Comparison of Skin Prick Tests with In Vitro Allergy Tests in the Characterization of Horses with Recurrent Airway Obstruction

We intended to identify relevant immunoallergic factors and to compare skin prick tests (SPTs) and in vitro allergy tests in the characterization of horses with recurrent airway obstruction (RAO), so as to ascertain that SPTs perform better. Forty Lusitano/cross-Lusitano horses (30 RAO cases and 10 healthy control horses)—a very valuable autochthonous breed—were studied. Clinical history, thoracic radiography, respiratory tract endoscopy, and bronchoalveolar lavage were used for diagnosis. Serum samples of all 40 horses and undiluted bronchoalveolar lavage fluid samples of 21 RAO horses and 6 control horses were submitted for evaluation by an allergen-specific immunoglobulin E (IgE) enzyme-linked immunosorbent assay. SPTs were performed on the 40 horses. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated for all diagnostic methods. Agreement between diagnostic methods was assessed by kappa statistic (Κ). Chi-square test with Yates correction showed SPT results from the RAO and control groups to be statistically different (P < .05). SPTs showed higher sensitivity, specificity, positive predictive value, and negative predictive value than both enzyme-linked immunosorbent assay tests. In human medicine, SPTs are considered to be the gold standard of allergy tests. Neither serum IgE nor bronchoalveolar lavage fluid IgE reliably detected allergen hypersensitivity, compared with SPT. SPTs performed significantly better overall than both in vitro tests. Low sensitivity of the in vitro assays indicates the need for continued study to elucidate a more sensitive specific IgE test.     

N-carbamoylglutamate improves lipid metabolism,inflammation, and apoptosis responses in visceral adipocytes of Japanese seabass (Lateolabrax japonicus), in vivo and in vitro

This study applied in vivo and in vitro methods to investigate the effect of dietary N-carbamoylglutamate (NCG) on lipid metabolism, inflammation and apoptosis related-gene expression in visceral adipose tissue and isolated adipocytes of Japanese seabass (Lateolabrax japonicus). A basal diet and a test diet supplemented with 720 mg/kg NCG were fed to the fish for 10 weeks. During the growth trial, no mortality and no significant differences in growth performance were observed in fish between the 2 groups (P > 0.05). Plasma Arg content and mRNA level of argininosuccinate synthetase (ASS) in adipose tissue were significantly increased, which indicated that NCG inclusion promoted endogenous Arg synthesis. Thereafter, the potential effects of NCG treatment on lipid metabolism-related genes expression were studied through in vivo and in vitro methods. In the present study, we successfully established a primary adipocytes culture system and isolated pre-adipocytes in vitro of Japanese seabass for the first time. Both the results in vivo and in vitro showed that NCG treatment decreased the mRNA levels of genes related to adipogenesis (fatty acid synthase, FASN), cholesterol synthesis (3-hydroxy-3-methylglutaryl-CoA reductase, HMGCR) and fat deposition (lipoprotein lipase [LPL] and leptin), which revealed the underlying mechanism of NCG on reducing fat deposition. The results of this study demonstrated that NCG inclusion reduced the expression of inflammatory and apoptosis cytokines markedly in vivo and in vitro. In conclusion, NCG did exert beneficial effects on ameliorating adipogenesis, inflammation and apoptosis via promoting Arg endogenous synthesis in Japanese seabass.     

FSH up-regulates angiogenic factors in luteal cells of buffaloes

Follicle-stimulating hormone has been widely used to induce superovulation in buffaloes and cows and usually triggers functional and morphologic alterations in the corpus luteum (CL). Several studies have shown that FSH is involved in regulating vascular development and that adequate angiogenesis is essential for normal luteal development. Angiogenesis is regulated by many growth factors, of which vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) have an established central role. Therefore, we have used a combination of in vitro and in vivo studies to assess the effects of FSH on the expression of VEGF and FGF2 and their receptors in buffalo luteal cells. The in vivo model consisted of 12 buffalo cows, divided into control (n = 6) and superovulated (n = 6) groups, and CL samples were collected on day 6 after ovulation. In this model, we analyzed the gene and protein expression of FGF2 and its receptors and the protein expression of VEGFA systems with the use of real-time PCR, Western blot analysis, and immunohistochemistry. In the in vitro model, granulosa cells were collected from small follicles (diameter, 4–6 mm) of buffaloes and cultured for 4 d in serum-free medium with or without FSH (10 ng/mL). To induce in vitro luteinization, LH (250 ng/mL) and fetal bovine serum (10%) were added to the medium, and granulosa cells were maintained in culture for 4 d more. The progesterone concentration in the medium was measured at days 4, 5, and 8 after the beginning of cell culture. Cells were collected at day 8 and subjected to real-time PCR, Western blot analysis, and immunofluorescence for assessment of the expression of FGF2, VEGF, and their receptors. To address the percentage of steroidogenic and growth factor-expressing cells in the culture, flow cytometry was performed. We observed that in superovulated buffalo CL, the FGF2 system mRNA expression was decreased even as protein expression was increased and that the VEGF protein was increased (P < 0.05). In vitro experiments with granulosa cells showed an increase in the mRNA expression of VEGF and FGF2 and its receptors 1 and 2 and protein expression of VEGF, kinase insert domain receptor, FGF receptor 2, and FGF receptor 3 in cells treated with FSH (P < 0.05), in contrast to the in vivo experiments. Moreover, the progesterone production by FSH-treated cells was elevated compared with untreated cells (P < 0.05). Our findings indicate that VEGF, FGF2, and their receptors were differentially regulated by FSH in vitro and in vivo in buffalo luteal cells, which points toward a role of CL environment in modulating cellular answers to gonadotropins.     

