently stained sperm are then sex-sorted using a specialized high speed sorter, MoFlo(®) SX XDP, and collected into biologically supportive media prior to reconcentration and cryopreservation in numbers adequate for use with artificial insemination for some specie...

Methods in molecular biology (Clifton, N.J.) August 17, 2020 Volume 2180 365-377 doi: 10.1007/978-1-0716-0783-1_15

Oldenhof H, Wolkers WF, Sieme H.In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow cooling from room temperature to 5 °C, packaging of insemina...

Methods in molecular biology (Clifton, N.J.) March 15, 2020 Volume 2123 355-360 doi: 10.1007/978-1-0716-0346-8_26

Chambers TM.Equine influenza virus (EIV) is a common respiratory pathogen of horses and other equids in most parts of the world. EIV are Type A influenza viruses and two subtypes are known: H3N8 and H7N7. Both are believed to have evolved from avian influenza virus ancestors. The H3N8 subtype circulates widely, but the H7N7 subtype is thought to be extinct. The clinical disease in horses, caused by either subtype, is an upper respiratory infection of varying severity depending upon the immune status of the individual animal. It is not normally life-threatening in itself except in very young foals; however...

Methods in molecular biology (Clifton, N.J.) July 14, 2017 Volume 1577 227-237 doi: 10.1007/7651_2017_49

Roth SP, Erbe I, Burk J.Reliable decellularization techniques applicable to tendon tissue play a critical role in the field of current tissue engineering. Particularly, an application as three-dimensional culture model for in vitro research and translational approaches to establish graft-based tendon repair as a routine clinical tool represent two main application fields for decellularized tendon scaffolds. Considering methodological issues of tendon decellularization, one of the major challenges lies in the preservation of the tendon-specific extracellular matrix (ECM) architecture to reflect natural tissue characte...

Methods in molecular biology (Clifton, N.J.) June 6, 2014 Volume 1161 365-370 doi: 10.1007/978-1-4939-0758-8_31

Chambers TM.Equine influenza virus (EIV) is a common respiratory pathogen of horses and other equids in most parts of the world. EIV are Type A influenza viruses and two subtypes are known: H3N8 and H7N7. Both are believed to have evolved from avian influenza virus ancestors. The H3N8 subtype circulates widely, but the H7N7 subtype is thought to be extinct. The clinical disease in horses, caused by either subtype, is an upper respiratory infection of varying severity depending upon the immune status of the individual animal. It is not normally life-threatening in itself except in very young foals; however...

Methods in molecular biology (Clifton, N.J.) July 19, 2006 Volume 329 59-79 doi: 10.1385/1-59745-037-5:59

Saito S, Sawai K, Minamihashi A, Ugai H, Murata T, Yokoyama KK.We describe here the isolation and maintenance of pluripotent embryonic stem (ES) cells from equine blastocysts that have been frozen and thawed. Equine ES cells appear to maintain a normal diploid karyotype in culture. These cells express markers that are characteristic of mouse ES cells, namely, alkaline phosphatase, stage-specific-embryonic antigen 1, STAT3, and Oct4. We also describe protocols for the induction of differentiation in vitro to neural precursor cells in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor, and platelet-derived growth factor and to he...

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Equine influenza culture methods.

Methods in molecular biology (Clifton, N.J.) June 6, 2014 Volume 1161 403-410 doi: 10.1007/978-1-4939-0758-8_35

Chambers TM, Reedy SE.Equine influenza viruses are cultured in embryonated hen eggs, or in mammalian cells, generally Madin-Darby canine kidney (MDCK) cells, using methods much the same as for other influenza A viruses. Mutations associated with host adaptation occur in both eggs and MDCK cells, but the latter show greater heterogeneity and eggs are the generally preferred host. Both equine-1 H7N7 and equine-2 H3N8 viruses replicate efficiently in 11-day-old eggs, but we find that equine-1 viruses kill the embryos whereas equine-2 viruses do not.

