A comparative evaluation of methods for estimating the colostrum quality in sows / Avaliação comparativa de métodos para estimar a qualidade do colostro em porcas

Colostrum is the main source of immunoglobulins (Ig) for neonate piglets and plays a crucial role within the health and growth of the piglet. Currently in pig farming, there are still no widespread practical methods for measuring the Ig concentration in colostrum at herd level. We evaluated sows' colostrum IgG concentration using an optical and a digital Brix refractometer and their performance was correlated to an IgG ELISA test, and flow cytometry. Colostrum concentrations of IgG and IgA averaged 74.05 ± 21.37mg/mL and 20.2 ± 5.32mg/mL respectively. The mean value of the Brix percentages for optical refractometer was 26.32%, and for digital was 28.32%. The Brix refractometer measurements of colostrum samples presented high correlation for IgG content analyzed by ELISA (Optical = 0.74, Digital = 0.87; P <0.001). Considering the immunophenotyping, the values for IgG and IgA lymphoblasts indicated a highly significant relationship to ELISA (IgG=0.77, IgA=0.84; P<0.001). The Brix refractometer can be considered a useful tool to be included in a colostrum monitoring program to improve potentially neonatal health. In addition, we demonstrated that flow cytometry can be an important tool to analyze and characterize the immunological potential of sow colostrum.(AU)

O colostro é a principal fonte de imunoglobulinas (Ig) para leitões recém-nascidos e desempenha um papel crucial na saúde e no crescimento dos leitões. Atualmente, na suinocultura, ainda não existem métodos amplamente utilizados na prática de produção para medir a concentração de imunoglobulinas no colostro suíno. Avaliou-se a concentração de IgG no colostro de porcas usando refratômetros Brix óptico e digital, e o desempenho foi comparado com ELISA e citometria de fluxo. As concentrações de IgG e IgA no colostro foram 74,05 ± 21,37mg/mL e 20,2 ± 5,32mg/mL, respectivamente. A percentagem de Brix média das amostras de colostro para o refratômetro óptico foi 26,32%, e para o digital foi 28,32%. As medições dos refratômetros de Brix apresentaram elevada correlação com a concentrações de IgG medidas por ELISA (óptico=0,74, digital=0,87; P<0,001). Considerando a imunofenotipagem, os valores dos linfoblastos IgG e IgA apresentaram alta correlação com o ELISA (IgG=0,77, IgA=0,84; P<0,001). O refratômetro Brix pode ser considerado uma ferramenta útil para ser incluída em um programa de monitoramento de colostro para melhorar a saúde neonatal. Além disso, foi demonstrado que a citometria de fluxo pode ser uma ferramenta importante para analisar e caracterizar o potencial imunológico do colostro de porcas.(AU)

Cryoprotective effect of melatonin supplementation on post-thawed rooster sperm quality.

Reactive oxygen species (ROS) and free radicals are one of the major detrimental factors that can negatively affect the quality of sperm during cryopreservation. Melatonin is an effective antioxidant and free radical scavenger in various cells. In this study, therefore, the aim was to evaluate the post-thawed quality of spermatozoa after cryopreservation of rooster semen in freezing extender supplemented with melatonin. Semen samples from seven Green-legged Partridge roosters were pooled and diluted with EK extender supplemented with 10-3, 10-6, or 10-9 M melatonin (control sample was prepared without supplementation with melatonin), and the pooled sample was subjected to cryopreservation. Post-thawed sperm motility was determined using the IVOS system, whereas plasma membrane status, acrosome integrity, mitochondrial activity, lipid peroxidation, chromatin status, and apoptotic-like changes were determined using fluorochromes and flow cytometry. Results, indicate post-thaw motile sperm cell count was greater (P < 0.05) in the frozen samples supplemented with melatonin (10-3 and 10-6 M) than the control sample. Although no significant differences were observed in post-thawed acrosomal integrity, plasma membrane integrity and mitochondrial activity were greater (P < 0.05) in samples frozen with melatonin (10-3 and 10-6 M) than that of the control sample. In addition, with supplementation of melatonin there was a decrease (P < 0.05) in the amount of lipid peroxidation, DNA fragmentation, and apoptotic-like changes after thawing. These results indicate there is a positive effect of melatonin supplementation in rooster semen freezing extenders on post-thaw sperm quality.

