Ovine Serum Research Articles

Concentration Dependences of Macromolecular Sizes in Aqueous Solutions of Albumins

On the basis of experimental data for the shear viscosity in the aqueous solutions of ovine serum albumin and using the cellular model describing the viscosity in aqueous solutions, the concentration dependences of the effective radius of ovine serum albumin macromolecules in the aqueous solutions within a concentration interval of 3.65–25.8 wt% and a temperature interval of 278–318 K at the constant pH = 7.05 are calculated. The concentration and temperature dependences of the effective radii of ovine, bovine, and human serum albumin macromolecules are compared. It is shown that they are partially similar for the solutions of ovine and human serum albumins within concentration intervals of 0.12–0.49 vol% and 0.18–0.48 vol%, respectively, provided an identical acid-base balance (pH) in those solutions. The following conclusions are drawn: (i) the concentration dependences of the effective radii of structurally similar macromolecules of various albumins are similar, but provided an identical pH, and (ii) the dependence of the volume concentration of aqueous albumin solutions on the temperature at the constant radius of a macromolecule confirms the hypothesis about the existence of a dynamic phase transition in aqueous solutions at a temperature of 42 ∘C, at which the thermal motion of water molecules significantly changes.

Short communication: Characterizing ovine serum stress biomarkers during endotoxemia

Breeding stress-resilient livestock is a potential strategy to help mitigate the negative effect of environmental and pathogenic stressors. The hypothalamic-pituitary-adrenal axis and immune system are activated during stress events and release mediators into the circulation that help restore physiological homeostasis. The purpose of this study was to assess a comprehensive set of circulatory mediators released in response to an acute immune stress challenge to identify candidate biomarkers that can be used for the selection of stress-resilient animals. Fifteen female lambs were stress challenged with an intravenous bolus of lipopolysaccharide (LPS; 400 ng/kg), and blood was collected from the jugular vein at 0, 2, 4, and 6 h after LPS challenge to identify and monitor candidate stress biomarkers; temperature was also recorded over time. Biomarker responses were evaluated with a repeated-measures model to compare time points with baseline values. As expected, all sheep had a monophasic febrile response to LPS challenge, and cortisol increased and returned to baseline by 6 h. The cytokines tumor necrosis factor-α, IL-6, IFN-γ (proinflammatory), and IL-10 (anti-inflammatory) increased, but only tumor necrosis factor-α returned to baseline during the monitoring period. The cytokines IL-1α, IL-1β, IL-17α (proinflammatory), and IL-4 (anti-inflammatory) did not respond to LPS challenge. All chemokines (CCL2, CCL3, CCL4, CXCL10, and IL-8) responded to LPS challenge; however, only CCL2, CCL3, CCL4, and CXCL10 increased over time, and only CCL3, CCL4, and CXCL10 returned to baseline during the monitoring period. MicroRNA (miR-145, miR-233, and miR-1246) also increased and remained elevated during the study. In summary, the LPS challenge induced a strong stress response in Rideau-Dorset sheep that could be monitored with a distinct profile of circulatory biomarkers.

Dietary supplementation with ovine serum immunoglobulin modulates correlations between mucin, microbiota and immunity proteins in the growing rat.

The objective of this study was to evaluate the relationship among the number of bacteria, number of goblet cells, gut mucin gene expression, mucin protein and immunity protein levels of rats fed a diet containing freeze-dried ovine Ig (FD). Sprague Dawley male rats were used in a 21-days study and were fed a basal control diet (BD; no Ig) and a test diet containing freeze-dried ovine Ig (FD). Diets were isocaloric and contained the same amount of the first limiting amino acids, methionine plus cysteine. Pearson's correlation analysis was conducted on the data (stomach, ileum and colon) obtained from individual rats (n=10) fed either casein-based diet (BD) or ovine serum Ig (FD) to evaluate the relationship between number of bacteria, number of goblet cells, gut mucin gene expression and gut mucin protein levels. Pearson's correlation analysis was then conducted with the data from the FD fed rats to evaluate the relationship among the above said variables. In the stomach content, a significant (p<.05) correlation was found between the Muc5Ac gene expression and mucosal mucin protein. In the ileum and colon, a significant (p<.05) correlation was observed among the mRNA levels of mucin (Muc2 and Muc4) genes. There was also evidence of a strong relationship (p<.05) between digesta mucin and mucosal mucin protein concentrations. A negative correlation of mucosal IgA protein concentration with total Lactobacillus (in ileum and colon) and total bacteria (in the ileum) was not evident with FD fed rats when compared to the results obtained using both BD and FD fed rats. In conclusion, this study suggests that feeding freeze-dried ovine Ig in growing rats results in a strong correlation between the number of bacteria, mucin and immunity proteins.