Advances in Holding and Cryopreservation of Equine Oocytes and Embryos

Methods for holding of oocytes and embryos during shipment as well as for their cryopreservation can greatly aid equine reproductive management. Oocytes can be held at room temperature overnight or at cooler temperatures for two nights without affecting maturation or embryo development after intracytoplasmic sperm injection. In contrast, methods for cryopreservation of equine oocytes that support high rates of embryo development have not yet been established. Equine embryos may be held overnight at temperatures from 5°C to 19°C without reduction in viability, but longer holding periods, or higher holding temperatures, may be detrimental. Small equine embryos (<300 μm), either in vivo derived or in vitro produced, can be slow frozen or vitrified successfully. In the last decade, methods have been developed to allow in vivo–derived expanded blastocysts, up to Day 8, to be vitrified successfully after blastocoele collapse. These methods of shipment and preservation allow mare owners in remote locations to have access to sophisticated assisted reproductive technologies.     

Development of a clinical prediction score for detection of suspected cases of equine grass sickness (dysautonomia) in France

Equine grass sickness (EGS) (equine dysautonomia) is a neurodegenerative condition of grazing equines. Pre-mortem diagnosis of EGS is a challenge for practitioners as definitive diagnosis requires ileal/myenteric lymph node biopsies. This study aimed to develop a clinical score that could be used by practitioners to improve the detection of acute or subacute EGS cases in the field. Suspected EGS cases were declared by veterinary practitioners. A case was classified as confirmed positive if ileal or rectal biopsy samples showed neuronal degeneration typical of EGS. A semi-quantitative scoring system, including epidemiological and clinical data, was created to attempt to classify suspected EGS horses into confirmed positive or negative cases. Each variable was weighted based on a boosted regression trees model, while taking into account its clinical relevance. Twenty-eight EGS cases were confirmed by biopsy during the entire study period. The best cut-off value for the score to have a high sensitivity while maximizing specificity was 8, with a sensitivity of 100% and a specificity of 53%. In our dataset, 77% of animals would be correctly classified with this cut-off value of 8. Highest sensitivity was chosen in order to detect the highest number of potential cases. Our score represents an inexpensive and useful tool to aid in the identification of suspected EGS cases in the field and selection for further diagnostics procedures to confirm or rule out the disease. Application of the score to larger populations of animals would be required to further adapt and refine the score.     

In Vitro Production of Equine Embryos

The development of methods to produce embryos in vitro in the horse has been delayed compared with other domestic species. Oocytes can be collected from excised ovaries or from the small or preovulatory follicles of live mares. Intracytoplasmic sperm injection is the only reliable method to fertilize equine oocytes in vitro. Intracytoplasmic sperm injection-produced embryos can be transferred into the oviducts of recipient mares or cultured to the morula or blastocyst stage of development for nonsurgical embryo transfers into recipients' uteri. Embryos cultured in vitro have some morphological differences compared with embryos collected from the mares' uteri. Most notably, the embryonic capsule does not form in culture, and the zona pellucida fails to expand completely. However, embryo produced in vitro can result in viable pregnancies and healthy offspring.     