Associations of environment, health history, T-zone lymphoma, and T-zone-like cells of undetermined significance: A case-control study of aged Golden Retrievers.

BACKGROUND: T-zone lymphoma (TZL), an indolent disease in older dogs, comprises approximately 12% of lymphomas in dogs. TZL cells exhibit an activated phenotype, indicating the disease may be antigen-driven. Prior research found that asymptomatic aged Golden Retrievers (GLDRs) commonly have populations of T-zone-like cells (phenotypically identical to TZL) of undetermined significance (TZUS). OBJECTIVE: To evaluate associations of inflammatory conditions, TZL and TZUS, using a case-control study of GLDRs. ANIMALS: TZL cases (n = 140), flow cytometrically diagnosed, were identified through Colorado State University's Clinical Immunology Laboratory. Non-TZL dogs, recruited through either a database of owners interested in research participation or the submitting clinics of TZL cases, were subsequently flow cytometrically classified as TZUS (n = 221) or control (n = 147). METHODS: Health history, signalment, environmental, and lifestyle factors were obtained from owner-completed questionnaires. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated using multivariable logistic regression, obtaining separate estimates for TZL and TZUS (versus controls). RESULTS: Hypothyroidism (OR, 0.3; 95% CI, 0.1-0.7), omega-3 supplementation (OR, 0.3; 95% CI, 0.1-0.6), and mange (OR, 5.5; 95% CI, 1.4-21.1) were significantly associated with TZL. Gastrointestinal disease (OR, 2.4; 95% CI, 0.98-5.8) had nonsignificantly increased TZL odds. Two shared associations for TZL and TZUS were identified: bladder infection or calculi (TZL OR, 3.5; 95% CI, 0.96-12.7; TZUS OR, 5.1; 95% CI, 1.9-13.7) and eye disease (TZL OR, 2.3; 95% CI, 0.97-5.2; TZUS OR, 1.9; 95% CI, 0.99-3.8). CONCLUSIONS AND CLINICAL IMPORTANCE: These findings may elucidate pathways involved in TZUS risk and progression from TZUS to TZL. Further investigation into the protective association of omega-3 supplements is warranted.

Melatonin enhances responsiveness to Dichelobacter nodosus vaccine in sheep and increases peripheral blood CD4 T lymphocytes and IgG-expressing B lymphocytes.

The immunomodulatory functions mediated by melatonin support its use as vaccine adjuvant. Previously, we have demonstrated that melatonin enhances antibody responses in sheep vaccinated against Dichelobacter nodosus. Here, we analyze the effect of melatonin on T and B lymphocyte subsets in peripheral blood of sheep vaccinated against D. nodosus. We also compare the use of melatonin in implants and in injections. Melatonin administration either as implants or by injection produced higher antibody titers against A1 and C serotypes compared to those animals that received only the vaccine. These results support the use of melatonin as an adjuvant in vaccination against D. nodosus. Firstly, melatonin induces higher antibody titer than the vaccine alone, secondly, melatonin increase IgG+ B lymphocytes and CD4+ T lymphocytes in vaccinated sheep. These results suggest that melatonin enhances T CD4 cell activation and subsequently secondary humoral immune responses. Further studies are required to determine the mechanism underlining the immunomodulatory role of melatonin in the context of vaccination.

Anticancer effects of high-dose ascorbate on canine melanoma cell lines.

