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Studying mononuclear phagocytes by flow cytometry is challenging due to their phenotypic similarities and the high plasticity of monocytic cells. Despite these challenges, significant progress has been made in cattle research through multicolor flow cytometry, transcriptomics of sorted subsets, and single-cell RNA-sequencing. Here, we provide an overview of established and proposed phenotypic classifications in the bovine mononuclear phagocyte system and discuss the challenges of marker discovery., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jayne C. Hope reports financial support was provided by Biotechnology and Biological Sciences Research Council (BBSRC). All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Chronic lymphoid leukemia (CLL) is a hematological disorder characterized by the clonal expansion of small mature lymphocytes that accumulate in the blood and bone marrow. CLL can arise from B-, T-, or natural killer cell clones. The cytological evaluation of blood smears is often the simplest and least invasive method for diagnosing lymphoid leukemia. Immunophenotyping is used to further subclassify the type of lymphoid leukemia., Case Presentation: A 15-year-old, 4.4-kg spayed female Shih Tzu was presented to the veterinary medical teaching hospital of Kangwon National University. Despite having a normal appetite and activity level, cervical and inguinal lymph node enlargement was noted on physical examination. Complete blood count revealed severe leukocytosis, severe lymphocytosis, and monocytosis. Splenomegaly, hepatomegaly, and lymph node enlargement were detected on radiographic and ultrasonographic examination. Immunophenotyping was performed using peripheral blood mononuclear cells (PBMCs). The majority of lymphocytes exhibited the following profiles: CD3 - CD79a - (97.5%), CD4 - CD8 - (98.6%), CD21 - CD79a - (98.4%), CD34 - (0.1%), CD45 + (99.6%), major histocompatibility complex class II + (99.5%), and CD14 - (0.5%). Based on the immunophenotyping results, possible differentials considered included the following: the majority of lymphocytes may be natural killer (NK) cell clones, plasma cell clones, or show aberrant expression or loss of CD21 marker due to the neoplastic nature of the cells. Further flow cytometry was performed using antibodies against CD3, CD5, CD94, and granzyme B. The combined results indicated that the predominant lymphocyte subset in the PBMCs was CD3 - CD5 - CD21 - CD94 - granzyme B - . To confirm monoclonality and exclude the aberrant loss of CD markers, a polymerase chain reaction for antigen receptor rearrangement (PARR) assay was conducted. The PARR assay, using DNA from blood and lymph node samples, showed B-cell monoclonality. Immunocytochemistry using PBMCs showed that the plasma cell marker Multiple Myeloma Oncogene 1 (MUM1) was not expressed. Therefore, the diagnosis was confirmed to be B-cell CLL., Conclusion: Immunophenotyping can help subclassify the type of lymphoid leukemia; however, as tumor cells can show aberrant expression or loss of the CD21 marker, combining immunophenotyping with the PARR assay could yield a more accurate diagnosis., (© 2024. The Author(s).)

In chicken, primordial germ cells (PGC) are crucial for the preservation and manipulation of genetic resources in poultry production. The HiS and FAcs culture systems are two important methods for the in vitro cultivation of chicken PGCs. The purpose of this study was to compare and analyze the two cultivation systems for PGCs (His and FAcs culture systems) to assess their efficacy and applicability in supporting PGC growth, maintaining PGC characteristics, and lineage transmission ability. The study found that both HiS and FAcs culture systems could maintain the basic biological characteristics of chicken PGCs, including the simultaneous expression of pluripotency and reproductive marker genes, as well as the presence of abundant glycogen granules. Subsequently, we identified 2,145 differentially expressed genes (DEG) through RNA sequencing. GO and KEGG analysis revealed a large number of DEGs enriched in the cell adhesion and calcium ion binding pathways, and the analysis found that these genes maintained a higher level in HiS-PGCs. Further personalized analysis found that the regulatory genes for maintaining PGC pluripotency were highly expressed in HiS-PGCs, while germ cell-related genes showed similar expression in both systems. Additionally, through RNA sequencing data and cell proliferation ability, it was found that PGCs in the FAcs system had a higher proliferation rate and a faster cell cycle. Finally, it was discovered that the expression of cell migration-related genes was maintained at a higher level in HiS-PGCs, but the migration efficiency of HiS-PGCs did not show a significant difference compared to FAcs-PGCs. These results suggest that both HiS and FAcs culture systems can maintain the proliferation and basic characteristics of chicken PGCs, but differences exist in cell proliferation, pluripotency regulation, and cell adhesion. These findings provide new information for optimizing PGC cultivation systems and are important for the preservation and genetic improvement of chicken PGCs., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Concurrent emerging and reemerging avian infectious diseases cause multiple risk factors in poultry. A body amount studies attempted to understand pathogen-associated immunity in chickens. Recent research has made progress in identifying immune functions in chicken, there are still gaps in knowledge, especially regarding immune responses during infectious diseases. A deeper understanding in chicken immune system is critical for improving disease control strategies and vaccine development., Results: This study proposes analytical method for chicken splenocytes, enabling the tracking changes in T cells, monocytes, and B cells across three ages. Optimized lymphocyte-activating conditions were suggested using concanavalin A and chicken interleikin-2, which facilitate immune cell activation and proliferation. Next, splenocytes from embryonic day 18, day 5, and day 30 were compared using surface markers and flow cytometry analysis. We observed an increase in T cell subsets, including activated T cells (CD4 + CD44 + and CD8 + CD44 + ), and B cells, along with a reduced monocyte population after hatching. However, morphological changes and genetic expression of functional immune molecules were limited., Conclusions: The present findings on chicken immune system development offer valuable insights into the avian immune system, including analytical methods and the phenotypic and functional changes in immune cells. Updated immune-boosting strategies during the early stages of life are crucial for developing preventive measures against major infectious diseases in the poultry industry., (© 2024. The Author(s).)

Intracytoplasmic sperm injection (ICSI) remains inefficient in cattle. One reason could lie in the injection of oocytes with sperm that have not undergone molecular changes associated with in vivo capacitation and fertilizing ability. This study aimed to enhance the efficiency of bovine intracytoplasmic sperm injection (piezo-ICSI) by employing fluorescent-activated cell sorting (FACS) to select the sperm population before injection based on capacitation markers. First, we evaluated the effects of incubating thawed sperm for 2 hours with different capacitating inductors: heparin, methyl-beta-cyclodextrin (MβCD), and dibutyryl cyclic AMP (dbcAMP), alone or in combinations in a basal capacitating (C) medium (Sp-TALP). Sperm capacitation and quality markers were evaluated by flow cytometry, revealing heparin as the most effective inducer of sperm capacitation changes. It, therefore, this treatment was chosen as the sperm pretreatment for FACS-piezo-ICSI. Two cell populations showing high capacitating levels (Heparin-HCL) and low capacitating levels (Heparin-LCL) of the markers associated with sperm capacitation i(Ca 2+ ) levels and acrosome integrity were selected by FACS and used for sperm injection. Pronuclear formation was significantly higher when ICSI was performed with Heparin-HCL sperm than with Heparin-LCL and the control group (Heparin unsorted) groups (50 %, 10 %, and 20 %, respectively). Furthermore, injecting Heparin-HCL sperm resulted in a higher blastocyst rate (22.5 %) than Heparin-LCL (10 %) and the control group (15.2 %). In conclusion, heparin treatment effectively induced changes associated with sperm capacitation. The combination of Heparin-HCL treatment and FACS enabled precise selection of capacitated sperm before ICSI, enhancing the efficiency of this technology in the bovine species., Competing Interests: Declaration of Competing Interest The authors declare to have no conflicts of interest to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The Tianzhu white yak, a globally rare species, holds immense value as a source for yak materials. While the Fas/FasL pathway is pivotal in granulosa cells apoptosis, its precise molecular workings remain enigmatic. This study endeavours to decipher the role of follicle-stimulating hormone (FSH) in suppressing ovarian granulosa cells (GC) apoptosis in the Tianzhu white yak. Utilizing advanced cell culture techniques, we employed the MTT method, flow cytometry, fluorescence labelling and RT-PCR to investigate the apoptotic effects of FSH on yak GCs. Our results reveal that FSH's inhibitory effect on GC apoptosis follows a normal distribution pattern, peaking at an FSH concentration of 100 ng/mL with an apoptosis inhibition rate of 89.31%. When serum was withdrawn, an FSH concentration of 2 × 10 6  ng/mL reduced apoptosis by 72.84%. Annexin V-FITC staining revealed membrane invaginations, bubble and protrusion formation on the cell surface, and alterations in membrane structure and cell morphology. Flow cytometry analysis further demonstrated that FSH administration prior to early granulosa cell apoptosis had a more profound effect than during gradual apoptosis, both showing a suppressive effect on early follicular granulosa cell apoptosis. A transcription-level analysis conducted 3 h prior to serum withdrawal, with the addition of 100 ng/mL FSH, revealed intricate regulations in the expression of Fas/FasL. Notably, we observed a gradual increase in FasL expression over time, yet the presence of FSH effectively down-regulated FasL expression to baseline levels, without notable changes in Fas expression. Immunocytochemical analysis further confirmed the presence of both Fas and FasL on the cell membrane, nucleus and cytoplasm, with varying intensities depending on the duration of FSH treatment. Our findings suggest that FSH may suppress the apoptotic pathway in follicular primarily by down-regulating FasL expression, indicating that Fas-regulated mitochondrial pathways play a more prominent role compared to death receptor pathways. This study offers a fresh perspective on the mechanism underlying follicular atresia in Tianzhu white yaks and lays a solid theoretical foundation for the expansion of this endangered species' population., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

