Evaluation of the contribution of the placentomal fusion during gestation in cattle

The placenta of mammals is a structure formed by the juxtaposition of the fetal membranes and the maternal tissues. The main function of the placenta is to regulate the physiological interchange between the fetus and the mother as well as to operate as an important endocrine organ during the gestation. The placentomal fusions were characterized throughout gestation of cattle using macroscopic, histological and flow cytometry analyses. Analyzing the cell cycle phases with a flow cytometry, a balance between the G2M phase and apoptosis was observed, suggesting that the placentomal fusions do not interfere in the placentary maturation process, which is a pre-requirement for the fetal-maternal disconnection and the release of fetal membrane. (C) 2011 Elsevier B.V. All rights reserved.

Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Delivery of cloned offspring: experience in Zebu cattle (Bos indicus)

The production of a healthy cloned calf is dependent on a multitude of successful steps, including reprogramming mediated by the oocyte, the development of a functional placenta, adequate maternal-fetal interaction, the establishment of a physiological metabolic setting and the formation of a complete set of well-differentiated cells that will eventually result in well-characterised and fully competent tissues and organs. Although the efficiency of nuclear transfer has improved significantly since the first report of a somatic cell nuclear transfer-derived animal, there are many descriptions of anomalies concerning cloned calves leading to high perinatal morbidity and mortality. The present article discusses some our experience regarding perinatal and neonatal procedures for cloned Zebu cattle (B. indicus) that has led to improved survival rates in Nellore cloned calves following the application of such `labour-intensive technology`.

Embryo Mitochondrial DNA Depletion Is Reversed During Early Embryogenesis in Cattle

The extensive replication of mitochondria during oogenesis and the wide variability in mitochondrial DNA ( mtDNA) copy numbers present in fully grown oocytes indicate that mtDNA amount may play an important role during early embryogenesis. Using bovine oocytes derived from follicles of different sizes to study the influence of mtDNA content on development, we showed that oocytes obtained from small follicles, known to be less competent in developing into blastocysts, contain less mtDNA than those originating from larger follicles. However, because of the high variability in copy number, a more accurate approach was examined in which parthenogenetic one-cell embryos were biopsied to measure their mtDNA content and then cultured to assess development capacity. Contrasting with previous findings, mtDNA copy number in biopsies was not different between competent and incompetent embryos, indicating that mtDNA content is not related to early developmental competence. To further examine the importance of mtDNA on development, one-cell embryos were partially depleted of their mtDNA (64% +/- 4.1% less) by centrifugation followed by the removal of the mitochondrial-enriched cytoplasmic fraction. Surprisingly, depleted embryos developed normally into blastocysts, which contained mtDNA copy numbers similar to nonmanipulated controls. Development in depleted embryos was accompanied by an increase in the expression of genes (TFAM and NRF1) controlling mtDNA replication and transcription, indicating an intrinsic ability to restore the content of mtDNA at the blastocyst stage. Therefore, we concluded that competent bovine embryos are able to regulate their mtDNA content at the blastocyst stage regardless of the copy numbers accumulated during oogenesis.

High genetic diversity in field isolates of Trypanosoma theileri assessed by analysis of cathepsin L-like sequences disclosed multiple and new genotypes infecting cattle in Thailand

In this study, we describe the first survey in Thailand of Trypanosoma theileri, a widespread and prevalent parasite of cattle that is transmitted by tabanid flies. Investigation of 210 bovine blood samples of Thai cattle from six farms by hematocrit centrifuge technique (HCT) revealed 14 samples with trypanosomes morphologically compatible to T. theileri. Additional animals were positive for T. theileri by PCR based on the Cathepsin L-like sequence (TthCATL-PCR) despite negative by HCT, indicating cryptic infections. Results revealed a prevalence of 26 +/- 15% (95% CI) of T. theileri infection. Additionally, 12 samples positive for T. theileri were detected in cattle from other 11 farms. From a total of 30 blood samples positive by HCT and/or PCR from 17 farms, seven were characterized to evaluate the genetic polymorphism of T. theileri through sequence analysis of PCR-amplified CATL DNA sequences. All CATL sequences of T. theileri from Thai cattle clustered with sequences of the previously described phylogenetic lineages TthI and TthII, supporting only two major lineages of T. theileri in cattle around the world. However, 11 of the 29 CATL sequences analyzed showed to be different, disclosing an unexpectedly large polymorphic genetic repertoire, with multiple genotypes of T. theileri not previously described in other countries circulating in Thai cattle. (C) 2011 Elsevier B.V. All rights reserved.