The use of the well-known powerful antioxidant ascorbate has recently become more widespread in human medicine. Intravenous administration of high-dose ascorbate has been demonstrated to exert anticancer effects. It has resulted in effective cell death in vitro and inhibition of tumour growth in vivo. The aim of the current study was to evaluate the effects of high-dose ascorbate on canine melanoma in vitro. Four canine melanoma cell lines, UCDK9M1, UCDK9M3, UCDK9M4 and UCDK9M5 were treated with ascorbate for 2 hours at a range of millimolar concentrations (0-20 mM) to investigate the resulting effects on cell viability. All four canine melanoma cell lines exhibited reduced viability in a dose-dependent manner. Further investigation demonstrated that high-dose ascorbate induced apoptosis via the activation of Bax. These findings suggest that high-dose ascorbate has an anticancer effect on canine melanoma cell lines in vitro. With regard to clinical application, further in vivo investigation should be conducted.

The impact of Aloe vera and licorice extracts on selected mechanisms of humoral and cell-mediated immunity in pigeons experimentally infected with PPMV-1.

BACKGROUND: The aim of the study was to evaluate the impact of herbal extracts on selected immunity mechanisms in clinically healthy pigeons and pigeons inoculated with the pigeon paramyxovirus type 1 (PPMV-1). For the first 7 days post-inoculation (dpi), an aqueous solution of Aloe vera or licorice extract was administered daily at 300 or 500 mg/kg body weight (BW). The birds were euthanized at 4, 7 and 14 dpi, and spleen samples were collected during necropsy. Mononuclear cells were isolated from spleen samples and divided into two parts: one part was used to determine the percentage of IgM+ B cells in a flow cytometric analysis, and the other was used to evaluate the expression of genes encoding IFN-γ and surface receptors on CD3+, CD4+ and CD8+ T cells. RESULTS: The expression of the IFN-γ gene increased in all birds inoculated with PPMV-1 and receiving both herbal extracts. The expression of the CD3 gene was lowest at 14 dpi in healthy birds and at 7 dpi in inoculated pigeons. The expression of the CD4 gene was higher in uninoculated pigeons receiving both herbal extracts than in the control group throughout nearly the entire experiment with a peak at 7 dpi. A reverse trend was observed in pigeons inoculated with PPMV-1 and receiving both herbal extracts. In uninoculated birds, increased expression of the CD8 gene was noted in the pigeons receiving a lower dose of the Aloe vera extract and both doses of licorice extracts. No significant differences in the expression of this gene were found between inoculated pigeons receiving both herbal extracts. The percentage of IgM+ B cells did not differ between any of the evaluated groups. CONCLUSIONS: This results indicate that Aloe vera and licorice extracts have immunomodulatory properties and can be used successfully to prevent viral diseases, enhance immunity and as supplementary treatment for viral diseases in pigeons.

Responses to dietary levels of methionine in broilers medicated or vaccinated against coccidia under Eimeria tenella-challenged condition.

BACKGROUND: Coccidiosis is a prevalent problem in chicken production. Dietary addition of coccidiostats and vaccination are two approaches used to suppress coccidia in the practical production. Methionine (Met) is usually the first limiting amino acid that plays important roles in protein metabolism and immune functions in chickens. The present study is aimed to investigate whether increasing dietary Met levels will improve the anticoccidial effects in broilers medicated or vaccinated against coccidia under Eimeria (E.) tenella-challenged condition. Two thousand male Partridge Shank broiler chicks were obtained from a hatchery. After hatch, birds were weighed, color-marked and allocated equally into two anticoccidial treatments, namely medicated and vaccinated groups. Chicks were either fed, from 1 d of age, diets containing coccidiostat (narasin) or diets without the coccidiostat but were inoculated with an anticoccidial vaccine at 3 d of age. At 22 d of age, 1080 chicks among them were randomly allocated evenly into 6 groups under a 2 × 3 treatment with 2 anticoccidial programs and 3 dietary methionine (Met) levels. Chicks medicated or vaccinated against coccidia were fed diets containing 0.45%, 0.56% or 0.68% of Met from 22 to 42 d of age. All chicks were orally introduced with an amount of 5 × 104 sporulated oocysts of E. tenella at 24 d of age. The growth performance, serum anti-oxidative indexes, intestinal morphology, cecal lesion scores, fecal oocyst counts and immune parameters were measured. RESULTS: The results showed increasing dietary Met level from 0.45% to 0.56% and 0.68% improved weight gain and feed conversion of broilers medicated against coccidia. In contrast, higher dietary levels of Met did not improve growth performance of the vaccinated chickens. Higher Met levels helped the medicated chickens resist E. tenella infection, as indicated by improved intestinal morphology and immune functions as well as decreased cecal lesion and fecal oocyst counts. CONCLUSIONS: Anticoccidial vaccination is a better strategy for controlling coccidiosis than feeding narasin, due to not only greater growth performance, but also the lower Met supplementation. Furthermore, higher dietary Met levels improved growth performance of chickens medicated rather than vaccinated against coccidia under E. tenella-challenged condition.

