Cytokine profiles in pregnant gilts experimentally infected with porcine reproductive and respiratory syndrome virus and relationships with viral load and fetal outcome.

In spite of extensive research, immunologic control mechanisms against Porcine Reproductive and Respiratory Syndrome virus (PRRSv) remain poorly understood. Cytokine responses have been exhaustively studied in nursery pigs and show contradictory results. Since no detailed reports on cytokine responses to PRRSv in pregnant females exist, the objectives of this study were to compare host cytokine responses between PRRSv-infected and non-infected pregnant gilts, and to investigate relationships between cytokine levels in infected gilts and viral load or fetal mortality rate. Serum samples and supernatants of peripheral blood mononuclear cells (PBMC) either stimulated with PRRSv or phorbol myristate acetate/Ionomycin (PMA/Iono) were analyzed for cytokines/chemokines: interleukins (IL) 1-beta (IL1ß), IL4, IL8, IL10, IL12, chemokine ligand 2 (CCL2), interferon alpha (IFNα) and interferon gamma (IFNγ). Three cytokines (IFNα, CCL2, IFNγ) in gilt serum differed significantly in inoculated versus control gilts over time. In supernatants of PRRSv stimulated PBMC from PRRSv-infected gilts, levels of IFNα were significantly decreased, while IL8 secretion was significantly increased. PRRSv infection altered the secretion of all measured cytokines, with the exception of IFNα, from PBMC after mitogen stimulation, indicating a possible immunomodulatory effect of PRRSv. IFNα, CCL2, and IFNγ in serum, and IFNγ in supernatants of PMA/Iono stimulated PBMC were significantly associated with viral load in tissues, serum or both. However, only IFNα in supernatants of PRRSv stimulated PBMC was significantly associated with fetal mortality rate. We conclude that of the eight cytokines tested in this study IFNα was the best indicator of viral load and severity of reproductive PRRSv infection.

Evaluation of protection in grazing lambs immunised with different doses of Haemonchus contortus gut membrane glycoproteins in Southern Brazil.

A vaccine containing doses ranging from 2 µg to 50 µg of integral membrane proteins from Haemonchus contortus intestinal cells (H11 and H-gal-GP complex) has been shown to be effective for lambs. A vaccine for H. contortus was tested in two-month old grazing Corriedale lambs during an eight-month trial on the outskirts of Bagé, Rio Grande do Sul, Brazil. The animals were kept in a single paddock and allocated to five similar groups according to weight, sex and faecal egg counts (FEC). Vaccinated lambs received 0, 2, 5, 10 or 50 µg of the same antigen diluted in QuilA adjuvant. Vaccine injections were given at days 0, 21, 49, 91, 133, and 175. Lambs were sampled weekly for FEC, packed cell volume (PCV), and plasma ELISA antibody titre. Lambs with PCVs ≤15 % were drenched with levamisole (7.5 mg/kg body weight) as a salvage treatment. During days 77-98 an artificial challenge was administered to increase parasite transmission. Post-artificial challenge (from day 98-217), the FEC of the vaccinated lambs were 59.3 % lower than those of the control lambs. Antigen dose correlated with changes in PCV (r = 0.387 p-value < 0.001). Vaccinated lambs also had higher PCV than controls (p < 0.001; contrast analysis). Salvage treatment was needed in 16.7 % of vaccinated lambs and 88 % of control lambs.

Effect of ageing on hormone secretion and follicular dynamics in sheep with and without the Booroola gene.

It has been suggested that ewes carrying the Booroola gene (Fec(B)) consistently ovulate more follicles because they recruit more primordial follicles and/or have a lower rate of atresia. If the former is correct, the pool of follicles would be depleted sooner in Fec(B) animals. We have studied follicular dynamics and endocrine function during follicular and early luteal phases of the estrous cycle of older ewes with or without the fecundity gene and compared this data with data obtained 6 yr previously in the same animals. Older sheep carrying the Booroola gene maintained a significantly higher ovulation rate than noncarrier ewes [4.2 +/- 0.8 vs. 2.2 +/- 0.6 corpora lutea (CL), respectively; P < 0.05], and in keeping with data from young animals, both ovulatory follicles and CL (4.7 +/- 0.3 vs. 6.9 +/- 0.7 mm and 12.8 +/- 0.5 vs. 16.7 +/- 0.8 mm, respectively) were smaller than those of noncarrier ewes (P < 0.05). The interval from luteolysis to the onset of the LH surge increased with age in all the animals (from 52.0 +/- 8.0 to 67.0 +/- 7.5 h in gene carrier sheep and from 56.0 +/- 2.0 to 79.5 +/- 9.6 h in noncarrier sheep, P < 0.05). The concentration of estradiol and inhibin A in the early luteal phase was lower in older noncarrier ewes (P = 0.08 and P < 0.05, respectively), and the level of inhibin A was inversely related to the level of FSH in aged sheep of both genotypes (P < 0.0001). In contrast, the number of developing follicles in older ewes of both genotypes was similar to the number found in younger ewes, suggesting that increased ovulation rate in sheep carrying the Fec(B) mutation is related to a reduced rate of atresia.