Methods in molecular biology (Clifton, N.J.) June 6, 2014 Volume 1161 393-402 doi: 10.1007/978-1-4939-0758-8_34

Balasuriya UB.Equine influenza (EI) is a highly contagious disease of horses caused by the equine influenza virus (EIV) H3N8 subtype. EI is the most important respiratory virus infection of horses and can disrupt major equestrian events and cause significant economic losses to the equine industry worldwide. Influenza H3N8 virus spreads rapidly in susceptible horses and can result in very high morbidity within 24-48 h after exposure to the virus. Therefore, rapid and accurate diagnosis of EI is critical for implementation of prevention and control measures to avoid the spread of EIV and to reduce the economi...

Methods in molecular biology (Clifton, N.J.) September 1, 2014 Volume 1213 215-228 doi: 10.1007/978-1-4939-1453-1_18

Brehm W, Burk J, Delling U.Stem cells in the form of mesenchymal stromal cells derived from various sources have been identified to have the potential of supporting the therapy of joint disease in the horse, and preliminary data has been published about the clinical application of stem cells in horses suffering from clinical joint disease. Furthermore, the horse is recognized to be the ideal large animal model for the preclinical study of cell therapy in joints. The advantage of this species in this respect is the size of the joints, which makes surgical applications practically feasible in analogy to human surgery. Add...

Methods in molecular biology (Clifton, N.J.) November 28, 2014 Volume 1257 277-287 doi: 10.1007/978-1-4939-2193-5_10

Sieme H, Oldenhof H.In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation secures future reproduction, and insemination doses can be easily shipped. Processing of semen for cryopreservation can be done with minimal efforts and relatively low costs. In this chapter we describe the entire cryopreservation process for stallion and bull sperm including dilution of sperm in primary and freezing extender, cooling and packaging in straws, freezing in liquid nitrogen vapor, cryogenic storage, and thawing. Special emphasis is given on preparation of commonl...

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Equine influenza serological methods.

Methods in molecular biology (Clifton, N.J.) June 6, 2014 Volume 1161 411-422 doi: 10.1007/978-1-4939-0758-8_36

Chambers TM, Reedy SE.Serologic tests for equine influenza virus (EIV) antibodies are used for many purposes, including retrospective diagnosis, subtyping of virus isolates, antigenic comparison of different virus strains, and measurement of immune responses to EIV vaccines. The hemagglutination-inhibition (HI), single radial hemolysis (SRH), and serum micro-neutralization tests are the most widely used for these purposes and are described here. The presence of inhibitors of hemagglutination in equine serum complicates interpretation of HI assay results, and there are alternative protocols (receptor-destroying enzy...

Methods in molecular biology (Clifton, N.J.) April 21, 2009 Volume 524 137-144 doi: 10.1007/978-1-59745-450-6_10

Heuzenroeder MW, Barton MD, Vanniasinkam T, Phumoonna T.The aim of this chapter is to provide a strategy for mapping linear antibody epitopes of protein antigens in order to discover candidates for vaccines or diagnostic tests. A set of overlapping peptides was designed and synthesised based upon a known amino acid sequence of the target protein, virulence-associated protein A (VapA) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals.The peptides were biotinylated and used in an ELISA to screen immune sera from foals. These biotinylated peptides were coated directly onto micro titre plates that had been pre-coated with Neut...

Methods in molecular biology (Clifton, N.J.) December 2, 2015 Volume 1330 189-201 doi: 10.1007/978-1-4939-2848-4_16

Johnson AK, Hinrichs K.There are few reports on the birth of foals resulting from equine adult somatic cell nuclear transfer (NT). On evaluation of reports of 28 live-born adult somatic-cell NT (clone) foals, 3 died within 2 weeks of birth of complications. Approximately 50 % of all reported cloned foals had complications, some requiring aggressive supportive care. The most common abnormalities reported were neonatal maladjustment syndrome, enlarged umbilical remnant, and angular deformity of the forelimbs, similar to problems described in cloned cattle. In contrast, large offspring syndrome and gross abnormalities ...