CD25, the interleukin-2 receptor α-chain, is expressed on cell surfaces of different immune cells and is commonly used for phenotyping of regulatory T cells (Tregs). CD25 has essential roles in the maintenance of hemostasis and immune tolerance and Treg cell involvement has been shown in human diseases and murine models for allergy, autoimmunity, cancer, chronic inflammation, and many others. In horses, a cross-reactive anti-human CD25 antibody has previously been used for characterizing Tregs. Here, we developed monoclonal antibodies (mAbs) to equine CD25 and compared their staining pattern with the anti-human CD25 antibody by flow cytometry. The comparison of the two reagents was performed by two separate analyses in independent laboratories. Overall, similar staining patterns for equine peripheral blood lymphocytes were obtained with the anti-human CD25 antibody and equine CD25 mAb 15-1 in both laboratories. Both reagents identified comparable CD4 + CD25 + and CD4 + CD25 + FOXP3 + percentages after stimulation of peripheral blood mononuclear cells (PBMC) with pokeweed mitogen. However, when compared to the anti-human CD25 antibody, the equine CD25 mAb 15-1 resulted in a better staining intensity of the equine CD25 + cells and increased the percentages of Tregs and other CD25 + cells ex vivo and after culturing of PBMC without stimulation. In summary, the equine CD25 mAbs provide new, improved reagents for Tregs and CD25 + cell phenotyping in horses., Competing Interests: Declaration of Competing Interest Nothing to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Mass Mortality Events (MMEs) affecting the noble pen shell Pinna nobilis have been reported since 2016. In this work, we used an in vitro flow cytometric assay to evaluate phagocytosis, coupled with cytology and Electron Microscopy (TEM), to define animal immunocompetence following infection by P. nobilis Picornavirus (PnPV). The study was performed on 27 animals in July 2021 and May 2022 on two natural population from the Ebro Delta (Catalonia, Spain) and animals maintained in captivity at facilities in Valencia and Murcia Aquarium. Hemolymph was collected in the field and in captivity as a non-destructive sampling method. Based on dimension and internal complexity, flow cytometry identified three haemocyte types, distinguished in granulocytes, hyalinocytes and a third type, biggest in size and with high internal complexity and granularity. Those cells corresponded at ultrastructure to hemocytes with advanced phases of PnPV infection and related to cytopathic effect of the replicating virus displaying numerous Double Membrane Vesicles (DMVs) and cells corpse fusion. The results showed that pen shell in captivity had significantly lower Total Hemocyte Count (THC) compared with natural population of Alfacs Bay (mean number of 7-9 x 10 4 vs 2-5 x 10 5  cells/mL, respectively). FACS (Fluorescence-activated cell sorting) based phagocytosis analysis demonstrate that animals in captivity at IMEDMAR-UCV and Murcia Aquarium, had scarce or absent ability to phagocyte the two stimuli (Staphylococcus aureus and Zymosan A) (10,2 % ± 1,7 of positives) if compared with the natural population in Alfacs Bay (28,5 % ± 5,6 of positive). Ultrastructure images showed that PnPV itself can lead to an alteration of the hemocyte cytoskeleton, impairing the capabilities to perform an active phagocytosis and an efficient phagolysosome fusion., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Interleukin-1β (IL-1β) is one of the key mediators of inflammation during innate immune responses. Mature bioactive IL-1β mediates essential host defense mechanisms but also has a mechanistic role in several autoinflammatory and degenerative diseases. In horses, specific and sensitive assays for IL-1β are crucial for immunological research on inflammatory processes and diseases. In this article, we describe the development of four monoclonal antibodies (mAbs) against equine IL-1β. The specificity of the new IL-1β mAbs was confirmed using a panel of equine recombinant cytokines and chemokines. The mAbs were validated for detection of native mature IL-1β in a fluorescent bead-based assay and for staining of IL-1β-producing immune cells by flow cytometry. The bead-based assay for equine IL-1β had a linear quantification range between 60 pg/ml to 960 ng/ml. Horse peripheral blood mononuclear cells (PBMC) secreted IL-1β after lipopolysaccharide (LPS) stimulation in time and dose dependent manner as quantified by the new equine IL-1β bead-based assay. A comparison of two commercial equine IL-1β ELISA kits with the new IL-1β fluorescent bead-based assay revealed that the bead-based assay improved the quantification of native equine IL-1β in LPS stimulated PBMC supernatants by detecting it with high intensity and a broad linear quantification range, while both ELISAs resulted in low signals and poor native IL-1β recognition. Intracellular staining and flow cytometric analysis confirmed that the main cellular source of IL-1β in equine PBMC after LPS stimulation were CD14 + monocytes. IL-1β secretion from PBMC was inhibited by a caspase inhibitor but protein translation within the cells was not, supporting the accumulation of pro-IL-1β within the cells even when proteolytic cleavage for IL-1β activation is missing. This confirmed the importance of specific mAbs for analyzing the biologically active, mature IL-1β in horses., Competing Interests: Declaration of Competing Interest Nothing to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Trypanosomosis due to Trypanosoma evansi (surra) is one of the most important diseases with a significant impact on camel health and production. Trypanosoma-induced immunosuppression mechanisms, which are key factors of disease pathogenesis, have been characterized in several animal species. The present study investigated, therefore, the impact of trypanosomosis on the immunophenotype of blood leukocytes in camels. For this, the relative and absolute values of blood leukocyte populations, their expression pattern of cell surface molecules, and the numbers of the main lymphocyte subsets were compared between healthy camels and camels with clinical symptoms of chronic surra and serological evidence of exposure to Trypanosoma infection. Leukocytes were separated from the blood of healthy and diseased camels, labeled with fluorochrome-conjugated antibodies, and analyzed by flow cytometry. Compared to healthy camels, the leukogram of diseased camels was characterized by a slightly increased leukocyte count with moderate neutrophilia and monocytosis indicating a chronic inflammatory pattern that may reflect tissue injury due to the long-lasting inflammation. In addition, the analysis of lymphocyte subsets revealed a lower number and percentage of B cells in diseased than healthy camels. In vitro incubation of camel mononuclear cells with fluorochrome-labeled T. evansi revealed a higher capacity of camel B cells than T cells to bind the parasite in vitro. Furthermore, cell viability analysis of camel PBMC incubated in vitro with T. evansi whole parasites but not the purified antigens resulted in Trypanosoma-induced apoptosis and necrosis of camel B cells. Here we demonstrate an association between trypanosomosis in camels and reduced numbers of blood B cells. In vitro analysis supports a high potential of T. evansi to bind to camel B cells and induce their elimination by apoptosis and necrosis., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Enhanced platelet responses have been demonstrated in heartworm-infected (HWI) dogs; however, the cause and clinical implications of altered platelet function have not been fully elucidated., Objective: This study evaluated platelet function in HWI dogs., Methods: Anticoagulated whole blood collected from eight HWI and eight uninfected dogs was evaluated using turbidometric platelet aggregometry, a platelet function analyzer (PFA-100), a total thrombus analysis system (T-TAS), tissue factor-activated and tissue plasminogen activator modified thromboelastography (TF- and tPA-TEG), CBC, von Willebrand Factor activity, and fibrinogen concentrations. Platelet activation state and the presence of reticulated platelets were assessed via flow cytometric expression of P-selection (CD-62P) and thiazole orange staining., Results: Platelet aggregation responses to adenosine diphosphate (ADP, 10 μM) or collagen (20 μg/mL), PFA-100 closure times, and T-TAS occlusion times did not differ between groups. TEG values TF-R, tPA-R, TF-K, and TF-LY60 were decreased (P = .025, P = .047, P  = .038, P  = .025) and TF-MA, tPA-MA, TF-G, tPA-G and TF-alpha angle were increased (P  < .04) in HWI dogs. HWI dogs had higher fibrinogen concentrations (465.6 ± 161 mg/dL vs 284.5 ± 38 mg/dL, P  = .008) and eosinophil counts (0.686 ± 0.27 × 10 3 /μL vs 0.267 ± 0.20 × 10 3 /μL, P  = .003). There was no difference in hematocrit, activation state, or percent of reticulated platelets. Non-activated reticulated platelets exhibited higher CD62P expression compared with mature platelets., Conclusions: Chronic canine heartworm disease was accompanied by hypercoagulability, hyperfibrinogenemia, and decreased fibrinolysis. Enhanced platelet activation was not identified in this group of HWI dogs., (© 2024 The Authors. Veterinary Clinical Pathology published by Wiley Periodicals LLC on behalf of American Society for Veterinary Clinical Pathology.)