Multiple factors affecting the efficiency of multiple ovulation and embryo transfer in sheep and goats.

This review offers an overview of the basic characteristics of in vivo embryo technologies, their current status, the main findings and the advances gained in recent years, and the outstanding subjects for increasing their efficiency. The use of superovulation and embryo transfer procedures remains affected by a high variability in the ovulatory response to hormonal treatment and by a low and variable number of transferable embryos and offspring obtained. This variability has been classically identified with both extrinsic (source, purity of gonadotrophins and protocol of administration) and intrinsic factors (breed, age, nutrition and reproductive status), which are reviewed in this paper. However, emerging data indicate that the main causes of variability are related to endocrine and ovarian factors, and so the number of studies and procedures addressing a better understanding and control of these factors may be increased in the future. The accomplishment of this objective, the improvement of procedures for embryo conservation and for the selection and management of recipient females, will allow further development and application of this technology.

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories.

Bead-based multiplex assays (BBMAs) are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several to 50-500 analytes within a single, small sample volume). Currently, few assays are commercially available for veterinary applications, but they are available to identify and measure various cytokines, growth factors and their receptors, inflammatory proteins, kinases and inhibitors, neurobiology proteins, and pathogens and antibodies in human beings, nonhuman primates, and rodent species. In veterinary medicine, various nucleic acid and protein-coupled beads can be used in, or for the development of, antigen and antibody BBMAs, with the advantage that more data can be collected using approximately the same amount of labor as used for other antigen and antibody assays. Veterinary-related BBMAs could be used for detection of pathogens, genotyping, measurement of hormone levels, and in disease surveillance and vaccine assessment. It will be important to evaluate whether BBMAs are "fit for purpose," how costs and efficiencies compare between assays, which assays are published or commercially available for specific veterinary applications, and what procedures are involved in the development of the assays. It is expected that many veterinary-related BBMAs will be published and/or become commercially available in the next few years. The current review summarizes the BBMA technology and some of the currently available BBMAs developed for veterinary settings. Some of the human diagnostic BBMAs are also described, providing an example of possible templates for future development of new veterinary-related BBMAs.

Features of follicle-stimulating hormone-stimulated follicles in a sheep model: keys to elucidate embryo failure in assisted reproductive technique cycles.

OBJECTIVE: To evaluate the individual functionality of gonadotropin-stimulated preovulatory follicles, for understanding embryo failure in assisted reproductive technique cycles, in a sheep model. DESIGN: Observational, model study. SETTING: Public research unit. ANIMAL(S): Fifteen adult Manchega ewes. INTERVENTION(S): Synchronization of the estrous cycle with intravaginal progestagens and ovarian stimulation with FSH; evaluation of reproductive activity, plasma sampling, ovarian ultrasonography, and ovariectomies. MAIN OUTCOME MEASURE(S): Determination of estrus behavior, plasma and intrafollicular concentrations of E(2) and inhibin A, number and size of ovarian follicles, and developmental competence of oocytes. RESULT(S): These results support the usefulness of serial measurements of plasma inhibin A for assessment of follicular growth during the FSH treatment, rather than of E(2) assays commonly used. Functionality of FSH-stimulated preovulatory follicles is clearly disturbed, as confirmed by a negative correlation between follicular size and intrafollicular concentrations of inhibin A and E(2) in preovulatory follicles after individual dissection; moreover, the ability of their oocytes to resume meiosis was diminished. CONCLUSION(S): Functionality of follicles in controlled ovarian stimulation (COS), and developmental competence of their oocytes, is disturbed by the high doses of gonadotropin supplied and finally determined by follicular sizes at starting FSH treatment.

Long-term suppression of reproductive function by a single dose of gonadotropin-releasing hormone antagonists in a sheep model.

OBJECTIVE: To determine the effect of single long-acting doses of GnRH antagonists on reproductive function in a sheep model. DESIGN: Observational, model study. SETTING: University-affiliated research unit. ANIMAL(S): Nine intact mature Merino sheep in experiment 1 and 12 mature Merino-crossed ewes with the ovary autotransplanted to the neck in experiment 2. INTERVENTION(S): Synchronization of estrous cycle either with intravaginal progestins or prostaglandin F2alpha analogues and treatment with a single dose of GnRH antagonist; evaluation of reproductive activity, plasma sampling, and ovarian ultrasonography. MAIN OUTCOME MEASURE(S): Determination of estrus behavior; plasma concentrations of P, FSH, LH, and inhibin A; and number and size of ovarian follicles. RESULT(S): In both experiments, the concentrations of FSH and LH were suppressed when compared with those in control ewes. In experiment 1, the ovulatory cycles were suppressed for > or = 55 days in treated sheep. In experiment 2, there were no follicles sized > or = 5 mm in treated ewes for 50 days. CONCLUSION(S): The suppression of the development of large follicles for > or = 30 days after a single injection of a long-acting GnRH antagonist provides a novel convenient method of pretreatment before COS.