Methods in molecular biology (Clifton, N.J.) February 13, 2021 Volume 2235 155-167 doi: 10.1007/978-1-0716-1056-5_11

Mesoangioblasts (MABs) are vessel-associated stem cells that express pericyte markers and are originally isolated from the embryonic dorsal aorta. From postnatal small vessels of skeletal muscle and heart, it is possible to isolate cells with similar characteristics to embryonic MABs. Adult MABs have the capacity to self-renew and to differentiate into cell types of mesodermal lineages upon proper culture conditions. To date, the origin of MABs and the relationship with other muscle stem cells are still debated. Recently, in a phase I-II clinical trial, intra-arterial HLA-matched MABs were pro...

Methods in molecular biology (Clifton, N.J.) August 23, 2023 Volume 2682 205-217 doi: 10.1007/978-1-0716-3283-3_15

Rockx B, Mire CE.Henipaviruses are emerging zoonotic viruses that can cause outbreaks of severe respiratory and neurological disease in humans and animals such as horses. The mechanism by which these viruses can cause disease remain largely unknown and to date there are no therapeutics or vaccines approved for use in humans. Nipah virus is listed on the World Health Organization R & D Blueprint list of epidemic threats. In order to advance the availability of effective therapeutics and vaccines and medicines that can be used to save lives and avert large scale crises, animal models are required which recap...

Methods in molecular biology (Clifton, N.J.) November 1, 2020 Volume 2178 301-310 doi: 10.1007/978-1-0716-0775-6_20

De-Simone SG, Provance DW.Affinity chromatography is a separation method based on a specific binding interaction between an immobilized ligand and its binding partner. An important class of ligands for the effective separation and purification of biotechnologically important substances is lectins, a group of naturally occurring molecules widely found in plants that display a range of specificities to bind different sugars. As sugars are often added to proteins through the process of glycosylation, ∼1/3 of all genetically encoded proteins are glycosylated, numerous cognate pairs of lectins with glycosylation groups ha...

Methods in molecular biology (Clifton, N.J.) November 18, 2010 Volume 702 61-75 doi: 10.1007/978-1-61737-960-4_6

Vidal MA, Lopez MJ.Equine adipose tissue-derived mesenchymal stem cells (ASCs) have only recently been investigated for their adipogenic, chondrogenic, and osteogenic differentiation potential. This chapter will briefly outline the molecular mechanisms leading to adipogenesis and the methods of equine adipose tissue harvest, ASC isolation, and adipogenic differentiation. The reader is also directed to other reported methods of adipogenesis for ASCs and mesenchymal stem cells (MSCs) from other tissues.

Methods in molecular biology (Clifton, N.J.) June 24, 2019 Volume 2006 219-227 doi: 10.1007/978-1-4939-9566-0_16

Carnevale EM, Maclellan LJ, Stokes JAE.Establishment of optimal methods for equine embryo culture has been slow when compared to some domestic species. In part, this delay was caused by the failure of standard in vitro fertilization techniques in horses. However, the development of intracytoplasmic sperm injection (ICSI) for the assisted fertilization of equine oocytes has resulted in a renewed interest in establishing optimal methods for embryo culture. Currently, ICSI-produced equine embryos are cultured using media designed for other species or other cell cultures and, typically, with the addition of serum. Although systems spec...

Methods in molecular biology (Clifton, N.J.) May 2, 2018 Volume 1785 121-128 doi: 10.1007/978-1-4939-7841-0_8

Vanniasinkam T, Barton MD, Das TP, Heuzenroeder MW.This chapter describes a strategy for mapping linear B-cell epitopes of proteins using synthetic biotinylated peptides in an ELISA.A set of overlapping peptides were designed based upon a known amino acid sequence of the target protein, VapA (Virulence-associated Protein A) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals. The peptides synthesized as biotinylated peptides were coated directly onto micro titer plates which had been pre-coated with NeutrAvidin™ and used to screen sera from foals confirmed to have R. equi disease. A linear B-cell epitope was identifie...