Development of a Lateral Flow Kit for Detection of IgG and IgM Antibodies Against Rift Valley Fever Virus in Sheep

Rift Valley Fever (RVF) is a viral zoonosis that primarily affects animals but also has the capacity to infect humans. The disease also results in significant economic losses due to death and abortion among RVF-infected livestock. A lateral flow device (LFD) was developed for the rapid detection of IgG and IgM anti-RVF antibodies in infected and vaccinated ovine sera. The conjugated RVF antigen with nano-gold was used as a detector antigen and was laid on a conjugated pad. The staphylococcal protein A was used as the capture complex (antibodies IgG-antigen conjugated with nanogold) at the first test line of device and anti-IgM antibodies were used as the capture complex (antibodies IgM-antigen conjugated with nanogold) at the second test line of device. When compared with RVF specific ELISA, The sensitivity, specificity, and accuracy of developed LFD for detection of IgG were found to be 88.2, 96.9 and 94.0%, respectively, while the same for detection of IgM was found to be 100, 96, and 96%, respectively.

A time-resolved fluorescence immunoassay for the detection of anti-Neospora caninum antibodies in sheep

Neospora caninum is a protozoan parasite (Phylum Apicomplexa) that has been recently suggested as a relevant cause of reproductive disorders in small ruminants. The aim of the present study is to develop and validate a new serological test based on time resolved fluorescency using N. caninum GRA7 recombinant antigen (GRA7-TRFIA) for the detection of N. caninum antibodies in sheep. A total of 346 serum samples (208 from experimentally infected sheep, 117 from a dairy farm with a previous history of Neospora-associated abortion, and 21 negative sera) were used. The validation of the new assay was performed by the evaluation of assay precision, analytical sensitivity (Se), accuracy and cross reactivity. In the experimentally infected sheep, antibody kinetics was compared between GRA7-TRFIA and an in house N. caninum tachyzoite soluble extract-based ELISA (NcSALUVET ELISA) by Wilcoxon matched-pairs signed rank test. The cut-off and diagnostic Se and specificity (Sp) of GRA7-TRFIA was estimated by ROC analysis with field samples. In addition, concordance and correlation between GRA7-TRFIA and a commercial ELISA and NcSALUVET ELISA were assessed by kappa value and Spearman correlation coefficient, respectively.Overall, GRA7-TRFIA showed an adequate precision, analytical Se and accuracy to detect anti-N. caninum antibodies in ovine serum, and no cross reactivity with the closely related protozoan Toxoplasma gondii. In naturally infected sheep, 100% Se and 95.35% Sp were obtained for a cut-off point of 62.68 Units of Fluorometry for N. caninum (UFN). Moreover, GRA7-TRFIA allowed earlier detection of N. caninum infection than NcSALUVET ELISA in experimentally infected sheep.

Impact of oral immunoglobulins on animal health-A review.

Immunoglobulin (Ig) is the one of the main anti-infective components of blood, colostrum and breast milk. It is the unique glycoprotein that defends the body from harmful bacteria, viruses and other environmental pathogens by either binding to them or by forming an encapsulating barrier. The expansion of antimicrobial and immunomodulatory products from natural sources for dietary supplementation in both animals and humans is an ever growing and thriving area of research. Purified Ig from sheep serum (ovine serum Ig) is one such candidate product. Recent work has shown the various biological effects of oral Ig in different animal models including its effect on growth, immunity, intestinal growth and gut barrier function. The objective of this paper is to review the results of recent studies demonstrating the effects of oral Ig in both pathogenic and non-pathogenic animal models and to suggest a possible mechanism of its action. Overall, purified oral Ig improves growth of healthy (and challenged) rats and defends against enteric infection by immunomodulation, mucin protein and/or modification of commensal microbial composition. The findings contribute to knowledge of how orally administered ovine Ig can influence and enhance key indicators of gut function and overall growth performance in an animal model.

