The aim of this study was to evaluate the effect of culture media and stage of development in the osmotic ability of in vitro-fertilized bovine embryos and the expression of aquaporin 3 (Aqp3) and Na/K ATPase isoform 1 (ATPAse1) genes in embryos (i) with different ability to undergo rehydration and (ii) following vitrification. In experiment 1, in vitro fertilized presumptive zygotes were co-cultured in SOFaac or modified CR2aa medium and embryos at blastocyst and expanded blastocyst stages at day 7 post-insemination were exposed to NaCl hypertonic medium (900 mOsm) for 5 min following 120 min of culture in isotonic medium in order to evaluate dehydration and rehydration, respectively. No difference (P>0.05) on blastocyst rate was found between CR2aa and SOFaac medium but embryos co-cultured in SOFaac medium underwent greater (P<0.05) dehydration. Embryos at expanded blastocyst stage underwent greater dehydration but slower rehydration than embryos at blastocysts stage (P<0.05). In the experiment 2, the amount of Aqp3 and ATPase1 transcripts were quantified in blastocysts with high or low rehydration after exposure to hypertonic medium. No difference (P>0.05) on relative amount of transcripts was found in either genes. In the experiment 3, expanded blastocysts produced in a co-culture system were vitrified, warmed and then cultured for 72 h for analysis of embryo survival and amount of Aqp3 and ATPase1 transcripts. Lower (P<0.05) embryo survival rate was found for vitrified-warmed embryos (57.9%) than for their fresh counterparts (84.6%). There was no difference on expression of ATPase1 gene but lower (P<0.01) amount of Aqp3 transcripts was found in the vitrified-warmed embryos. In conclusion, embryo ability to undergo shrinkage and swelling is influenced by medium used in a co-culture system and by embryo stage. Rehydrating ability of embryos after exposure to NaCl hypertonic medium is not associated with variations on expression of Aqp3 and ATPase1 genes, but the vitrification can alter gene expression of in vitro-fertilized bovine embryos produced in a co-culture system.

SummaryApoptosis of macrophages has been reported as an effective host strategy to control the growth of intracellular pathogens, including pathogenic mycobacteria. Tumour necrosis factor-a (TNF-a) plays an important role in the modulation of apoptosis of infected macrophages. It exerts its biological activities via two distinct cell surface receptors, TNFR1 and TNFR2, whose extracellular domain can be released by proteolysis forming soluble TNF receptors (sTNFR1 and sTNFR2). The signalling through TNFR1 initiates the majority of the biological functions of TNF-a, leading to either cell death or survival whereas TNFR2 mediates primarily survival signals. Here, the expression of TNF-a receptors and the apoptosis of alveolar macrophages were investigated during the early phase of infection with attenuated and virulent mycobacteria in mice. A significant increase of apoptosis and high expression of TNFR1 were observed in alveolar macrophages at 3 and 7 days after infection with attenuated Mycobacterium bovis but only on day 7 in infection with the virulent M. bovis. Low surface expression of TNFR1 and increased levels of sTNFR1 on day 3 after infection by the virulent strain were associated with reduced rates of apoptotic macrophages. In addition, a significant reduction in apoptosis of alveolar macrophages was observed in TNFR1 À/À mice at day 3 after bacillus Calmette-Gu erin infection. These results suggest a potential role for TNFR1 in mycobacteria-induced alveolar macrophage apoptosis in vivo. In this scenario, shedding of TNFR1 seems to contribute to the modulation of macrophage apoptosis in a strain-dependent manner.

ABSTRACT. The bovine tick Rhipicephalus microplus is responsible for severe economic losses in tropical cattle production. Bos indicus breeds are more resistant to tick infestations than are Bos taurus breeds, and the understanding of the physiological mechanisms involved in this difference is important for the development of new methods of parasite control. We evaluated differences in the transcript expression of genes related to the immune response in the peripheral blood of cattle previously characterized as resistant or susceptible to tick infestation. Crossbreed F2 Gir x Holstein animals (resistant, N = 6; susceptible, N = 6) were artificially submitted to tick infestation. Blood samples were collected at 0, 24, and 48 h after tick infestation and evaluated for transcript expression of the CD25, CXCL8, CXCL10, FoxP3, interleukin (IL)-10, and tumor necrosis factor alpha (TNFα) genes. Gene expression of CD25 (6.00, P < 0.01), IL-10 (31.62, P < 0.01), FoxP3 (35.48, P < 0.01), and CXCL10 (3.38, P < 0.05) was altered in the resistant group at 48 h compared with samples collected before infestation. In the susceptible group, CXCL8 (-2.02, P < 0.05) and CXCL10 (2.20, P < 0.05) showed altered expression 24 h after infestation. CXCL8 (-5.78, P < 0.05) also showed altered expression at 48 h after infestation when compared with samples collected before infestation. We detected a correlation between T γδ cell activity and the immunological mechanisms that result in a higher resistance to R. microplus in cattle.