Deep insemination with sex-sorted Cashmere goat sperm processed in the presence of antioxidants.

Flow cytometrically sex-sorted sperm have been widely used for improving reproductive management in the dairy industry. However, the industrial application of this technology in other domestic species is largely limited by the lower fertility after insemination. The aim of this study was to investigate effects of antioxidant supplementation during the sex-sorting and freezing process on the quality and functions of sorted sperm from Liaoning Cashmere goats. We tested the effects of antioxidant supplementation during sex-sorting and freezing process, including ascorbic acid-2-glucoside AA-2G, glutathione, melatonin and vitamin C (VC), on the quality and functions of sex-sorted fresh and frozen-thawed sperm. Based on these experiments, we performed deep insemination with sex-sorted sperm using our improved strategy, in comparison to unsorted sperm. In Experiment 1, compared with control group and other antioxidants, AA-2G supplementation significantly alleviated the degradation of motility and viability of fresh sperm after sorting and showed the highest percentage of sperm with normal morphology. In addition, AA-2G supplementation showed an evident protection against the sorting process-induced membrane and acrosome damage. In Experiment 2, AA-2G supplementation was most effective in protecting motility, while melatonin supplementation appears to facilitate the degradation of quality of frozen-thawed sex-sorted sperm. In Experiment 3, we performed deep insemination with sperm that were sorted and frozen in the presence of AA-2G and obtained a satisfying pregnancy rate comparable to that from unsorted sperm. The results showed that AA-2G supplementation efficiently protects quality and function of both fresh and frozen-thawed sex-sorted sperm of Cashmere goats, thus obtaining a satisfying pregnancy outcome.

Randomized placebo-controlled study of the effects of Yunnan Baiyao on hemostasis in horses.

OBJECTIVE To determine effects of oral administration of Yunnan Baiyao on platelet activation, coagulation, and fibrinolysis in healthy horses. ANIMALS 12 healthy adult horses. PROCEDURES In a randomized blinded crossover study that included a 4-week washout period between treatments, horses were orally administered a paste containing Yunnan Baiyao (15 mg/kg) or placebo at 12-hour intervals for 3 days. Blood samples were collected before start of treatment (time 0) and at 24 and 72 hours for a CBC, measurement of fibrinogen concentration, coagulation screening tests, and a panel of assays to assess platelet activation (including ADP- and collagen-induced aggregation and closure times, flow-cytometric variables of platelet-leukocyte aggregates, platelet membrane P-selectin and phosphatidylserine expression, and microparticle release), von Willebrand factor (vWF) concentration, and cofactor activity. In addition, thrombelastography was used to evaluate fibrin formation in tissue factor-activated whole blood and plasma and to assess tissue plasminogen activator-induced plasma fibrinolysis. For each treatment, values obtained before and 72 hours after start of administration were compared by use of Wilcoxon signed rank tests. RESULTS Yunnan Baiyao treatment had no significant effect on any hemostatic variable, compared with results for the placebo treatment. CONCLUSIONS AND CLINICAL RELEVANCE Administration of Yunnan Baiyao at a dosage typically used in clinical practice had no effect on in vitro measures of platelet or vWF function and no enhancement of fibrin-clot formation or stability. Any hemostatic actions of Yunnan Baiyao may require higher dosages or result from cell-surface interactions at sites of vascular and tissue injury not examined in this study.