Epidemiological Investigation of Toxoplasma gondii Infections in Commercial Sheep Flock in an Endemic Area for Ocular Toxoplasmosis in Southern Brazil.

Toxoplasma gondii is a widely distributed parasite and of great importance to human and animal health. The objective of this study was to assess the prevalence of T. gondii antibodies and risk factors associated with the infection in sheep in the Northwest region of the State of Rio Grande do Sul, Brazil; this region has a very high rate of human ocular toxoplasmosis. Ovine sera were tested by the modified agglutination test (cut-off 1:25). T. gondii antibodies were detected in 70.2% (224 of 319). According to the logistic regression, the most significant factors associated were age and cat access to food stock facility. Preventive measures are discussed to reduce the risk of transmission of this zoonosis.

Preparation of Anti‐Nonspecific Adsorption Chitosan‐Based Bovine Serum Albumin Imprinted Polymers with Outstanding Adsorption Capacity and Selective Recognition Ability Based on Magnetic Microspheres

AbstractBovine serum albumin (BSA)‐imprinted magnetic microspheres are successfully fabricated by a precipitation copolymerization, in which 2‐hydroxyethyl acrylate (HEA) and water‐soluble chitosan separately work as functional monomers and cross‐linking agents. Hydroxyl derived from HEA as well as hydroxyl, amino, and carboxyl groups, originating from chitosan on the surface of the prepared imprinted microspheres, can form hydrogen bonds and extra electrostatic interaction with proteins. The sulfobetaine methacrylate (SBMA) is introduced into the imprinted polymer as an anti‐protein segment to reduce the nonspecific adsorption and improve the selectivity. Detailed characterization demonstrates that the imprinted layer is about 10 nm, which is extremely beneficial for BSA identification and quality transmission. Furthermore, these microspheres also display an excellent selective adsorption of BSA (IF = 5.0). The results reveal that the prepared imprinted materials can be used as BSA biosensor or separation materials.

Extension of coarse-grained UNRES force field to treat carbon nanotubes

Carbon nanotubes (CNTs) have recently received considerable attention because of their possible applications in various branches of nanotechnology. For their cogent application, knowledge of their interactions with biological macromolecules, especially proteins, is essential and computer simulations are very useful for such studies. Classical all-atom force fields limit simulation time scale and size of the systems significantly. Therefore, in this work, we implemented CNTs into the coarse-grained UNited RESidue (UNRES) force field. A CNT is represented as a rigid infinite-length cylinder which interacts with a protein through the Kihara potential. Energy conservation in microcanonical coarse-grained molecular dynamics simulations and temperature conservation in canonical simulations with UNRES containing the CNT component have been verified. Subsequently, studies of three proteins, bovine serum albumin (BSA), soybean peroxidase (SBP), and α-chymotrypsin (CT), with and without CNTs, were performed to examine the influence of CNTs on the structure and dynamics of these proteins. It was found that nanotubes bind to these proteins and influence their structure. Our results show that the UNRES force field can be used for further studies of CNT-protein systems with 3–4 order of magnitude larger timescale than using regular all-atom force fields.Graphical abstractBovine serum albumin (BSA), soybean peroxidase (SBP), and α-chymotrypsin (CT), with and without CNTsᅟ

Development of recombinase polymerase amplification assays for the rapid detection of peste des petits ruminants virus