Methods in molecular biology (Clifton, N.J.) December 22, 2023 Volume 2749 175-184 doi: 10.1007/978-1-0716-3609-1_16

Falk J, Donadeu FX.Groundbreaking work by Takahashi and Yamanaka in 2006 demonstrated that non-embryonic cells can be reprogrammed into pluripotent stem cells (PSCs) by forcing the expression of a defined set of transcription factors in culture, thus overcoming ethical concerns linked to embryonic stem cells. Induced PSCs have since revolutionized biomedical research, holding tremendous potential also in other areas such as livestock production and wildlife conservation. iPSCs exhibit broad accessibility, having been derived from a multitude of cell types and species. Apart from humans, iPSCs hold particular med...

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Polyclonal Peptide Antisera.

Methods in molecular biology (Clifton, N.J.) October 2, 2015 Volume 1348 103-107 doi: 10.1007/978-1-4939-2999-3_11

Pihl TH, Illigen KE, Houen G.Polyclonal antibodies are relatively easy to produce and may supplement monoclonal antibodies for some applications or even have some advantages. The choice of species for production of (peptide) antisera is based on practical considerations, including availability of immunogen (vaccine) and animals. Two major factors govern the production of antisera: the nature of adaptive immune responses, which take place over days/weeks and ethical guidelines for animal welfare. Here, simple procedures for immunization of mice, rabbits, sheep, goats, pigs, horses, and chickens are presented.

Methods in molecular biology (Clifton, N.J.) October 1, 2011 Volume 792 39-45 doi: 10.1007/978-1-61779-325-7_3

Hagen J, Hartnett C, Houchins JP, Giguère S, Kalyuzhny AE.Human and mouse immune system cells are the most frequently used specimens in ELISPOT assays. In an effect to expand the application of ELISPOT assay to other species, we developed matched antibody pairs for ready-to-use kits designed for studying the frequency of equine IFNγ- and IL-4-secreting peripheral blood mononuclear cells (PBMCs). Equine PBMCs were stimulated with either concanavalin A (Con A) or calcium ionomycin mixed with phorbol 12-myristate 13-acetate (CaI + PMA). We found that Con A, in general, had a more profound stimulating effect than CaI + PMA on IL-4 secretion, whereas bot...

Methods in molecular biology (Clifton, N.J.) June 6, 2014 Volume 1161 371-377 doi: 10.1007/978-1-4939-0758-8_32

Chambers TM, Reedy SE.In horses, presumptive diagnosis of equine influenza is commonly made on the basis of clinical signs. This alone is insufficient for confirmation of equine influenza, because other equine infectious respiratory diseases can in some degree have similar clinical presentations. Surveillance and control of equine influenza also necessitate detection of subclinical cases. Effective diagnosis of equine influenza virus infection is critically dependent on obtaining adequate specimens of virus-containing respiratory secretions for testing. These specimens are also valuable as sources for isolation of ...

Methods in molecular biology (Clifton, N.J.) March 15, 2020 Volume 2123 361-367 doi: 10.1007/978-1-0716-0346-8_27

Chambers TM, Reedy SE.In horses, presumptive diagnosis of equine influenza is commonly made on the basis of clinical signs. This alone is insufficient for confirmation of equine influenza, because other equine infectious respiratory diseases can in some degree have similar clinical presentations. Surveillance and control of equine influenza also necessitate detection of subclinical cases. Effective diagnosis of equine influenza virus infection is critically dependent on obtaining adequate specimens of virus-containing respiratory secretions for testing. These specimens are also valuable as sources for isolation of ...