In vitro susceptibility of Brachyspira hyodysenteriae to a commercial citrus fruit extract.

Brachyspira hyodysenteriae is the main etiological agent of swine dysentery (SD). Nowadays, treatment and control of SD is increasingly difficult due to the emergence of antimicrobial resistance together with the restrictions on the use of antibiotics in veterinary practice. The aim of this study was to evaluate, as an alternative in the control of this disease, the antimicrobial activity and the main mechanism of action of BIOCITRO, a citrus extract commercialized as raw material and used as feed additive, against B. hyodysenteriae. Ten isolates of B. hyodysenteriae were used to assess the minimum inhibitory and minimum bactericidal concentrations (MIC and MBC) of BIOCITRO by broth microdilution method. Moreover, stationary phase cultures of two B. hyodysenteriae strains were subjected for 90min to four different concentrations of BIOCITRO and compared with the untreated controls by flow cytometry (FC), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The results showed that BIOCITRO has a relevant bacteriostatic and bactericidal effect against B. hyodysenteriae with MIC and MBC values ranging from 32 to 128partspermillion (ppm). It induces damage in at least 35% and 76% of the bacterial cells when exposed to 128 and 256ppm of BIOCITRO respectively as revealed by the intake of propidium iodide by FC. Relevant changes in the structure of the bacterial cells were observed by SEM and confirmed by FTIR. According to these results, BIOCITRO seems to be a satisfactory alternative to the use of antibiotics in the control of SD.

Molecular markers of putative spermatogonial stem cells in the domestic cat.

Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43 days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.

Comparison of gene expression profiles of T cells in porcine colostrum and peripheral blood.

OBJECTIVE To compare gene expression patterns of T cells in porcine colostrum and peripheral blood. ANIMALS 10 multiparous sows. PROCEDURES Cytotoxic and CD4-CD8 double-positive T cells were separated from porcine colostrum and peripheral blood. Total RNA was extracted. The cDNA prepared from RNA was amplified, labeled, fragmented, and competitively hybridized to DNA microarray slides. The DNA microarray data were validated by use of a real-time reverse-transcription PCR assay, and expression of the genes FOS, NFKBI, IFNG, CXCR6, CCR5, ITGB2, CCR7, and SELL was assessed. Finally, DNA microarray data were validated at the protein level by use of flow cytometry via expression of c-Fos and integrin ß-2. RESULTS Evaluation of gene expression profiles indicated that in contrast to results for peripheral blood, numerous cell-signaling pathways might be activated in colostrum. Profile analysis also revealed that FOS and NFKBI (genes of transcription factors) were involved in most cell-signaling pathways and that expression of these genes was significantly higher in colostral T cells than in peripheral blood T cells. Furthermore, CCR7 and SELL (genes of T-cell differentiation markers) in colostral T cells had expression patterns extremely similar to those found in effector or effector memory T cells. CONCLUSIONS AND CLINICAL RELEVANCE All or most of the T cells in colostrum had an effector-like phenotype and thus were more activated than those in peripheral blood. This gene expression profile would enable T cells to migrate to mammary glands, be secreted in colostrum, and likely contribute to passive immunity provided by sows to newborn pigs.

Apoptosis inhibitor of macrophage (AIM) reduces cell number in canine histiocytic sarcoma cell lines.

Apoptosis inhibitor of macrophage (AIM) is initially reported to protect macrophages from apoptosis. In this study, we determined the effect of AIM on the macrophage-derived tumor, histiocytic sarcoma cell lines (HS) of dogs. Five HS and five other tumor cell lines were used. When recombinant canine AIM was applied to non-serum culture media, cell numbers of all the HS and two of other tumor cell lines decreased dose-dependently. The DNA fragmentation, TUNEL staining and flow cytometry tests revealed that AIM induced both of apoptosis and cell cycle arrest in the HS. Although AIM is known as an apoptosis inhibitor, these results suggest that a high dose of AIM could have an opposite function in HS and some tumor cell lines.