Peste des petits ruminants (PPR) is a severe infectious disease of small ruminants caused by PPR virus (PPRV). Rapid and sensitive detection of PPRV is critical for controlling PPR. This report describes the development and evaluation of a conventional reverse transcription recombinase polymerase amplification (RT-RPA) assay and a real-time RT-RPA assay, targeting the PPRV N gene. Sensitivity analysis revealed that the conventional RT-RPA assay could detect 852 copies of standard PPRV RNA per reaction at 95% probability within 20 min at 41 °C, and the real-time RT-RPA assay could detect 103 copies of RNA molecules per reaction at 95% probability. Specificity analysis showed that both assays have no cross-reactivity with nucleic acid templates prepared from other selected viruses or common pathogens. Clinical evaluation using 162 ovine and hircine serum and nasal swab samples showed that the performance of both the real-time RT-RPA assay and the conventional RT-RPA assay were comparable to that of real-time RT-PCR. The overall agreements between real-time RT-PCR and real-time RT-RPA, and conventional RT-RPA were 99.4% (161/162) and 98.8% (160/162), respectively. The R2 value of real-time RT-RPA and real-time RT-PCR was 0.900 by linear regression analysis. Our results suggest that both RT-RPA assays have a potential application in the rapid, sensitive and specific detection of PPRV.

Seroprevalence of Brucellosis in Small Ruminants Assayed by The Rose Bengal Test, Al-Jabal Al-Akhdar Libya

This study aimed at determining the prevalence of anti- Brucella antibodies in small ruminants in Al- Jabal Al- Akhdar area, Libya. Nine regions were selected for the investigation (Al- Goba, Al- Wasata, Side Kahled, Lamloda, Al-Hesha, Marawa, Al-Gagab, Gandola and Ain Mara). Seroprevalence was assayed using the Rose – Bengal Plate Test (RBPT). Four hundred blood samples were collected randomly from 247 sheep and 153 goats with a history of abortion and reproductive disorders, during the period from January 2015 to June 2016. Approximately 10 ml blood sample was taken from each animal, in vacutainers. Serum samples were separated and subjected to examination by the RBPT. Samples showing visible agglutination within 4 mins. were regarded as positive for anti- Brucella antibodies. Data were analyzed statistically by the Chi- square test using the SpSS software, at p ≤ 0.05 level of significance. Out of the 400 ovine and caprine sera tested, 125 (38%) were positive for anti – Brucella antibodies by the RBPT (Table 3). The rate of seropositivity was higher in goats (69.3%) than in sheep (18.6%) (Table 2). There were variations in seroreactivity from different regions. For instance, sera from Al- Hesha and Gandola exhibited 100% positivity, whereas those from both species in Al- Gagab were remarkably sero-negative (0%) (Table 3). Striking differences were shown by the sera from Gandola and Ain- Mara. Where all the caprine sera from Gandola were positive for anti – Brucella antibodies, all the 18 sera from Ain- Mara were serologically negative. Serum reactivity from both goats and sheep in other regions ranged between 60 and 83.3% in goats and 11.5 and 23.3% in sheep (Table 3). It can be concluded that the prevalence of anti- Brucella antibodies is high in small ruminants of Al- Jabal Al- Akhdar, Libya and may indicate a possible existence of Brucellosis in goats and sheep.

Validation of an improved Anaplasma antibody competitive ELISA for detection of Anaplasma ovis antibody in domestic sheep.

An accurate and simple-to-perform new version of a competitive ELISA (cELISA) kit that became commercially available in 2015 for testing of cattle for antibody to Anaplasma marginale was validated for detection of Anaplasma ovis antibody in domestic sheep. True positives and negatives were identified using nested PCR (nPCR) as the gold standard. Negative bovine control sera supplied with the kit were used to calculate % inhibition (%I), designated bovine control ELISA (BcELISA), and this was compared to %I calculated from negative ovine sera derived from hand-raised, pathogen-free sheep, designated ovine control ELISA (OcELISA). The receiver operating characteristics area under the curve was 1.0 with a p value <0.001 regardless of the source of the control sera. The cutoff values for negative BcELISA and OcELISA were <30%I and <27%I, respectively. Our work confirmed that this Anaplasma antibody cELISA kit version 2 can be used with the serum controls supplied in the kit to test for A. ovis antibody in domestic sheep. Furthermore, this work confirmed the historically high infection prevalence (>93%) at the U.S. Sheep Experiment Station (Dubois, Idaho), in spite of efforts to reduce the possibility for iatrogenic transmission there, suggesting high levels of tick-borne transmission.