Not all boar sperm samples survive cryopreservation well. A method of eliminating damaged sperm might enable more cryopreserved boar semen to be used for pig breeding. In this study we investigated the use of Magnetic Activated Cell sorting (MACS) to eliminate damaged sperm from thawed boar semen samples. The thawed samples were mixed with Dead cell removal particles and were applied to the column in a SuperMACS II. Different fractions were collected: Original sample (O), Flow-through (FT), and Eluate (E). Sperm membrane integrity, mitochondrial membrane potential and reactive oxygen species were evaluated by flow cytometry after staining with SYBR 14 and propidium iodide, or 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide, or hydroethidine and dichlorodihydrofluorescein diacetate, respectively. The FT samples had increased membrane integrity, a greater proportion of sperm with high mitochondrial membrane potential and a greater proportion of sperm negative for hydrogen peroxide than O samples (P<0.0001), which in turn had increased membrane integrity than E samples (P <0.0001). However, differences were seen between boars. The FT samples had increased values of live, superoxide positive sperm than O samples (P <0.0001) and O samples had greater values than E samples (P <0.0001), while there was no effect of boar. Sperm quality was best in the FT fraction, comprising approximately 32% of the sperm sample. In conclusion, although there were differences between boars, MACS separation can improve sperm quality in thawed semen samples. It would be interesting to see if this improvement is reflected in fertility outcomes., Competing Interests: Declaration of Competing Interest none, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Isolation of genomic DNA of blood parasites in birds, reptiles, amphibians, and fishes is a challenging task, given that their red blood cells are nucleated; for that reason, parasite genomic DNA is only a fraction of the total extracted DNA, and it is challenging to obtain concentrated high-quality genetic material. Percoll Density Gradient (PDG) and flow cytometry are tools for separating and analyzing cell populations or even a single cell, and both represent potent approaches for isolating avian haemosporidians parasites. Our experimental design included several steps seeking to concentrate the parasite´s DNA. We used blood samples from a Rock pigeon infected with Haemoproteus columbae. After inducing parasite exflagellation and gametogenesis in vitro, we subjected the samples to a Percoll Density Gradient to separate the parasites from the rest of the blood cells. Following centrifugation, the layer containing extracellular parasites underwent a flow cytometry and cell sorting process, during which we selected two different subpopulations of cells for analysis. Based on qPCR analyses, we demonstrate parasite DNA enrichment in Percoll Density Gradient and flow cytometry samples; simultaneously, these samples showed the lowest concentration of Columba livia DNA. However, the concentration of parasite DNA was higher in the PDG than in the cell sorting sample. This study reports the concentration of the Haemoproteus parasite by flow cytometry without DNA-intercalating dyes, and this methodology can serve as a technique for DNA enrichment of blood parasites infecting nucleated red blood cells to improve techniques that allow obtaining complete genomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Current diagnostic methods for Johne's disease in cattle allow reliable detection of infections with Mycobacterium avium ssp. paratuberculosis (MAP) not before animals are 2 years of age. Applying a flow cytometry-based approach (FCA) to quantify a MAP-specific interferon-gamma (IFN-γ) response in T cell subsets, the present study sought to monitor the kinetics of the cell-mediated immune response in experimentally infected calves. Six MAP-negative calves and six calves, orally inoculated with MAP at 10 days of age, were sampled every 4 weeks for 52 weeks post-inoculation (wpi). Peripheral blood mononuclear cells (PBMC) were stimulated with either purified protein derivatives (PPD) or whole cell sonicates derived from MAP (WCSj), M. avium ssp. avium or M. phlei for 6 days followed by labeling of intracellular IFN-γ in CD4 + and CD8 + T cells. No antigen-specific IFN-γ production was detectable in CD8 + cells throughout and the responses of CD4 + cells of MAP-infected and control calves were similar up to 12 wpi. However, the mean fluorescence intensity (MFI) for the detection of IFN-γ in CD4 + cells after WCSj antigen stimulation allowed for a differentiation of animal groups from 16 wpi onwards. This approach had a superior sensitivity (87.8%) and specificity (86.8%) to detect infected animals from 16 wpi onwards, i.e., in an early infection stage, as compared to the IFN-γ release assay (IGRA). Quantification of specific IFN-γ production at the level of individual CD4 + cells may serve, therefore, as a valuable tool to identify MAP-infected juvenile cattle., (© 2024. The Author(s).)

Background: Dysregulated T lymphocyte response is thought to play a key role in chronic intestinal inflammation (CIE)., Objectives: To evaluate the presence of changes in peripheral and intestinal T lymphocyte subsets and to describe potential immune and inflammatory biomarkers in dogs with CIE., Animals: Sixteen healthy dogs and 26 dogs were diagnosed with CIE., Methods: Prospective case-control study evaluating peripheral and intestinal T lymphocytes using flow cytometry and inflammatory markers obtained from complete blood cell counts., Results: Dogs with CIE had higher peripheral activated T helper (Th) lymphocytes (87/μL [18-273] CIE, 44/μL [16-162] healthy control (HC, P = .013) and regulatory T cells (Treg; 108/μL [2-257] CIE, 34/μL [1-114] HC, P = .004). In the intestinal epithelium, CIE dogs presented lower percentages of Th (4.55% [1.75-18.67] CIE, 8.77% [3.79-25.03] HC, P = .002), activated Th cells (0.16% [0.02-0.83] CIE, 0.33% [0.05-0.57] HC, P = .03) and CD4/CD8 ratio (0.08 [0.02-0.39] CIE, 0.21 [0.07-0.85] HC, P = .003). Conversely, higher percentage of activated T cytotoxic cells (20.24% [3.12-77.12] CIE, 12.32% [1.21-39.22] HC, P = .04) and interferon-gamma (IFN-γ) producing T lymphocytes (7.36% [0.63-55.83] CIE, 1.44% [0.00-10.56] HC, P = .01) within the epithelium was observed. In the lamina propria the percentage of Treg lymphocytes was higher (6.02% [1.00-21.48] CIE, 3.52% [0.18-10.52] HC, P = .02)., Conclusions and Clinical Importance: Systemic and intestinal immune alterations occur in dogs with CIE suggesting that blood IFN-γ producing T lymphocytes and the systemic immune-inflamation index (SII) could potentially serve as biomarkers for the disease., (© 2024 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

The limited availability of canine-reactive monoclonal antibodies restricts the analyses of immune cell subsets and their functions by flow cytometry. The PrimeFlow™ RNA Assay may serve as a potential solution to close this gap. Here we report a blood immunophenotyping method utilizing combined protein- and RNA-based flow cytometry to characterize canine T cell activation and proliferation within individual cells. In this assay, CD69 expression was detected by an RNA probe and CD25 and Ki67 were detected by antibodies. Canine peripheral blood mononuclear cells (PBMCs) were stimulated with three agents with different modes of action, anti-CD3/CD28 antibodies, phytohemagglutinin, or phorbol myristate acetate /ionomycin. Robust T cell activation (CD25+ and/or CD69+) and proliferation (Ki67+) were detected. Both CD69 and CD25 appear to be robust and sensitive T cell activation markers with early induction and low background expression. Upon stimulation, T cell proliferation occurred later than T cell activation and was associated with CD25 expression. This canine T cell activation and proliferation immunophenotyping method was evaluated in 5 independent experiments using PBMCs from 10 different beagle dogs with satisfactory assay performance. This method can greatly facilitate the evaluation of immune disease pathogenesis and immunotoxicity risk assessment in nonclinical drug development in canine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