Reduced glutathione addition improves both the kinematics and physiological quality of post-thawed red deer sperm.

The potential protective effect of reduced glutathione (GSH) and trolox (TRX), an analogue of vitamin E, supplementation during in vitro culture (2h, 39°C) of electroejaculated frozen/thawed red deer sperm was investigated. Cryopreserved sperm were thawed and incubated with no additive (Control) and 1mM or 5mM of each antioxidant to find out whether these supplementations can maintain the sperm quality, considering the use of thawed samples for in vitro techniques such as in vitro fertilisation (IVF), sperm sex sorting or refreezing. The effect of GSH on sperm motility was positive compared to TRX which was negative (P<0.001). After 2h of incubation at 39°C, use of GSH improved motility while TRX supplementation reduced sperm motility compared with Control samples without antioxidant. Use of TRX at both concentrations (1 and 5mM; TRX1 and TRX5) resulted in lesser percentages of apoptotic sperm (12.4±1.1% and 11.7±0.9%) than GSH1, GSH5 (15.2±1% and 14.6±1.1%) and Control samples (16.9±1.2%) (P<0.001). Use of GSH at both concentrations (1 and 5mM) resulted in greater mitochondrial activity as compared with findings for the Control, TRX1 and TRX5 groups. Results of this study indicate that GSH is a suitable supplement for electroejaculated red deer sperm. It would be necessary to conduct fertility trials (in vivo and in vitro), to assess whether GSH supplementation of thawed red deer sperm could improve fertility rates.

Hepatoprotective effects of a Chinese herbal formulation, Yingchen decoction, on olaquindox-induced hepatopancreas injury in Jian carp (Cyprinus carpio var. Jian).

In order to identify effective hepatoprotective herbs for clinical application in fish farming, 200 mg/kg olaquindox (OLA) was added to a basal diet (group 1, control) to form OLA diet (group 2), then 1.35, 2.7 and 5.4 % (w/w) of a Chinese herbal formulation, Yingchen decoction (YCD), were added to the OLA diet to form three additional diets for groups 3, 4 and 5, respectively. A total of 375 juvenile Jian carp (Cyprinus carpio var. Jian) (52.12 ± 2.95 g/tail) were divided into five groups (triplicates per group) and fed the five diets mentioned above, respectively, for 6 weeks. At the termination of feeding experiment, serum biochemical indexes, viability of hepatocytes and the hepatopancreas microstructure for each group were detected and observed. The results showed that serum ALT and AST in group 2 were significantly higher than the control (P < 0.05). Plasma membranes hepatocyte nuclei in group 2 were found to be mostly indistinct, compared to group 1, and gradually recovered with the increasing supplementation of YCD in group 3, 4 and 5. The viability of isolated hepatocytes in group 2 was the lowest and gradually recovered with the increasing supplementation of YCD in group 3, 4 and 5. The results suggest that YCD protected the Jian carp hepatopancreas against injury from OLA, and that 5.4 % YCD would be the optimum dosage in a Jian carp diet.

The phenotype and activation status of T and NK cells in porcine colostrum suggest these are central/effector memory cells.