Polychlorinated biphenyl and polybrominated diphenyl ether profiles in serum from cattle, sheep, and goats across California

It has been previously been shown by our lab and others that persistent organic pollutants, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs), are contaminants in milk produced for human consumption. To further this research we determined the concentration of 21 PCB and 14 PBDE congeners in livestock serum, mainly bovine, across California. Congeners were extracted from serum using solid phase extraction (SPE), cleaned up by silica cartridge and quantified using gas chromatography-triple quadruple mass spectrometry. We detected significant differences among species and the production class of cattle (beef or dairy). The sum of all 21 PCB congeners (ΣPCBs) in caprine and ovine sera had a mean value of 9.26 and 9.13 ng/mL, respectively, compared to 3.98 ng/mL in bovine sera. The mean value for the sum of all 14 PBDE congeners (ΣPBDEs) in caprine and ovine sera was 2.82 and 2.39 ng/mL, respectively, compared to 0.91 ng/mL in bovine sera. Mean ΣPCBs in dairy cattle was 5.92 ng/mL compared to 2.70 ng/mL in beef cattle. Mean ΣPBDEs in dairy cattle was 1.33 ng/mL compared to 0.70 ng/mL in beef cattle. There were no regional differences in the ΣPCBs or ΣPBDEs in cattle distributed across California. These results highlight the fact that livestock are still being exposed to these pollutants yet little is known about where this exposure may be coming from.

Multiple response optimization of heat shock process for separation of bovine serum albumin from plasma

ABSTRACTBovine serum albumin produced by heat shock from bovine plasma has been researched for the effects of sodium caprylate concentration [Cap], temperature (T), and pH on yield (Yield%) and purity (BSA%). Response surface methodology and desirability function approaches were applied to optimize its process. The best compromise solution was found with BSA% = 95.0 and Yield% = 28.5 for a [Cap] = 2 mM, T = 67.9°C, and pH = 5. A Monte Carlo simulation showed that it is possible to obtain excellent values for each individual response. However, a techno-economic feasibility study must be carried out to determine which one is the best option.

Replacement of serum with sericin in in vitro maturation and culture media: Effects on embryonic developmental competence of Sanjabi sheep embryo during breeding season

Sericin is a water-soluble component of silk and has been used as a biomaterial due to its antibacterial and ultraviolet radiation-resistant properties. This study was designed to evaluate the effect of sericin supplementation, as a serum replacement, in maturation and culture media on the meiotic competence of oocytes or in vitro culture of ovine embryos. In experiment 1, oocytes were matured in the presence of 10% fetal ovine serum (FOS), 0.1% polyvinyl alcohol (PVA) and different concentrations of sericin (0.1, 0.5, 1 and 2.5%), for 24 h. The addition of 0.5% sericin to maturation medium increased the rates of maturation to metaphase II of oocytes compared with those in cultures with 0.1% PVA. Following fertilization, blastocyst development was higher for oocytes matured with 0.5% of sericin compared with 0.1% PVA. However, the rates of nuclear maturation of oocytes and blastocyst development under FOS and 0.5% sericin were not significantly different. In experiment 2, presumptive zygotes were cultured in the presence of 10% FOS, 0.1% PVA and different concentrations of sericin (0.1, 0.5, 1 and 2.5%), for 7–8 days. The addition of 0.5% sericin to culture medium increased the blastocyst rate compared with those in cultures without sericin or addition of 0.1% PVA and 10% FOS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM and IVC in ovine oocytes and zygotes.