Orally Administered Pediococcus acidilactici and Saccharomyces boulardii–Based Probiotics Alter Select Equine Immune Function Parameters

To investigate the effects of a combination of Pediococcus acidilactici and Saccharomyces boulardii, the following experiments were performed. Initially, an in vitro experiment was performed in which the culture supernatant of S. boulardii and P. acidilactici was added to the culture media of isolated peripheral blood mononuclear cells (PBMCs), and the proliferative response to cellular stimulants was assayed. After this initial experiment, an in vivo experiment was performed in which 12 horses were used and assigned to one of two groups of six horses each: placebo controls or principle-treated horses. After a period of treatment, various end points were determined to test the effects of test article on (1) proliferative responses of cultured PBMCs; (2) serum immunoglobulin (Ig) concentrations; (3) lymphocyte phenotype subsets; (4) white blood cell count; and (5) response to vaccination. Results of the in vitro testing demonstrated a substantial reduction (23%) in proliferation of stimulated PBMCs. Results of in vivo testing demonstrated enhanced proliferation on day 72 in cells stimulated with phorbol (P = .04). On study day 37, the segmented neutrophil number was reduced and IgG concentration increased (mean, 329.0 vs. 185.9 ng/mL; P = .029). Results demonstrate that the test article did have some effects on systemic immunity, specifically proliferative responses, immunoglobulin G concentrations, and neutrophil numbers. Based on the findings of this study, further evaluation of these probiotics for equine wellness or disease modulation is warranted.     

The role of seaweed as a potential dietary supplementation for enteric methane mitigation in ruminants: Challenges and opportunities

Seaweeds are macroalgae, which can be of many different morphologies, sizes, colors, and chemical profiles. They include brown, red, and green seaweeds. Brown seaweeds have been more investigated and exploited in comparison to other seaweed types for their use in animal feeding studies due to their large sizes and ease of harvesting. Recent in vitro and in vivo studies suggest that plant secondary compound-containing seaweeds (e.g., halogenated compounds, phlorotannins, etc.) have the potential to mitigate enteric methane (CH₄) emissions from ruminants when added to the diets of beef and dairy cattle. Red seaweeds including Asparagopsis spp. are rich in crude protein and halogenated compounds compared to brown and green seaweeds. When halogenated-containing red seaweeds are used as the active ingredient in ruminant diets, bromoform concentration can be used as an indicator of anti-methanogenic properties. Phlorotannin-containing brown seaweed has also the potential to decrease CH₄ production. However, numerous studies examined the possible anti-methanogenic effects of marine seaweeds with inconsistent results. This work reviews existing data associated with seaweeds and in vitro and in vivo rumen fermentation, animal performance, and enteric CH₄ emissions in ruminants. Increased understanding of the seaweed supplementation related to rumen fermentation and its effect on animal performance and CH₄ emissions in ruminants may lead to novel strategies aimed at reducing greenhouse gas emissions while improving animal productivity.     

Effect of different methods for conserving rice grain on in situ ruminal degradation and in vivo nutrient digestion and rumen fermentation in steers

We investigated the effects of different rice conservation techniques on in situ ruminal degradation and in vivo nutrient digestibility and rumen fermentation in steers. Raw rice grain was dried before crushing (DRY), ensiled after crushing (ENS‐A), or ensiled before crushing (ENS‐B). Six ruminally cannulated steers were used in a replicated 3 × 3 Latin square design with three dietary treatments: diets containing DRY, ENS‐A, or ENS‐B at 36% of the dietary dry matter. The in situ rapidly degradable fraction and effective ruminal degradability were higher for ensiled rice than for DRY, and higher for ENS‐A than for ENS‐B. The ruminal pH was lower and the lactic acid and total volatile acid concentrations were higher for the steers fed ensiled rice than those fed the DRY diet, but a treatment effect was not observed in the comparison between ENS‐A and ENS‐B. The whole‐tract digestibility of crude protein and ether extract was improved when the rice grain was ensiled, but there were no differences in nutrient digestibility between ensiling methods. These results show that ensiling treatment can be a strategy to improve the nutrient value of rice grain, but the ensiling method has little impact on in vivo digestion.     

Sucrose assists selection of high‐quality oocytes in pigs

We investigated whether high‐quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis‐shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p < .05) developmental competence than that of mis‐shapened oocytes in terms of blastocyst rate and total cell number in blastocysts obtained after 6 days of in vitro culture. We also confirm that round aged oocytes are higher in quality than mis‐shapened aged oocytes. In an attempt to find out why high‐quality oocytes maintain a round shape whereas poorer oocytes become mis‐shapened following sucrose treatment, we examined the arrangement of actin microfilaments and microtubules. Abnormal organization of these cytoskeletal components was higher (p < .05) in mis‐shapened oocytes compared to round oocytes after 52 hr of IVM. In conclusion, sucrose treatment helps selection of high‐quality oocytes, including aged oocytes, in pigs. Abnormal cytoskeleton arrangements partly explain for low developmental competence of mis‐shapened oocytes.