T cell lymphomas are a diverse group of tumors found in both dogs and humans, originating from various normal T cell types. Identifying the origin of neoplastic lymphocytes can offer valuable insights into the pathogenesis and clinical behavior of these tumors. T zone lymphoma (TZL) in dogs is characterized by the absence of CD45 expression, a strong breed predilection, and its association with adult-onset demodicosis-a condition believed to be linked to immunosuppression. In this study, our aim was to employ transcriptomic and functional data to determine the normal counterpart of TZL. Identifying the normal counterpart may help us understand both how these tumors arise and explain their clinical behavior. Gene expression profiling using NanoString and RNA seq was used to compare the transcriptome between neoplastic T zone cells, normal canine T cells and publicly available gene sets using Gene Set Enrichment Analysis. Mitogen, anti-CD3 stimulation and PMA/ionomycin stimulation were used to assess T cell proliferation in vitro, and intracellular cytokine production was measured by flow cytometry. Gene expression profiling revealed that TZL is most likely derived from an activated or memory alpha-beta T cell but the cells do not fall cleanly into an effector subtype. TZL cells express CD4-specific transcription factors GATA3 and THPOK, even though TZL cells more commonly express CD8, or neither CD4 nor CD8. TZL cells produce high levels of interferon gamma and tumor necrosis factor alpha when stimulated, further supporting the hypothesis that they are derived from an antigen experienced T cell. TZL cells do not proliferate when stimulated through the T cell receptor but will divide when the T cell receptor is bypassed with PMA and ionomycin. The observation that these cells are derived from a mature, previously activated T cell is the first step in understanding the genesis of this unique T cell tumor., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Recently, there has been a growing interest in stem cells for human medicine. Limited feline endometrial mesenchymal stem cell (fEM-MSC) research in veterinary medicine necessitates reporting for future feline disease research and therapy., Objectives: This study aimed to isolate fEM-MSCs from feline endometrial tissues and evaluate their morphology, proliferative ability, differentiation ability, and immunophenotype., Methods: Feline endometrial tissues were obtained from the ovariohysterectomies of healthy cats and isolated using an enzymatic method. The morphology and proliferative ability of the isolated cells were assessed using a doubling time (DT) assay from passages 3 to 6 (P3 - P6). We measured pluripotency gene expressions of cells in P2 using quantitative real-time polymerase chain reaction (qRT-PCR). To investigate MSC characteristics, a trilineage differentiation assay was conducted in P4, and cells in P4 were immunophenotyped using flow cytometry., Results: fEM-MSCs showed a typical spindle-shaped morphology under a microscope, and the DT was maintained from P3 to P6. fEM-MSCs could differentiate into adipocytes, osteoblasts, and chondrocytes, and expressed three pluripotency markers (OCT4, SOX2, and NANOG) by qRT-PCR. Immunophenotypic analysis showed that the fEM-MSCs were CD14 - , CD34 - , CD45 - , CD9 + , and CD44 + ., Conclusions: In this study, the feline endometrium was a novel source of MSCs, and to the best of our knowledge, this is the first report on the isolation method and characteristics of fEM-MSCs., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Korean Society of Veterinary Science.)

Peripheral T-cell lymphoma (PTCL) is highly aggressive in dogs and demonstrates a poor response to traditional chemotherapy. The aim of this retrospective study was to assess the prognostic significance of peripheral blood (PB) and bone marrow (BM) infiltration evaluated by flow cytometry (FC) in dogs with treatment-naïve and histologically confirmed PTCL. To be included, dogs had to undergo complete staging, including FC on lymph nodes, PB and BM samples. Additionally, dogs had to receive an alkylating-rich protocol and have a complete follow-up. Treatment response was evaluated based on RECIST criteria at each chemotherapy session, and the end-staging was conducted at the completion of treatment. Endpoints were time to progression (TTP) and lymphoma-specific survival (LSS). The relationship between TTP/LSS and the percentage of PB and BM infiltration, categorized as > 1%, > 3%, > 5%, > 10%, > 15% and > 20% was investigated. Fifty dogs were included: based on imaging and FC, 78.0% had stage 5 disease, 14.0% had stage 4, 6.0% had stage 3 and 2.0% had stage 1. By multivariable analysis, the CD4-negative phenotype was the only factor associated with a shorter TTP (P = 0.049), while BM infiltration was significantly associated with LSS (P = 0.037). Dogs with BM infiltration > 5% had shorter median LSS (114 days; 95%CI: 0-240) compared to dogs with BM infiltration ≤ 5% (178 days; 95%CI: 145-211). Lack of complete response (P = 0.039) and administration of corticosteroids before chemotherapy (P = 0.026) also significantly worsened LSS. BM flow cytometric evaluation could be considered an essential part of staging work-up for dogs with PTCL and has prognostic relevance., Competing Interests: Conflict of interest statement None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Canine seminal plasma is a complex fluid containing proteins, peptides, enzymes, hormones as well as extracellular vesicles that are involved in many physiological and pathological processes including reproduction. We examined the expression of the extracellular vesicles surface antigens Aminopeptidase-N (CD13) and Dipeptidyl peptidase IV (CD26) by flow cytometry. For this study, third fraction of the ejaculate, from fertile adult male German Shepherd dogs, was manually collected twice, two days apart. FACS analyses revealed that CD13 and CD26 are co-expressed on the 69.3 ± 3.7% of extracellular vesicles and only a 2.0 ± 0.5% of extracellular vesicles express CD26 alone. On the other hand, 28.6 ± 3.6% of seminal EVs express CD13 alone. Our results agree with the hypothesis that CD26 needs to be co-expressed with other signal-transducing molecules, while CD13, can perform functions independently of the presence or co-expression of CD26. The results obtained in normal fertile dogs could represent physiological expression of these enzymes. Therefore, it would be interesting to carry out further studies to evaluate the expression of CD13 and CD26 on extracellular vesicles as biomarker for prostate pathological condition in dogs., (© 2023. The Author(s).)

Flow-cytometry has become increasingly popular to assess the haemocytes morphology and functions of marine molluscs. Indeed, haemocytes are the first line of defence of the immune system in molluscs and are used as a proxy for oyster health. Authors publishing in the field of flow-cytometry and molluscs health seemed to utilise the same methods for all model species used, independently of their geographical location in the world (temperate, tropical, etc.). Hence, this paper dived into flow-cytometry methodology and investigated if using different plates, different thresholds, different incubation times and temperatures as well as different fluorochromes concentrations affected the results. This study revealed that the cell count did not change when using different thresholds on the FSC-H parameter of the instrument but was affected by the plate type, the temperature of incubation, and the time of incubation. Indeed, non-adherent plates yielded the highest cell count and lower cell counts were associated with a higher temperature and a longer time of incubation. Furthermore, the haemocytes functions such as the phagocytosis, the lysosomal content, the intracellular oxidative activity, and the mitochondria activity were also affected by the temperature and the time of incubation. An increase in the phagocytosis capacity, lysosomal content and mitochondria activity was observed with a higher temperature. At the exception of the phagocytosis rate, all the other parameters such as the phagocytosis capacity, the intracellular oxidative activity, and the lysosomal content increased with a longer incubation time. We also showed that it is best to optimise the amount of fluorochromes used to avoid unnecessary background or non-specific staining., (Copyright © 2023. Published by Elsevier Ltd.)

Differentiating immune-mediated causes from other causes of anemia and thrombocytopenia can be challenging. Flow cytometry can detect surface-associated immunoglobulin (sIg) on red blood cells (RBC) and platelets (PLT) in dogs and horses. Sample storage parameters for ideal assay performance has not been evaluated in horses. The study objective is to identify optimal storage time and temperature of equine whole blood for the detection of RBC-sIg and PLT-sIg via flow cytometry. Both assays were performed on samples at time 0, 4, 24, 48, and 72 h post collection. RBC-sIg samples were stored at 4 °C and PLT-sIg samples were stored at 4 °C and room temperature. RBC-sIg percentages were stable up to 72 h storage. Platelet surface-associated IgG percent positive platelets increased above baseline at all timepoints and percent positive platelets were inconsistent across timepoints for IgM and IgA. PLT-sIg testing should ideally be performed within 4 h of collection. In instances where this is not feasible, samples should be stored at 4 °C and analyzed no later than 24 h after collection. Whereas cutoff values for RBC-sIg remained similar across timepoints, results for PLT-sIg should be compared to time-specific cutoff or reference intervals established by the laboratory running the test., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Bovine Trichomonosis (BT), a sexually transmitted disease endemic in countries with extensive cattle farming and natural service, is one of the most common causes of reproductive failure. 5-nitroimidazoles and their derivatives are used for its treatment, mainly metronidazole. The emergence of drug resistance mechanisms and treatment failures raise the need to investigate the effectiveness of new active compounds that contribute to parasite control. In this regard, extracts of Lantana camara (Verbenacea) have shown high biocidal potential against isolates of Trypanosoma cruzi and Leishmania braziliensis in vitro assays, although their effect on Tritrichomonas foetus has not been demonstrated yet. The available information on in vitro susceptibility of trichomonicidal drugs comes from the use of a diversity of methodologies and criteria, especially the observation of parasite motility under the optical microscope to assess their viability. Recently, in our laboratory, the use of flow cytometry has been described for the first time as a rapid and efficient method to evaluate the viability of T. foetus against metronidazole. The present study aimed to evaluate the cytostatic effect of L. camara extracts against T. foetus isolates by flow cytometry. Under aerobic conditions, IC 50 values of 22.60 µg/mL were obtained on average. Under anaerobic conditions, the IC 50 oscilated around 29.04 µg/mL. The results obtained allowed describing the susceptibility exhibited by these protozoa, being a valuable information for the development of potential BT treatments., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Lymphocyte immunophenotyping can be useful for evaluating immune competence and predicting the disease prognosis. It is essential to gain knowledge about canine lymphocyte immunophenotypes in various conditions. The study deals with the characteristics of lymphopenia in dogs, with an emphasis on lymphocyte immunophenotyping by flow cytometry. Blood samples from 44 dogs with lymphopenia were included in the study. All lymphopenias sent from veterinary clinics to the diagnostic laboratory were analyzed. The hematological and biochemical abnormalities were investigated, as well as the effect of the age. Lymphopenias were classified according to the level of C-reactive protein (CRP). The percentage of T cells, B cells, T h cells and T c cells, and T/B and T h /T c ratios were determined by flow cytometry. Lymphopenias often occurred in dogs over 7 years of age (79.5 %). The most common were postoperative lymphopenia (31.8 %) and inflammatory diseases (29.5 %), most commonly affecting the gastrointestinal tract. Frequent abnormalities were monocytosis (56.8 %), increased CRP (72.7 %) and decreased albumin/globulin ratio (50.0 %). The percentage of T h lymphocytes was significantly lower in the group with elevated CRP than in the group with basal CRP (P = 0.0329). A negative correlation was found between the level of CRP and the percentage of T h lymphocytes (r = -0.3278, P = 0.0390). This study provided new insights into the appearance, incidence and classification of canine lymphopenia., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