42 THE EFFECT OF VARIOUS CRYOPROTECTIVE AGENTS AND SLOW COOLING RATE ON VIABILITY OF SHEEP OVARIAN TISSUE

Priority objects of protection in agrobiocenoses are grades of cultural plants and local breeds of the cultivated animals. The most general criteria for preservation of local breeds are viability, adaptability, a state of health, reproductive abilities, and unique genetic polymorphism at the molecular and morphological levels. Therefore, the aim of this study was to compare the effectiveness of different cryoprotectors on morphology of ovine ovarian tissue cryopreserved by a passive cooling method. Ovarian tissue from 10 indigenous Chuyi breed was transported to the laboratory within 30 min at 32 to 34°C, divided into smaller pieces (2.0 × 1.2 × 1.0 mm), and randomly distributed into 4 groups: (1) control (fresh tissue), (2) pieces after freezing/thawing with 1.5 M ethylene glycol (EG); (3) pieces after freezing/thawing with 1.5 M propanediol (PROH); (4) pieces after freezing/thawing with 1.5 M dimethyl sulfoxide (DMSO). The ovarian pieces were placed in a cryovial and equilibrated sequentially in freezing medium containing 0.25, 0.75, and 1.5 M cryoprotectors with 0.5 M sucrose (5 min each), precooled at 4°C, and stored in a −80°C freezer for 24 h. Then, the cryovials containing the ovarian pieces were placed in liquid nitrogen and stored (for 2 months) until thawing. The ovarian pieces were cultured in vitro for 7 days in TCM-HEPES+ 10% native ovine serum (NOS) (Seisenbayeva et al. 2015 Reprod. Fertil. Dev. 28, 193–194). After 7 days of culture, we evaluated the effects of passive cooling methods with different cryoprotectors on ovarian tissue morphology by light microscopy after hematoxylin and eosin staining of tissue sections. The number of viable and damaged follicles was counted. All morphologically normal primordial, primary, and secondary follicles had healthy and intact oocytes, each containing a round nucleus and clearly visible nucleolus surrounded by well-organised granulosa cells without a pyknotic nucleus. Integrity rate of tissue after treatment was evaluated by Student’s test. In groups 1, 2, 3, and 4, the mean densities of follicles per 1 mm3 was 17.0 ± 4.6a, 16.2 ± 7.2b, 13.0 ± 5.1c, and 11.9 ± 4.8d, respectively (ab, ac, ad P &gt; 0.05). For these groups, respectively, 69.2 ± 8.2%a , 61.7 ± 8.6%b , 52.4 ± 8.8%c and 48.5 ± 8.8%d preantral follicles were morphologically normal (ab, ac, ad P &gt; 0.05). We did not find significant differences between groups. The analysis of comparative histology shows that 1.5 M EG is more effective on viability of ovarian follicles than 1.5 M DMSO or 1.5 M PROH.

Effect of degradation of poly(epsilon-caprolactone) films on functional properties for tissue engineering bioreactors

Event Abstract Back to Event Effect of degradation of poly(epsilon-caprolactone) films on functional properties for tissue engineering bioreactors Nazely Diban1, Sandra Sanchez-González1, Inmaculada Ortiz1 and Ana M. Urtiaga1 1 University of Cantabria, Department of Chemical and Biomolecular Engineering, Spain The regeneration of the central nervous system (CNS) is one of the major challenges that regenerative medicine faces nowadays. In order to promote in vitro neural cell regeneration, the use of artificial substrates may provide support for the neural cell growth and axon regeneration [1]. The scaffolds used in the tissue engineering bioreactors must provide sufficient mechanical stability and high transport of nutrients towards the cells and good removal of the metabolites during the whole lifetime of the in vitro cultures. The scaffold porous structure and hydrophilic character are important properties determining the mechanical and mass transport performance and the functionality of the scaffolds for this application. Scaffolds of biodegradable and biocompatible polymers have been proven as promising substrates to promote cell adhesion and proliferation [2]. The scaffolds must provide an adequate functionality in the bioreactor during suficient time so the regeneration of the tissue occurs simultaneously with the lost of functional properties of the scaffold due to degradation. In our previous work [2], flat Poly(ε-caprolactone) (PCL) films with adequate morphology to allow neuronal tissue growth were fabricated using a phase inversion casting technique and isopropanol (IPA) as coagulant (Figure 1). PCL is a polyester that degrades through a characteristic hydrolitical mechanism. In this work, we explore the effect of degradation on the structural (aspect, porosity, morphology), mechanical and transport properties (water and model ovine serum albumim solution transport) of the PCL films under the typical bioreactor conditions (37 ºC in a Phosphate Buffer Solution (PBS) at pH 7.4). The adequacy of the change of the functional properties to match the neural celular proliferation rate will be studied. Figure 1. SEM images of the cross section and surface of the PCL films and confocal images of U87 human glioblastoma static cell culture after 48 hours on the PCL films. All cell nuclei are shown in blue, and the actin cytoskeletal architecture is show in green. Financial support of the Cantabria Explora call through project JP03.640.69 is gratefully acknowledged