In pigs, the epitheliochorial placenta does not allow transfer of maternally derived antibodies or immune cells to the fetus. Thus, piglets are dependent on intake of colostrum for acquisition of passive immunity during the neonatal period. As well as immunoglobulin G (IgG), cellular components of colostrum, mainly lymphocytes, can enter the systemic circulation and secondary lymphoid organs of the neonate. In order to understand the function and immunological role of these cells, a flow cytometric study was undertaken to characterise the cellular profile and phenotype of T cells and NK cells present in porcine colostrum. The results indicated that the greatest numbers of lymphocytes were found on the first day of lactation. The predominant cell types in colostrum were CD8(+) single positive T cells (53.6%), followed by CD4(+)CD8(+) double positive T cells (21.1%), CD2(+)CD8(+) γδ T cells (15.0%) and NK cells (13.5%). CD4(+) single positive T cells (4.4%) and other γδ T cell subpopulations (1.8% CD2(-)CD8(-) and 0.4% CD2(+)CD8(-)) were present in colostrum at low levels. Although the profile of the T cell subpopulations during the first 3 days of lactation remained constant, the absolute numbers of T and NK cells decreased significantly in the first few hours of lactation. Expression of CCR7, CD11b, CD25, CD45RA and MHC class II was used to assess the activation status of T and NK cells in colostrum. T cell subpopulations expressed markers consistent with an effector memory phenotype, indicating that these were antigen-experienced cells. The phenotype of colostral T and NK cells suggests a role in mucosal immunity and potentially in transfer of passive immunity from sow to piglet.

Development of a method for isolating bovine colostrum mononuclear leukocytes for phenotyping and functional studies.

The present study reports a method for isolating bovine colostrum mononuclear cells (CMC) for phenotyping and functional studies. As well as being an important source of immunoglobulins, colostrum also contains leukocytes that may be of greater importance for passive immunity than has previously been thought. Different protocols have been reported for isolating leukocytes from bovine colostrum, although none of these have been validated, and phenotypic analysis of cell populations has not always been performed. In this study, bovine CMC were isolated by density gradient centrifugation. Cell populations were identified by flow cytometry using antibodies against selected bovine cell surface markers and the proliferative capacity of these cells was determined using a (3)H-thymidine proliferation assay. The mean cell count of isolated CMC was 3 × 10(4) and 1 × 10(5) per mL colostrum for the samples used in the flow cytometric assay and the proliferation assay, respectively. A mean of 25.4 ± 17.1% CMC were identified as T lymphocytes, 2.9 ± 3.0% as B lymphocytes and 32.7 ± 13.7% as macrophages. In terms of proliferation, the mean counts per minute were 4.3 × 10(3) and 1.8 × 10(4) for cells cultured in medium only or in the presence of concanavalin A, respectively, showing that CMC are viable and capable of responding to mitogen stimulation. Isolation of CMC and the subsequent phenotypic analysis of the different subpopulations were repeatable, with agreement indices varying between 0.5 and 1.0. Agreement indices for the proliferation assay were estimated at 0.8.

Immunization with recombinant 3-1E protein in AbISCO®-300 adjuvant induced protective immunity against Eimeria acervulina infection in chickens.

Immunostimulating complexes (ISCOMs), a kind of novel antigen presenting system, could enhance immune protection by antigen presentation. AbISCO®-300 comprising purified saponin, cholesterol and phosphatidyl choline is an effective ISCOM adjuvant. To evaluate the immune protection of recombinant 3-1E protein against Eimeria acervulina infection, chickens were immunized with recombinant 3-1E protein in combination with AbISCO®-300 or recombinant 3-1E protein alone in this study. The protective immunity was assessed with body weight gain, fecal oocyst output, detection of intestinal IgA positive cells and percentages of CD3(+), CD4(+) or CD8(+) intestinal intraepithelial lymphocytes (IELs). Chickens vaccinated with different doses of recombinant 3-1E protein plus AbISCO®-300 showed higher percentages of CD3(+), CD4(+), and CD8(+) intestinal IELs, increased positive expression rate of intestinal IgA, increased body weight gains and decreased oocyst shedding compared with recombinant 3-1E protein-only vaccinated groups. The results showed that immunization with various doses of the recombinant 3-1E protein in AbISCO®-300 adjuvant enhanced immune protection against avian coccidiosis.

Validation of Sysmex XT-2000iV generated quantitative bone marrow differential counts in untreated Wistar rats.