As science and technology continue to advance, the use of flow cytometry is becoming more widespread. It can provide important information about cells in the body by detecting and analysing them, thereby providing a reliable basis for disease diagnosis. In the diagnosis of bovine epidemic diseases, flow cytometry can be used to detect bovine viral diarrhoea, bovine leukaemia, bovine brucellosis, bovine tuberculosis, and other diseases. This paper describes the structure of a flow cytometer (liquid flow system, optical detection system, data storage and analysis system) and its working principles for rapid quantitative analysis and sorting of single cells or biological particles. Additionally, the research progress of flow cytometry in the diagnosis of bovine epidemic diseases was reviewed in order to provide a reference for future research and application of flow cytometry in the diagnosis of bovine epidemic diseases.

This review presents information on biochemical features of spermatozoa bearing X or Y chromosome, enabling production of a sperm fraction with pre-defined sex chromosome. The almost only technology currently used for such separation (called sexing) is based on the fluorescence-activated cell sorting of sperm depending on DNA content. In addition to the applied aspects, this technology made it possible to analyze properties of the isolated populations of spermatozoa bearing X or Y chromosome. In recent years, existence of the differences between these populations at the transcriptome and proteome level have been reported in a number of studies. It is noteworthy that these differences are primarily related to the energy metabolism and flagellar structural proteins. New methods of sperm enrichment with X or Y chromosome cells are based on the differences in motility between the spermatozoa with different sex chromosomes. Sperm sexing is a part of the widespread protocol of artificial insemination of cows with cryopreserved semen, it allows to increase proportion of the offspring with the required sex. In addition, advances in the separation of X and Y spermatozoa may allow this approach to be applied in clinical practice to avoid sex-linked diseases.

In drug development, the dog is often used as a model for non-rodent preclinical safety studies. In particular, immunophenotyping in dogs can be important to characterize the toxicological profile of a test item. A wide range of antibodies specific to surface antigens is needed, however, commercially available antibodies to dog are scarce. To date, numerous studies have reported the cross-reactivity of human monoclonal antibodies with canine peripheral blood mononuclear cells (PBMC). In this study, we aimed to increase the number of canine-specific antibodies and took a rather novel approach to further determine cross-reactivity of 378 human recombinant antibodies lacking Fc regions to surface antigens on canine PBMC. The screening resulted in 30 human monoclonal antibodies well reactive to canine PBMC. Sequence homology of the targeted human and canine antigens was analyzed with Basic Local Alignment Search Tool. Thirteen human cross-reactive antibodies of interest were analyzed with cells from canine whole blood in combination with lineage markers. Finally, ten antibodies were identified as useful markers for the application in dog. Except for CD27, the remaining nine antibodies are already commercially available human cross-reactive antibodies. This study provides a new source for all ten antibodies described here., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Eighty-five sperm samples were cryopreserved and SYBR14/PI, MitoTracker Deep Red FM, FITC-PSA/PI and chlortetracycline were used for imaging flow cytometry evaluation of sperm viability, mitochondrial membrane potential (MMP), acrosome integrity and sperm capacitation, respectively. Sperm motility was also registered. Sperm motility (46.1 ± 7.7 vs. 24.1% ± 6.5%), sperm viability (49.8 ± 11.5 vs. 32.3% ± 9.6%) and high MMP (49.8% ± 12.4% vs. 34.9% ± 9.9%) decreased significantly (p < .05) during cryopreservation process, in contrast to acrosome-reacted in viable spermatozoa (1.0% ± 1.6% vs. 1.0% ± 1.0%) and sperm capacitation (10.0 ± 9.8 vs. 8.2% ± 12.4%) that were similar (p > .05) before and after cryopreservation. Positive correlations were found between sperm motility versus high MMP (r = .63), sperm motility versus sperm viability (r = .67) and sperm viability versus high MMP (r = .88). In conclusion, cryopreservation of alpaca spermatozoa is related to a decrease in sperm motility, sperm viability and high MMP, meanwhile acrosome integrity and sperm capacitation are not affected., (© 2023 Wiley-VCH GmbH.)

Sex-control technology have great economic value and is one of the hot topics in livestock research. To produce more milk, dairy farmers prefer female offspring. X/Y sperm separation is an effective method for offspring sex control. Currently, the major commercial production method for sperm separation is flow cytometry sorting in cattle. However, flow cytometry requires expensive equipment and long sorting times. So, a simple and inexpensive method for producing a higher number of dairy cows is required. In this study, R848 activates toll-like receptor 7/8 (TLR7/8), thereby separating X from Y sperm. The results showed TLR7/8 is expressed in the tail of X sperm. Immunofluorescence (IF) of testes, epididymis, and ejaculate shows that the number of TLR7 + /8 + sperm cells is up to 50 %. Furthermore, TLR7/8 agonist (R848) affects mitochondrial function through the PI3K/GSK3α/β/hexokinase and PI3K/NFκB/hexokinase signalling pathways, inhibiting X sperm motility, while the motility of Y-sperm remains unchanged. The difference in sperm motility causes Y sperm (with high motility) to move to the upper layer and X-sperm (with low motility) to the lower layer allowing the separation of X and Y sperm. Based on this study, we reveal a simple and effective method for enriched X/Y sperms from cattle., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Although immunotherapy is becoming a standard approach of human cancer treatment, only a small but critical fraction of patients responds to the therapy. It is therefore required to determine the sub-populations of patients who will respond to immunotherapies along with developing novel strategies to improve efficacy of anti-tumor immune reactions. Current development of novel immunotherapies relies heavily on mouse models of cancer. These models are important for better understanding of mechanisms behind tumor immune escape and investigation of novel strategies to overcome it. Nevertheless, the murine models do not necessarily represent the complexity of spontaneously occurring cancers in humans. Dogs spontaneously develop a wide range of cancer types with an intact immune system under similar environment and exposure to humans, which can serve as translational models in cancer immunotherapy research. To date though, there is still a relatively limited amount of information regarding immune cell profiles in canine cancers. One possible reason could be that there are hardly any established methods to isolate and simultaneously detect a range of immune cell types in neoplastic tissues. To date only a single manuscript describes characterization of immune cells in canine tumour tissues, concentrating solely on T-cells. Here we describe a protocol for multi-color flow cytometry to distinguish immune cell types in blood, lymph nodes, and neoplastic tissues from dogs with cancer. Our results demonstrate that a 9-color flow cytometry panel enables characterization of different cell subpopulations including myeloid cells. We also show that the panel allows detection of minor/aberrant subsets within a mixed population of cells in various neoplastic samples including blood, lymph node and solid tumors. To our knowledge, this is the first simultaneous immune cell detection panel applicable for solid tumors in dogs. This multi-color flow cytometry panel has the potential to inform future basic research focusing on immune cell functions in translational canine cancer models., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Parys et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: A novel method using a moving threshold (r-PLTmt) to determine canine r-PLTs (reticulated platelets) has been introduced for ADVIA 2120 software v6.11.7., Objectives: We aimed to evaluate absolute (ar-PLTmt) and percent (%r-PLTmt) prior to and after visual inspection of scattergrams (ar-PLTmtv, %rPLTmtv) compared with flow cytometry (flow) and to determine reference intervals (RIs) in 120 dogs., Methods: For method comparison, 42 blood samples of healthy and thrombocytopenic dogs were included. Calculation of Spearman's rho, Bland-Altman, and Passing-Bablok analysis was performed. Coefficients of variation (CVs) were determined for three concentration levels., Results: Moderate correlations between %r-PLTmt and %r-PLTmtv (r s 0.75-0.76) were seen compared with flow cytometry. The CV for medium %r-PLTs counts assessed with flow cytometry was 12.9%. Comparable CVs were obtained for ar-PLTmt (14.4%) and %r-PLTmt (15.7%), and ar-PLTmtv and %r-PLTmtv (10.9% and 12.9%, respectively). At low and high concentration levels, CVs for % and absolute r-PLTmt/rPLTmtv ranged between 23%-30% and 15%-20%. In patients with microcytic hypochromic erythrocytes, CVs for ar-PLTmt and %r-PLTmt were 36%-66%. Visual inspection of scattergrams resulted in a marked decrease in CV ranging between 15% and 20%. A proportional bias of 10.8% between %r-PLTmt and flow cytometry became lower (9.7%) after visual validation of scattergrams. Passing-Bablok analysis showed proportional and constant error. RIs for r-PLTmt and r-PLTmtv were 0.2%-3.8% and 0.6-10.2 × 10 9 /L and 0.3%-4.5% and 1.1-10.3 × 10 9 /L, respectively. Median values for %r-PLTmtv were higher in young adults (≤2 years) than in older dogs (P = 0.03)., Conclusions: r-PLTmt and r-PLTmtv were moderately correlated with flow cytometry. Visual inspection of scattergrams is recommended., (© 2022 The Authors. Veterinary Clinical Pathology published by Wiley Periodicals LLC on behalf of American Society for Veterinary Clinical Pathology.)