126 EFFECTIVE METHOD FOR IN VITRO CULTURE OF CRYOPRESERVED OVINE OVARIAN TISSUE

Today, ovarian tissue cryopreservation is used for preserving the reproductive function of women, as well as the genetic material of rare and endangered species or domestic animals breeds. In the last 20 years genetic diversity of farm animals breeds suffered considerable losses in Kazakhstan; therefore, genetic preservation of valuable local breeds is desirable. The aim of this study was to compare the effectiveness of different in vitro culture media on morphology of ovine ovarian tissue cryopreserved by a slow-freezing protocol with 1.5 M dimethyl sulfoxide (DMSO). Ovaries were collected from indigenous Chuyi breed and immediately transported to the laboratory at 30°C within 1 h. Ovaries were rinsed several times in PBS supplemented with antibiotics (75 mg L–1 of penicillin-G, 50 mg L–1 of streptomycin sulfate). In Hepes-buffered medium 199, halved and the medulla removed with curved iris. Using a scalpel, the cortex was cut into 5- × 3- × 1-mm strips. Ovarian strips were equilibrated sequentially in freezing medium containing 0.25, 0.75, and 1.5 M DMSO with 0.5 M sucrose (5 min each). Then, ovarian strips were frozen in plastic straws using a programmable freezer Planer Kryo-360 3,3 (Planer, UK) and cooled as follows: stabilised at 20°C for 5 min, cooled from 20°C to –70°C at 5°C min–1, seeded to the temperature –7°C, cooled again to –30°C at 0.3°C min–1, cooled to –150°C at 35°C min–1, and finally plunged into liquid nitrogen and stored for 10 days. The straws were thawed at room temperature for 1 min, and then immersed in a water bath at 37°C for 2 min, warmed at room temperature with Dulbecco’s PBS (DPBS), supplemented with 10% FCS and 0.75 M sucrose (15 min), then DPBS + 10% FCS (30 min), and finally placed in the culture media for 10 min. Fresh and frozen tissue pieces were randomly distributed into 12 groups for further culture: 1) TCM 199 + 10% FBS; 2) TCM 199 + 10% native ovine serum (NOS); 3) TCM-Hepes + 10% FBS; 4) TCM-Hepes + 10% NOS; 5) DMEM + 10% FBS; 6) DMEM + 10% NOS; with and without 7.5 mg mL–1 of FSH. After 7 days of culture, the effects of different culture media on ovarian tissue morphology was evaluated by light microscopy after hematoxylin and eosin staining of tissue sections. The best result was observed when frozen ovarian tissue was cultured in the presence of FSH. The best result was observed in group 3 and 4 with FSH. The percentages of normal primordial, primary, and preantral follicles were: 1) TCM 199 + 10% FBS + FSH = 53.5 ± 3.1, 39.7 ± 3.8a, 28.5 ± 3.2; 2) TCM 199 + 10% NOS + FSH = 49.4 ± 2.3a, 36.7 ± 3.3a, 25.3 ± 4.1; 3) TCM-Hepes + 10% FBS + FSH = 66.3 ± 2.5, 45.7 ± 3.9, 35.1 ± 3.8; 4) TCM-Hepes + 10% NOS + FSH = 86.5 ± 3.8b, 75.4 ± 4.2b, 45.7 ± 3.5; 5) DMEM + 10% FBS + FSH = 42.1 ± 3.5a, 33.7 ± 2.9a, 21.3 ± 4.9, 20.7 ± 3.9; 6) DMEM + 10% NOS + FSH = 41.3 ± 3.9a, 32.9 ± 2.5a; control group = 98.2 ± 1.1, 93.7 ± 1.7, 90.3 ± 1.9 (ab, P &lt; 0.01). The majority of follicles in groups without FSH were degenerated. In group 4) TCM-Hepes + 10% NOS without FSH, a damaged structure of primordial follicles was observed.