Characterization of spliced leader genes of Trypanosoma (Megatrypanum) theileri: phylogeographical analysis of Brazilian isolates from cattle supports spatial clustering of genotypes and parity with ribosomal markers

Trypanosoma (Megatrypanum) theileri from cattle and trypanosomes of other artiodactyls form a clade of closely related species in analyses using ribosomal sequences. Analysis of polymorphic sequences of a larger number of trypanosomes from broader geographical origins is required to evaluate the Clustering of isolates as suggested by previous studies. Here, we determined the sequences of the spliced leader (SL) genes of 21 isolates from cattle and 2 from water buffalo from distant regions of Brazil. Analysis of SL gene repeats revealed that the 5S rRNA gene is inserted within the intergenic region. Phylogeographical patterns inferred using SL sequences showed at least 5 major genotypes of T. theileri distributed in 2 strongly divergent lineages. Lineage TthI comprises genotypes IA and IB from buffalo and cattle, respectively, from the Southeast and Central regions, whereas genotype IC is restricted to cattle from the Southern region. Lineage Tth II includes cattle genotypes IIA, which is restricted to the North and Northeast, and IIB, found in the Centre, West, North and Northeast. PCR-RFLP of SL genes revealed valuable markers for genotyping T. theileri. The results of this study emphasize the genetic complexity and corroborate the geographical structuring of T. theileri genotypes found in cattle.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha 1 (residues Gly-9 to Arg-21), alpha 2 (residues Glu-27 to Asn-40), alpha 3 (residues Arg-44 to Thr-54), alpha 4 (residues Leu-57 to Tyr-64), and alpha 5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Diagnoses of fipronil resistance in Brazilian cattle ticks (Rhipicephalus (Boophilus) microplus) using in vitro larval bioassays

Fipronil is a phenylpyrazolic insecticide that is widely used in agriculture and has been recently used to control the cattle tick, Rhipicephalus (Boophilus) microplus. Because of the serious problems associated with resistance to the available acaricides, this product has been used as an important alternative to control acaricide-resistant ticks. The objective of this work was to analyse the fipronil sensitivity of ticks that were collected from farms with a history of fipronil use by larval bioassays. A total of 11 Brazilian tick populations were studied: one population from Rio Grande do Sul, one population from Mato Grosso do Sul and nine populations from Sao Paulo. To validate the assays, susceptible reference strains, POA (Porto Alegre, Brazil) and Mozo (Dilave, Uruguay), and ticks from six different farms that never used fipronil were tested. The resistance of various tick populations to technical grade fipronil (95.3%) was primarily evaluated using the larvae immersion test (LIT) and the larval packet test (LPT), when a sufficient number of larvae was collected. Using the LIT, the resistance ratios (RR(50)) of the tick populations from Rio Grande do Sul and Mato Grosso do Sul were 14.9 and 2.6, respectively, and the populations derived from Sao Paulo had RR(50)s ranging from 2.5 to 6.9. Four populations were evaluated with the LPT, and two populations displayed lower RR(50), while other populations displayed higher RR(50) than those determined by the LIT. This article reports the first cases of fipronil resistance in Brazil and highlights the LIT as a more sensitive technique for the evaluation of fipronil resistance in R. (B) microplus ticks. We suggest the use of the LIT as an evaluation tool for monitoring fipronil resistance in the control programmes of R. (B) microplus. (C) 2010 Elsevier B.V. All rights reserved.