Monocytes are innate immune system key players with pivotal roles during infection and inflammation. They migrate into tissues and differentiate into myeloid effect cells (macrophages, dendritic cells) which orchestrate inflammatory processes and are interfaces between the innate and adaptive immune responses. Their clinical relevance to health and disease of cattle (Bos taurus) and water buffalo (Bubalus bubalis), two of the most important livestock species, has been highlighted in physiologic (pregnancy) and pathologic (mastitis, metritis, and viral infections) conditions. The existence of three different monocyte subsets in cattle was established by flow cytometry (FC), as follows: classical (cM; CD14 ++ CD16 -/low ), intermediate (intM; CD14 ++/+ CD16 + ), and non-classical (ncM; CD14 -/low CD16 ++ ) monocytes. FC applications for studying the immune system of cattle and water buffalo still have significant limitations. In this article, we describe some practical approaches to overcome these limitations and, in particular, allow the identification and enumeration of cM, intM, and ncM subpopulations in cattle and buffalo peripheral blood. Indeed, we propose the new procedure lyse/wash/no-centrifugation (L/W/NC) that can be combined with the FC absolute counting procedures and can overcome specific issues of the lyse/no-wash protocols (L/NW). Finally, for the first time, we demonstrated the existence of cM, intM, and ncM monocyte subsets also in the water buffalo, showing some interesting differences with cattle, such as the bubaline cM are mainly CD14 +/++ /CD16 + . These subtle differences may influence inflammatory disease regulation in, for example, mastitis and metritis. The upregulation of CD16 expression on cM may reveal different monocyte priming, leading to different functional features of macrophages/dendritic cells in tissues after infection. © 2023 Wiley Periodicals LLC. Basic Protocol: Absolute count of cM, intM, and ncM without compensation Alternate Protocol: Absolute count of cM, intM, and ncM for single laser platform Support Protocol 1: In-house monoclonal antibody labeling using a Pacific Blue ™ kit Support Protocol 2: In-house monoclonal antibody labeling using an Alexa Fluor ® 647 kit Support Protocol 3: Titration of fluorochrome-conjugated antibodies., (© 2023 Wiley Periodicals LLC.)

Objectives: The aim of this study was to isolate feline dental pulp stem cells (fDPSCs) and characterize their clonogenic and proliferative abilities, as well as their multipotency, immunophenotype and cytogenetic stability., Methods: Dental pulp was isolated by explant culture from two cats <1 year old at post mortem. Their clonogenicity was characterized using a colony-forming unit fibroblast assay, and their proliferative ability was quantified with a doubling time assay in passages 2, 4 and 6 (P 2 , P 4 and P 6 , respectively). Multipotency was characterized with an in vitro trilineage differentiation assay in P 2 , and cells were immunophenotyped in P 4 by flow cytometry. Chromosomic stability was evaluated by cytogenetic analysis in P 2 , P 4 and P 6 ., Results: The fDPSCs displayed spindle and epithelial-like morphologies. Isolated cells showed a marked clonogenic capacity and doubling time was maintained from P 2 to P 6 . Trilineage differentiation was obtained in one sample, while the other showed osteogenic and chondrogenic differentiation. Immunophenotypic analysis showed fDPSCs were CD45 - , CD90 + and CD44 + . Structural and numerical cytogenetic aberrations were observed in P 2 -P 4 ., Conclusions and Relevance: In this study, fDPSCs from two cats were isolated by explant culture and immunophenotyped. Cells displayed clonogenic and proliferative ability, and multipotency in vitro, and signs of chromosomic instability were observed. Although a larger study is needed to confirm these results, this is the first report of fDPSC isolation and in vitro characterization.

This article summarizes the current applications of flow cytometry in clinical veterinary medicine, which is largely restricted to the diagnosis of hematopoietic neoplasms (lymphomas and leukemias) of domestic dogs, cats, and horses. A brief background on the technique of flow cytometry and fundamentals of data interpretation are included. Major emphasis is placed on clinical indications for flow cytometry, principles of sample collection and submission, and awareness of diagnostic and prognostic utility. Expectations regarding both the benefits and limitations of flow cytometry in a clinical setting, and its complementary nature with other types of testing, are also reviewed., Competing Interests: Disclosure The author declares no commercial or financial conflicts of interest related to this work. No funding sources apply., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Low levels of intracellular reactive oxygen species (ROS) are essential for normal sperm function and are produced by sperm mitochondria as a byproduct of metabolism, but in excess, ROS can cause catastrophic cellular damage and has been correlated with infertility, poor sperm motility and abnormal morphology in humans. Stallion sperm motility is fueled predominantly by oxidative phosphorylation-produced ATP, requiring high basal rates of mitochondrial function. Consequently, whether elevated ROS production by stallion sperm is an indicator of dysfunctional or highly motile cells has been debated by researchers over the last decade. The objective of this study was to evaluate the relationship between various sperm morphologies and ROS production in fresh and cooled stallion semen by employing the novel method of imaging flow cytometry for stallion semen assessment. For evaluation of fresh semen, single ejaculates (n = 5) were collected from four resident stallions at the University of California, Davis. For the evaluation of 24-h cool-stored semen, single ejaculates were collected from stallions at Texas A&M University (n = 5) and shipped to the University of California, Davis overnight for evaluation. Ejaculate volume, sperm concentration and motility parameters were recorded. Samples were co-stained for viability and ROS detection with SytoxGreen™ and dihydroethidium (DHE), respectively, and evaluated with the Amnis® ImageStream® system (Luminex Corporation). Antimycin, an electron transport chain inhibitor that triggers ROS production (1 μM), was used as a positive control for DHE, while dead cells (2× snap frozen in liquid nitrogen) served as a positive control for SytoxGreen™. Unstained samples were also evaluated as controls. Imaging flow cytometric analysis was performed with the ideas® software (Luminex Corporation). Evaluated morphologies included abnormal head (AH), abnormal midpiece (AM), abnormal tail (AT), proximal cytoplasmic droplet (PD), or distal cytoplasmic droplet (DD), and morphologically normal (MN) cells. For fresh semen, an additional abnormality, coiled tail and midpiece (CTM) was assessed; 24-h cool-stored semen did not contain enough viable CTM cells for analysis. Only cells with obvious, single abnormalities were selected for the first portion of analysis to minimize subjectivity. Mixed effects modelling was used to evaluate the relationship between each morphologic classification and the corresponding DHE fluorescence intensity. Compared to the MN population, ROS production was significantly higher in viable cells with AH, PD and AM (p < .0001) in both fresh and cooled semen. CTM cells had significantly higher levels of ROS production compared to MN cells in fresh semen (p < .0001). There was no significant difference in ROS levels between MN cells and AT and DD cells in either fresh or cooled semen (p > .05). These results suggest that ROS generation is indicative of abnormal cell morphology and function and confirm that imaging flow cytometry is a valuable tool for the assessment of stallion semen., (© 2022 Wiley-VCH GmbH.)