BackgroundMost studies on granulosa cell (GC) function in cattle have been performed using GC and follicular fluid (FF) samples collected from slaughterhouse ovaries. Using this approach, the follicular developmental stage and functional status are unknown and indirectly inferred, limiting data interpretation. Ultrasound-guided follicle aspiration has previously been used to recover GC or FF samples, but this was mostly carried out in large follicles or pools of small follicles, without recording the efficiency of recovery. The present study was aimed at adapting and evaluating an ovum pick-up (OPU) system for the in vivo recovery of FF and GC from individual follicles of different diameters.MethodsIn the first trial, the losses of fluid inside the tubing system were calculated using a conventional or an adapted-OPU system. Blood plasma volumes equivalent to the amount of FF in follicles of different diameters were aspirated using a conventional OPU Teflon circuit. The OPU system was then adapted by connecting 0.25 mL straws to the circuit. A second trial evaluated the efficiency of FF recovery in vivo. Follicles ranging from 4.0 to 16.8 mm in diameter were aspirated individually using the conventional or adapted-OPU systems. A third trial assessed the in vivo recovery of GC and the subsequent amount of RNA obtained from the follicles of different diameters from Holstein and Gir cattle.ResultsIn Trial I, the plasma recovery efficiency was similar (P > 0.05) for the volumes expected for 12 and 10 mm follicles, but decreased (P < 0.05) for smaller follicles (45.7+/−4.0%, 12.4+/−4.3% and 0.0+/−0.0% for 8, 6, and 4 mm follicles, respectively). Using the adaptation, the losses intrinsic to the aspiration system were similar for all follicle diameters. In Trial II, the expected and recovered volumes of FF were correlated (r = 0.89) and the efficiency of recovery was similar among follicles <12 mm, while larger follicles had a progressive increase in FF losses that was not related to the tubing system. In Trial III, the number of GC and amount of RNA obtained were not affected (P > 0.05) by follicle size, but differed according to breed (615,054+/−58,122 vs 458,095+/−36,407 for Holstein and Gir, respectively; P < 0.05).ConclusionsThe adapted-OPU system can be successfully used for the in vivo collection of FF and GC from follicles of different diameters. This will enable further endocrine, cellular, and gene expression analyses.

No abstract

Objective:In vitro maturation has been shown to influence gene expression in oocytes, but a common shortcoming in reports on the matter has been the use of different donors in each experimental group thus disregarding donor effects. This study aimed to investigate the abundance of mRNA in oocytes matured in vivo and in vitro obtained from the same group of donors.Methods:A bovine model was used to assess the relative abundance of specific transcripts in in vitro-matured (IN VITRO-OPU) and in vivo-matured (IN VIVO-OPU) oocytes collected from the same donors by transvaginal ovum pick-up (OPU). Transcript abundance in oocytes from the IN VIVO-OPU group and oocytes matured in vitro but retrieved from different cows slaughtered at a commercial abattoir (IN VITRO-Abattoir group) was also compared. Total RNA was extracted from denuded oocytes and cDNA was produced via reverse transcription using an oligo(dT) primer for relative quantification of eight target transcripts by real-time PCR.Results:Oocytes in the IN VITRO-OPU group had lower (p<0.05) abundance of peroxiredoxin 1 (Prdx1), heat shock protein 70.1 (Hsp70.1), growth and differentiation factor 9 (Gdf9), and maternal antigen that embryo requires (Mater) transcripts than the oocytes in the IN VIVO-OPU group, all obtained from the same pool of donor cows. Similar results were seen in the comparisons involving the IN VIVO-OPU and IN VITRO-Abattoir groups (p<0.05).Conclusion:In vitro maturation affected the abundance of polyadenylated transcripts in the oocyte cytoplasm when compared to in vivo maturation induced by exogenous hormones in oocytes collected from the same donor pool.

ABSTRACT. Pitcairnia albiflos is a Bromeliaceae species endemic toBrazil that has been included as data-deficient in the extinction risk list of Brazilian flora. We analyzed genetic variability in P. albiflos populations using RAPD markers to investigate population structure and reproductive mechanisms and also to evaluate the actual extinction risk level of this species. Leaves of 56 individuals of P. albiflos from three populations were collected: Urca Hill (UH, 20 individuals), Chacrinha State Park (CSP, 24 individuals) and Tijuca National Park (TNP, 12 individuals). The RAPD technique was effective in characterizing the genetic diversity in the P. albiflos populations since it was possible to differentiate the populations and to identify exclusive bands for at least two of them. Even if there is low genetic diversity among them (CSP-UH = 0.463; CSP-TNP = 0.440; UH-TNP = 0.524), the populations seem to be isolated according to the low genetic diversity observed within them (H pop CSP = 0.060; H pop UH = 0.042; H pop TNP = 0.130). This fact might be the result of clonal and self-reproduction predominance and also from environmental degradation around the collection areas. Consequently, it would be important to protect Genetic variability of Pitcairnia albiflos (Bromeliaceae) all populations both in situ and ex situ to prevent the decrease of genetic variability. The low genetic variability among individuals of the same population confirms the inclusion of this species as critically endangered in the risk list for Brazilian flora.

No abstract