Methods in molecular biology (Clifton, N.J.) November 16, 2019 Volume 2087 43-59 doi: 10.1007/978-1-0716-0154-9_4

Siemsen DW, Kirpotina LN, Malachowa N, Schepetkin IA, Porter AR, Lei B, DeLeo FR, Quinn MT.The development of new advances in understanding the role of neutrophils in inflammation requires effective procedures for isolating and purifying neutrophils. Methods for isolating human neutrophils are fairly standard, and some are covered in other chapters of this volume and previous editions. However, procedures for isolating neutrophils from nonhuman species used to model human diseases vary from those used in isolating human neutrophils and are not as well developed. Since neutrophils are highly reactive and sensitive to small perturbations, the methods of isolation are important to avo...

Methods in molecular biology (Clifton, N.J.) March 15, 2020 Volume 2123 383-392 doi: 10.1007/978-1-0716-0346-8_29

Balasuriya UBR.Equine influenza (EI) is a highly contagious disease of horses caused by the equine influenza virus (EIV) H3N8 subtype. EI is the most important respiratory virus infection of horses and can disrupt major equestrian events and cause significant economic losses to the equine industry worldwide. Influenza H3N8 virus spreads rapidly in susceptible horses and can result in very high morbidity within 24-48 h after exposure to the virus. Therefore, rapid and accurate diagnosis of EI is critical for implementation of prevention and control measures to avoid the spread of EIV and to reduce the econom...

Methods in molecular biology (Clifton, N.J.) August 21, 2013 Volume 1035 35-42 doi: 10.1007/978-1-62703-508-8_4

Campos-Junior PH, Costa GM, de Avelar GF, Segatelli TM, Lacerda SM, Aponte PM, de França LR.Morphometry is a classical quantitative method often used in biology to provide a data basis for functional interpretations/interactions of a particular organ or system. Herein we took advantage of this valuable approach to evaluate the spermatogonial stem cell niche using the horse testis and immunocytochemical localization of GFRA1 [glial cell line-derived neurotrophic factor receptor produced by Sertoli cells)] as an example. Using the NIH ImageJ free software, we describe in detail all the necessary steps to investigate this specific and crucial microenvironment. Based on several recently ...

Methods in molecular biology (Clifton, N.J.) January 1, 1995 Volume 45 55-60 doi: 10.1385/0-89603-308-2:55

Perryman LE, Mason PH.Monoclonal antibodies (MAbs) have been successfully used to evaluate immune responses in horses, and to target important antigens of equine infectious agents to which protective immune responses may be directed (1–5). Most of these studies are performed with murine MAb produced by fusing spleen cells from immunized mice with an appropriate myeloma cell line, as described in Chapter 3. However, there are experimental questions for which murine MAb are not adequate. These include: 1.Definition of microbial epitopes recognized by the infected host; 2.Identification of immunodominant epito...

Methods in molecular biology (Clifton, N.J.) June 6, 2014 Volume 1161 379-392 doi: 10.1007/978-1-4939-0758-8_33

Balasuriya UB.The primary goals of this chapter are to discuss common viral RNA isolation and purification methods that are routinely used by various diagnostic laboratories, to highlight the advantages and drawbacks of each method, and to identify the most suitable and reliable method to increase the sensitivity and specificity of RT-PCR assays for the detection of equine influenza virus (EIV) in clinical specimens. Our experiences and review of literature show that magnetic bead-based nucleic extraction methods (manual and automatic) work well for isolation and purification of EIV RNA from nasal swab spec...

Methods in molecular biology (Clifton, N.J.) March 24, 2004 Volume 253 49-58 doi: 10.1385/1-59259-744-0:049

Merkies K, Buhr MM.No abstract available

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ISSN:	1064-3745 (Print) 1940-6029 (Electronic) 1064-3745 (Linking)
NLM ID:	9214969
(OCoLC):	24839341
LCCN:	sn 92005585
Classification:	W1 ME9616J

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