For a more practically applicable analysis of different sperm characteristics, this study aimed to develop a 5-color flow cytometry (FC) panel to concurrently analyze four sperm parameters in liquid boar and stallion semen, using also a DNA-marker for selecting sperm cell events. From each of thirty extended boar semen doses and twelve stallion semen doses, six aliquots were taken. For evaluating mitochondrial activity (A), degree of lipid disorder of plasma membrane (B), integrity of plasma membrane (C), acrosomal status (D) and marking DNA (E), five aliquots were individually stained with Rhodamine 123, Merocyanine 540, Propidium Iodide, PNA-Alexa Fluor 647, and Hoechst 33342, respectively. The sixth aliquot was stained with all the five fluorochromes simultaneously, whereas spectral overlap was corrected by a compensation matrix. Strong correlations were found between the single and 5-color staining assays for boar sperm (A: 0.99, B: 0.96, C: 0.93, D: 0.98, E: 0.99; P < 0.01). Furthermore, moderate and substantial Concordance Correlation Coefficients (CCC) were presented by all these parameters (0.99, 0.96, 0.92, 0.98, and 0.99, respectively). For stallion sperm, the correlation coefficients between the assays were also strong (A: 0.99, B: 0.98, C: 0.99, D: 0.99, E: 0.95; P < 0.01) and substantial CCC were observed for all of them (0.99, 0.97, 0.99, 0.99, and 0.90, respectively). For both species, the mean difference between the methods (d̅) did not overtake 0.84. The results confirmed that this 5-color panel could be successfully implemented for analyzing boar and stallion sperm quality in a single, practical and quick FC assay., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Horses have played a prominent role in shaping our modern world, with important effects on health. Unfortunately, better characterization of the horse immune system is still needed. In this report, using flow cytometry techniques, four monoclonal antibodies against horse CD11c integrin were characterized and described for their ability to provide a positive recognition signal in peripheral blood mononuclear cells. Further immune cell phenotype experiments were performed using MHC-II, CD14, TLR4 and the specific anti-horse CD11c monoclonal antibody (1C4). With this staining panel, it was possible to detect a cell population defined by CD11c + MHC-II + TLR4 + CD14 low , which could be considered as putative dendritic cells. This manuscript shows that a new monoclonal antibody (1C4) can be used for the characterization of dendritic cells and their different lineages, opening the possibility of better understanding the mechanisms of immunity in horses., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Meloxicam is a commonly used analgesic in rabbits. However, its possible impact on lymphocyte subpopulations remained unknown. The aim of the study was to investigate a possible effect of long-term administration of meloxicam on rabbit lymphocyte subpopulations. The study included 8 rabbits given meloxicam orally once daily (1 mg/kg BW) for 14 days and 8 rabbits as a control group. Peripheral blood samples were collected on day 0 (before the first dose of meloxicam), day 3, 7 and 14. Samples were evaluated with a haematology analyser and a flow cytometer. A significant decrease in T: B cell ratio was found in all samples taken during meloxicam administration compared to day 0, as well as in comparison with the control group (P < 0.01). A significant increase (P < 0.05) in proportion of CD5 +CD8 + lymphocytes occurred by day 3. Subsequently, although the values slightly decreased, they still remained elevated throughout all the experiment compared to the values from day 0 (P < 0.05). A slight decrease in T and B cell activation (CD5 +CD25 + and IgM+CD25 +) noticed by day 3, declined during the next days of administration and became more and more significant (finally, P = 0.0078). Since a high significant decrease (P < 0.01) in both T and B cell activation as well as a significant increase (P < 0.05) in CD5 +CD8 + T cells proportion were observed after meloxicam administration, a predicted effect of long-term administration of meloxicam on rabbit lymphocytes was confirmed., Competing Interests: Declaration of interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Flow cytometry can be used to evaluate many sperm attributes and Dr. Duane Garner was influential in developing assays to understand sperm physiology and function. We review some of Dr. Garner's work and describe experiments that evaluate sperm capacitation using Dr. Garner's philosophy. In exploratory experiments, boar sperm were cryopreserved in lactose egg yolk (LEY) or Beltsville Freezing Extender 5 (BF5) and incubated in one capacitating medium. In another experiment, frozen-thawed bull sperm were incubated in TALP-Ca or CFDM1 capacitating media. In both experiments, sperm viability and capacitation were evaluated using multiple probes. Boar sperm frozen in LEY had greater survival rates (38%) than sperm frozen in BF5 (22%; P < 0.05) but did not capacitate as effectively as sperm in BF5 (P < 0.05). In Experiment 2, bull sperm survived to a greater extent when incubated in TALP-Ca than in CFDM1 (P < 0.05) and had greater capacitation for most parameters (P < 0.05). Of particular interest, 77% of sperm incubated in TALP-Ca had activated second messenger systems involved in capacitation, compared with < 5% of sperm incubated in CFDM1. The results indicate different freezing and capacitating media induce different responses to sperm capacitation and functions. If only sperm viability and acrosomal integrity were evaluated, these results would be interpreted very differently. Dr. Garner's philosophy of evaluating multiple sperm parameters was an impetus to determine unique treatment differences which help in understanding sperm capacitation, and design further experiments to determine how media content causes sperm physiology differences., Competing Interests: Declaration of Competing Interest The authors declare that we have no competing interests., (Published by Elsevier B.V.)

Somatic cell count (SCC) in milk is an essential indicator for defining and managing udder health. However, analyzing differential SCC (dSCC) can be helpful in determining the type or evolution stage of mastitis. A high abundance of polymorphonuclear cells (PMN) is associated with acute mastitis; however, the status of a chronic disease is less well characterized. A method capable of analyzing SCC and dSCC can prove to be a helpful tool for monitoring the status of evolution of mastitis disease in a better way. Therefore, a new direct-flow cytometry method was developed to count and differentiate somatic cells in milk without the steps of centrifugation or washing, avoiding variabilities that occur due to enrichment or loss of specific cell types. In this new method, SCC is analyzed using the method of DNA staining with Hoechst stain, whereas dSCC are analyzed using specific antibodies targeting 2 main cell types associated with mastitis: PMN cells and antigen-presenting cells, which are associated with innate and adaptive immunity. Equivalent SCC values were obtained between the new method and the routine ISO 13366-2 method in a comparison of 240 raw milk samples. Furthermore, dSCC results were confirmed by microscopy after May-Gründwald-Giemsa staining in 165 quarter milk samples from healthy and diseased cows. The method was verified with fluorescence microscopy on the 2 targeted cell types and in raw milk samples. The newly developed method is independent of any instrument and can be further designed to differentiate other cell types and animal species by selecting appropriate antibodies., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Artificial insemination using semen from genetically superior sires remains one of the most effective biotechnologies ever commercialized for animal breeding purposes. Genetic progress, however, cannot begin until conception occurs. Processing laboratories that provide cryopreserved bull semen for commercial use depend on in vitro assays of semen quality to identify samples that are expected to result in less than desirable conception rates. These identified samples are discarded, rather than released to salable inventories, with the desired effect of minimizing variance in field fertility among both sires and individual collections. Although the industry was successfully founded on subjective assessment of motility and acrosome integrity, flow cytometric and computer-assisted sperm analysis offer more objective and repeatable measures of sperm quality attributes. Albeit more expensive to implement, the increased precision and repeatability when using these objective assays lends to greater confidence in the accuracy of decisions for individual collections and (or) bulls. The efficacy of a quality control program is evidenced by the range in sire fertility estimates calculated from field fertility data, which have historically indicated >90% of all sires achieve fertility deviation within ±3% points of the breed average. This impressive precedent implies somewhat limited opportunity for transition to objective assessments to have a meaningful impact on an already narrow range of fertility distributions. Nonetheless, flow cytometric assessments of novel attributes of sperm quality hold promise for detection of truly sub-fertile sires (deviations < -3) that presently elude detection with use of existing semen bioassays., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Respiratory diseases are among the most common and expensive to treat diseases in camels with a great economic impact on camel health, welfare, and production. Bronchoalveolar lavage fluid (BALF) has been proven as a valuable sample for investigating the leukocyte populations in the respiratory tract of several species. In the present study, fluorescent antibody labeling and flow cytometry were used to study the immune cell composition of BALF in dromedary camels. Animals with clinical respiratory diseases (n = seven) were compared with apparently healthy animals (n = 10). In addition, blood leukocytes from the same animals were stained in parallel with the same antibodies and analyzed by flow cytometry., Results: Camel BALF macrophages, granulocytes, monocytes, and lymphocytes were identified based on their forward and side scatter properties. The expression pattern of the cell markers CD172a, CD14, CD163, and MHCII molecules on BALF cells indicates a similar phenotype for camel, bovine, and porcine BALF myeloid cells. The comparison between camels with respiratory disease and healthy camels regarding cellular composition in their BALF revealed a higher total cell count, a higher fraction of granulocytes, and a lower fraction of macrophages in diseased than healthy camels. Within the lymphocyte population, the percentages of helper T cells and B cells were also higher in diseased than healthy camels. The elevated expression of the activation marker CD11a on helper T cells of diseased camels is an indication of the expansion of helper T cells population due to infection and exposure to respiratory pathogens. The higher abundance of MHCII molecules on BALF macrophages from diseased camels indicates a polarization toward an inflammatory macrophage phenotype (M1) in respiratory diseased camels. No significant differences were observed in the systemic leukogram between healthy and diseased animals., Conclusions: Collectively, the current study represents the first report on flow cytometric analysis of immune cell composition of bronchoalveolar lavage fluid (BALF) in dromedary camels., (© 2022. The Author(s).)

Three dogs under 12 months old were diagnosed with atypical multiple myeloma (MM), having an aggressive multifocal anaplastic round cell sarcoma in bone marrow, viscera, and/or peripheral blood, which were confirmed by cytology and immunohistochemistry to be of plasma cell origin. The intramedullary sarcomas caused myelophthisis, osteolysis, and hypercalcemia. Complete or free light chain monoclonal gammopathy in the serum and/or urine was demonstrated by protein electrophoresis and immunofixation. The polymerase chain reaction for antigen receptor rearrangement assay performed on 2 cases identified a clonally rearranged immunoglobulin gene. Neoplastic cells lacked expression of CD45, CD3, CD18, CD21, CD34, and MHCII by flow cytometry. Immunohistochemistry revealed MUM1 immunoreactivity of the neoplastic cells. Combining all data, the diagnosis was MM. An aggressive form of MM in young dogs should be a differential diagnosis for patients with an immunoglobulin-productive, B cell-clonal, CD45-negative, MUM1-positive discrete cell neoplasm arising from the bone marrow.