BACKGROUND: Preclinical drug trials frequently require assessment of bone marrow toxicity in animals to evaluate hematopoietic safety. Since the gold standard, cytologic evaluation, is time consuming and requires highly trained individuals, automated methods remain intriguing. OBJECTIVE: The Sysmex XT-2000iV hematology analyzer allows user-developed customizable gating. This study was conducted to validate the gating of bone marrow cell populations in Sysmex cytograms from untreated rats. METHODS: B- and T-lymphocytes and myeloid cells were experimentally depleted from Charles River Wistar Han IGS (CRL: WI [Han]) rat whole bone marrow suspension using a magnetic cell sorting (MACS) method. The positively and negatively selected populations were used to verify select gates within the Sysmex cytogram. Intra- and inter-animal precision, comparability between right and left femur, as well as agreement with microscopic myelograms based on 500 counted cells, were determined. RESULTS: Intra-sample precision and right-to-left femur comparability confirmed that gating was reproducible and stable. In 50 tested rats, myeloid to erythroid ratios (M:E) were 1.32 ± 0.33 in males and 1.38 ± 0.29 in females by Sysmex compared to 1.36 ± 0.32 in males and 1.42 ± 0.32 in females by microscopic evaluations. Bland-Altman differences between methods was ≤ ± 0.35 units for M:E, ≤ 5.4% for maturing myeloid cells, ≤ 3.4% for proliferating myeloid cells, ≤ 6.0% for maturing myeloid cells, ≤ 3.4% for proliferating myeloid cells, and ≤ 4.1% for lymphocytes. CONCLUSIONS: In untreated control Charles River Wistar Han IGS (CRL: WI [Han]) rats, the Sysmex XT-2000iV produced an automated M:E and 5-part differential count equivalent to microscopic differential counts.

Effect of vitamin C on growth of caprine spermatogonial stem cells in vitro.

The genetic manipulation of spermatogonial stem cells (SSCs) can be used for the production of transgenic animals in a wide range of species. However, this technology is limited by the absence of an ideal culture system in which SSCs can be maintained and proliferated, especially in domestic animals like the goat. The aim of this study therefore was to investigate whether the addition of vitamin C (Vc) in cell culture influences the growth of caprine SSCs. Various concentrations of Vc (0, 5, 10, 25, 40, and 50 µg/mL(-1)) were added to SSC culture media, and their effect on morphology and alkaline phosphatase activity was studied. The number of caprine SSC colonies and area covered by them were measured at 10 days of culture. The expression of various germ cell and somatic cell markers such as VASA, integrins, Oct-4, GATA-4, α-SMA, vimentin, and Thy-1 was studied to identify the proliferated cells using immunostaining analyses. Further, the intracellular reactive oxygen species (ROS) level was measured at the 3rd, 6th, and 9th day after culture, and expression of Bax, Bcl-2, and P53, factors involved in the regulation of apoptosis, were analyzed on the 7th day after culture using reverse transcription polymerase chain reaction and quantitative real-time polymerase chain reaction. The results showed that the SSCs formed compact colonies and had unclear borders in the different Vc-supplemented groups at 10 days, and there were no major morphologic differences between the groups. The number and area of colonies were both the highest in the 40 µg/mL(-1) Vc group. Differential expression of markers for germ cells, undifferentiated spermatogonia, and testis somatic cells was observed. Cultured germ cell clumps were found to have alkaline phosphatase activity regardless of the Vc dose. The number of Thy-1- and Oct-4-positive cells was the most in the 40 µg/mL(-1) Vc group. Moreover, the level of ROS was dependent on the Vc dose and culture time. The Vc dose 40 µg/mL(-1) was found to be optimum with regard to decreasing ROS generation, and increasing the expression of the antiapoptotic gene Bcl-2 and decreasing the expression of the proapoptotic genes Bax and P53. In conclusion, the addition of 40 µg/mL(-1) Vc can maintain a certain physiological level of ROS, trigger the expression of the antiapoptosis gene Bcl-2, suppress the proapoptotic gene P53 and Bax pathway, and further promote the proliferation of caprine SSCs in vitro.