Evaluation of a bovine pregnancy-associated glycoprotein enzyme-linked immunosorbent assay kit for serological diagnosis of pregnancy in sheep

ABSTRACT: Pregnancy diagnosis is an important tool for farm management. Ultrasonography is the main technique used for pregnancy diagnosis in ewes. As an alternative, radioimmunoassay (RIA) allows accurate and early detection of pregnancy-associated glycoproteins (PAGs) in sheep blood. However, radioactive-based techniques, as RIA, have been increasingly inadvisable due to environmental risk. Homology between ovine and bovine PAGs is high, and ELISA kits used for PAGs detection in cattle are safer than RIA. Thus, this study aimed to evaluate the feasibility of PAGs detection for pregnancy diagnosis in sheep serum samples using an ELISA kit produced for cattle. The sensitivity and specificity of the ELISA kit were 93.5% and 98.9%, respectively, whereas positive and negative predictive values were 99.0% and 93.1%, respectively, in comparison to ultrasonography diagnostic (control). PAGs reached consistently detectable concentrations in ovine serum around 33 days after mating. Accuracy of the ELISA test was 96.1% from 33 days of pregnancy until lambing. After parturition, PAGs were still detectable seven days post-lambing. However, from 21 days post-parturition, PAGs from the previous pregnancy were no longer detected in serum samples. In conclusion, the bovine ELISA kit can accurately detect pregnancy in sheep 33 days following mating, while PAGs levels from the previous gestation are no longer detected from 21 days post-partum. The evaluated ELISA test is a reliable tool for pregnancy diagnosis in sheep at random stages and as a complementary exam at early gestation.

Selenium speciation in paired serum and cerebrospinal fluid samples of sheep

This study was performed to characterise selenium (Se) and Se species in cerebrospinal fluid (CSF) of sheep and its relation to the respective Se concentrations in serum. Paired samples from 10 adult sheep were used for the study. Five sheep were fed a diet with a marginal Se concentration of <0.05mg Se/kg diet dry weight (dw, Se–), and five animals were fed the same diet supplemented with sodium selenite revealing a concentration of 0.2mg Se/kg diet dw (Se+). The feeding strategy was conducted for two years; The results on metabolic effects were published previously. At the end of the feeding period, paired samples of serum and CSF were collected and analysed using ion exchange chromatography inductively coupled plasma–dynamic reaction cell–mass spectrometry (IEC-ICP-DRC-MS) technique for total Se concentration and concentrations of Se species. Albumin concentrations were analysed additionally.The feeding strategy caused significant differences (p<0.01) in serum Se concentrations with 33.1±5.11μg Se/l in the Se– group and 96.5±18.3μg Se/l in the Se+ group, respectively. The corresponding total Se concentrations in CSF were 4.38±1.02μg Se/l and 6.13±1.64μg Se/l in the Se– and the Se+ group, respectively, missing statistical significance (p=0.077). IEC-ICP-DRC-MS technique was able to differentiate the Se species selenoprotein P-bound Se (SePP), selenomethionine, glutathione peroxidase-bound Se (Se-GPx), selenocystine, thioredoxin reductase-bound Se, ovine serum albumin-bound Se (Se-OSA), SeIV and SeVI in ovine serum and CSF. Quantitatively, SePP is the main selenoprotein in ovine serum followed by Se-GPx. The CSF/blood ratio of albumin (QAlbumin) reflected a physiological function of the blood-CSF barrier in all sheep. QSe-species were higher than QAlbumin both feeding groups, supporting the hypothesis of local production of Se species in the brain. Significant positive regression lines for CSF vs. serum were found for albumin and Se-OSA only, suggesting a role of albumin to convey Se across the blood-CSF barrier. The ovine model, together with the IEC-ICP-DRC-MS technique to characterise the Se species, might be a worthwhile model for further studies as repeated sample collection as well as modification of the nutritional status is feasible and effective.