Canine acute leukaemia is a heterogeneous neoplasm with multiple phenotypes. Criteria to subtype acute leukaemia by flow cytometry have not been validated. The goal of this study was to develop a panel of antibodies and objective antigen expression criteria for the assignment of lymphoid or myeloid lineage by flow cytometry. We isolated mRNA from the blood of 45 CD34+ acute leukaemia cases and measured expression of 43 genes that represent lymphoid and myeloid lineages using NanoString technology. We determined differentially expressed genes between major groups identified by unsupervised hierarchical clustering. We then evaluated the expression of antigens by flow cytometry to determine if cases could be assigned to a lineage. Two groups were identified by gene expression. Group 1/LYMPH overexpressed lymphoid-associated genes (ex. DNTT) and had a higher percentage of CD5 + CD3- cells by flow cytometry. Group 2/MYELO overexpressed myeloid-associated genes (ex. ANPEP/CD13) and had a higher percentage of class II major histocompatibility complex (MHCII)- CD14+ and/or CD18 + CD4- cells. We proposed that >12.5% CD5 + CD3- cells in the blood was indicative of lymphoid lineage, and > 3.0% CD14 + MHCII- cells or > 18% CD18 + MHCII-CD4- cells was indicative of myeloid lineage. 15/15 cases that met the proposed criteria for acute lymphocytic leukaemia were in LYMPH group and 12/15 cases that met the proposed criteria for acute myeloid leukaemia were in MYELO group. The majority of CD34+ cases that did not meet either immunophenotyping lineage criterion (12/13) clustered within the LYMPH group. In conclusion, currently available antibodies can be useful for determining canine acute leukaemia subtypes., (© 2022 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Background: Nodal small cell B-cell lymphoma subtypes in dogs cannot be distinguished by flow cytometry and information regarding treatment, prognosis, and outcome are limited., Hypothesis/objectives: Objectives were to describe outcome in dogs with nodal small cell B-cell lymphoma diagnosed by flow cytometry and correlate clinical and laboratory data with survival. We hypothesized that B-cell Ki67 expression measured by flow cytometry is associated with shorter progression free survival (PFS) and overall survival (OS)., Animals: Forty-nine dogs with nodal small cell B-cell lymphoma, defined by >80% CD21+ B-cells by flow cytometry and small-sized B-cells by forward scatter., Methods: Retrospective study reviewing treatment and outcome data extracted from medical records. Percentage of Ki67-expressing B-cells was measured by flow cytometry. Clinical, laboratory, and flow cytometry data were assessed for association with outcome., Results: Median percentage of B-cell Ki67 was 41% (range, 3%-97%). Median PFS was 119 days and median OS was 222 days (n = 49). Among cases treated with CHOP-based chemotherapy (n = 32), median PFS was 70 days, median OS was 267 days, and 50% of cases achieved complete response. Low percentage of B-cell Ki67 (≤11%) was associated with prolonged OS by univariable analysis. Greater age, substage B, high B-cell CD25 expression and low B-cell CD21 and class II major histocompatibility complex expression by flow cytometry were independently associated with shorter OS., Conclusions and Clinical Importance: Most nodal small cell B-cell lymphoma cases had aggressive disease. Low Ki67 expression can help identify cases with better prognosis. Age, substage, and flow cytometry variables are useful prognostic factors., (© 2022 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

The canine transmissible venereal tumor (CTVT) is a transplantable cancer with the ability evade the immune system, despite strict immune surveillance of the host; in this context, the relationship between inflammatory infiltrate and CTVT prognosis is not entirely understood. Natural canine transmissible venereal tumors of 22 dogs were evaluated for tumor/host interaction through clinical and epidemiological data, cyto-histopathological and cytogenetic findings and, mainly, cell-mediated immune response. We performed analysis on dogs with naturally acquired disease to provide information from the study of CTVT biology in its natural course, as the clinical evolution of the natural tumor in the host is not yet as well known as in the laboratory. Populations for T cell labeling (CD3 + CD4 + CD8 + ), B cells, NK cells, and macrophages were analyzed by flow cytometry in blood and tumor samples and expressions of MHC class I and class II molecules were quantified by immunohistochemistry and compared mainly between the phases of progression and regression in the natural CTVT. Dogs were also treated with vincristine sulfate and evaluated for chemotherapeutic response. Chemotherapy was effective in 88% of cases and there was no recurrence of the disease 12 months after the cure. Tumor cells displayed a numerical chromosomal variation between 54 and 72, not correlating with the host genotype. Although a greater expression of MHC molecules [18.6 ± 5.8% class I (P < 0.004) and 38.5 ± 6.5% class II (P < 0.003)] was observed in the regression phase, no significant effect was observed between the clinical phase of the tumor and cellular immune response in the analysis by flow cytometry (P > 0.05). We also found no correlation between cytological subtype of the tumor (plasmacytoid, lymphocytoid and mixed) and cellular immune response, suggesting that there is no difference in tumor immunogenicity. Here, we found no immunological evidence to support the theory of the immune-induced complete spontaneous regression in CTVT., (Copyright © 2022. Published by Elsevier Ltd.)

Many aspects of the bovine immune system remain poorly characterized, which poses an obstacle to improving dairy cow health. Herein, we describe two flow cytometry panels that included antibodies against CD8α, CD4, TCR-δ, CD172α, CD14, MHCII, CD21, CD62L, and CD11b. These panels were used to characterize the phenotype of leukocyte subpopulations from the peripheral blood of 30-day old Holstein calves and Holstein cows at 260 d of gestation and calving. No leukocyte subset differences were found between the pre- and post-partum cows. However, calf leukocytes presented a higher proportion of CD3 + lymphocytes, γδ T-cells, CD8 + γδ T-cells, and monocytes when compared with mature cows. Conversely, cow lymphocytes had a higher proportion of CD4 + and CD8 + T-cells, and B-cells than calf lymphocytes. The proportion of CD4 + T-cells and B-cells expressing CD62L was greater in calves than in cows, while cow B-cells expressed greater levels of CD11b than calf B-cells. In contrast, calf polymorphonuclear cells (PMN) and monocytes expressed greater levels of CD11b compared to cows. Moreover, calf monocytes expressed higher levels of MHCII compared with those of cows. Collectively, our data provides a resource to better understand the bovine immune system as well as immune-related diseases that affect dairy cattle., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Sheep are an important livestock species worldwide and an essential large-animal model for animal husbandry and veterinary research. Understanding fundamental immune indicators, especially T-lymphocyte parameters, is necessary for research on sheep diseases and vaccines, to better understand the immune response to bacteria and viruses for reducing the use of antibiotics and improving the welfare of sheep. We randomly selected 36 sheep of similar ages to analyze cell-related immune indicators in peripheral blood mononuclear cells (PBMCs). The proportions of CD4 + and CD8 + T cells in PBMCs were detected by flow cytometry. We used Concanavalin A (Con A) and Phorbol-12-myristate-13-acetate (PMA)/Ionomycin to stimulate PBMCs, and measured the expression of IFN-γ, IL-4, and IL-17A using enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISpot). Simultaneously, PMA/Ionomycin/brefeldin A (BFA) was added to PBMCs, then the expression of IFN-γ, IL-4, and IL-17A was detected by flow cytometry after 4 h of culturing. In addition, we observed the proliferation of PBMCs stimulated with Con A for 3, 4, and 5 days., Results: The proportions of CD4 + T lymphocytes (18.70 ± 4.21%) and CD8 + T lymphocytes (8.70 ± 3.65%) were generally consistent among individuals, with a CD4/CD8 ratio of 2.40 ± 0.79. PBMCs produced high levels of IFN-γ, IL-4, and IL-17A after stimulation with PMA/Ionomycin and Con A. Furthermore, PMA/Ionomycin stimulation of PBMC yielded significantly higher cytokine levels than Con A stimulation. Flow cytometry showed that the level of IFN-γ (51.49 ± 11.54%) in CD8 + T lymphocytes was significantly (p < 0.001) higher than that in CD4 + T lymphocytes (14.29 ± 3.26%); IL-4 (16.13 ± 6.81%) in CD4 + T lymphocytes was significantly (p < 0.001) higher than that in CD8 + T lymphocytes (1.84 ± 1.33%), There was no difference in IL-17A between CD4 + (2.83 ± 0.98%) and CD8 + T lymphocytes (1.34 ± 0.67%). The proliferation of total lymphocytes, CD4 + T lymphocytes, and CD8 + T lymphocytes continued to increase between days 3 and 5; however, there were no significant differences in proliferation between the cell types during the stimulation period., Conclusions: Evaluating primary sheep immune indicators, especially T lymphocytes, is significant for studying cellular immunity. This study provided valuable data and theoretical support for assessing the immune response of sheep to pathogens and improving sheep welfare., (© 2022. The Author(s).)