Effect of Jersey type on thermoregulatory responses during exercise in a warm humid environment

The thermoregulatory responses of subjects wearing two different forms of rugby league jersey, one with plastic sponsorship recognition and numbering (trial Gl) and one without (trial G2), and a lightweight alternative (trial G3), were compared with a trial without any form of upper body garment (trial GO). Ten male volunteers, mean age 20.9 (±2.3) years, height 179.8 (±4.7) cm, weight 80.2 (±8.9) kg, and body surface area 1.99 (±0.13) m2, participated in this study. Subjects had a mean maximal oxygen uptake capacity of 56.0 (±6.3) ml.kg.min-1 and a sum of 8 skinfolds of 80.6 (±23.8) mm. Subjects were exercised at approximately 50% of maximal oxygen uptake in a warm humid environment for 50 minutes. Mean ambient temperature was 27.6°C (±0.32) with a relative humidity of 64.7% (±1.44). Measurements of core and skin (7 sites) temperature, heart rate, oxygen uptake, plasma volume, peak lactate concentration, and pre- and post-trial body weight, hematocrit and garment weight were recorded. The statistical results showed that all subjects experienced significant (p ≤.0001) decreases in body weight representing a percentage decrease ranging from 1.2-1.3%. No significant difference was found between trials with respect to body weight change. No significant effect of garment type was found on pre- and post-trial hematocrit, plasma volume changes or peak blood lactic acid concentration. However, mean peak lactate was highest for trial Gl (5.6 mmol.L-1 ±2.2) and lowest for trial G3 (4.6 mmol.L-1 ±1.27). Post-trial core temperature was significantly (p≤ .0001) higher than the resting value; no significant difference was found between trials. The mean absolute increase for all experimental trials was 0.9°C. A significant (p≤.005) difference between mean total (7 sites) skin temperature was found with a post-hoc test revealing that trials Gl and G2 were significantly higher than trial GO; no significant difference was found when comparing trial G3 with trial GO or when comparing the garments between each other. Mean skin temperature under the garment (4 sites) was found to be significantly (p≤.05) higher for all trials involving a garment when compared with mean skin temperature outside (3 sites) the garment; no significant difference was found between trials. Mean oxygen uptake was significantly different between trials (p≤.005), with trial Gl and G3 found to be significantly lower than trial GO; no difference was found when comparing the garments with each other. Post-trial garment weights were significantly (p≤.001) heavier than pre-trial and were significantly (p≤.0001) different when compared with each other. There was no significant effect on heart rate, haematocrit, plasma volume changes, peak blood lactic acid concentration, or core temperature due to garment type. However, differences in skin temperature suggest that the garment used in trial G3 may have a benefit. Further research should consider the impact of increased exercise intensity and/or environmental temperature and humidity on the measured parameters while wearing the garments described in this study.

Wear attributes of cashmere single jersey knitted fabrics blended with high and low crimp superfine merino wool

In a replicated experiment, we investigated the impact of cashmere in blends with superfine wools on the wear attributes of single jersey knitted fabrics. We also investigated the relative performance of low crimp/low fiber curvature superfine wool when compared with cashmere and also when compared with traditional high crimp/high fiber curvature superfine wool in pure and blended knitted fabrics. Wool type, blend ratio and fabric structure affected fabric air permeability, resistance to pilling and change in appearance, relaxation shrinkage, hygral expansion, and dimensional stability during laundering. The responses to variation in fiber crimp were much greater than previously reported. The fabric properties of low crimp wool differed significantly from those made from high crimp wool, and low crimp wool fabric properties differed significantly from, but were closer to, the fabric properties of cashmere, compared with high curvature wool.

The effect of humidity and temperature on Wool Comfort Meter assessment of single jersey wool fabrics

The Wool ComfortMeter provides an objective measurement of the fabric-evoked prickle discomfort rating provided by wearers. This work aimed to quantify the sensitivity of the Wool ComfortMeter over a range of different temperature and humidity conditions to determine the recommended test conditions for its operation. The design was: three temperatures (notionally 20, 25 and 30°C) at three relative humidities (RHs, notionally 50, 65 and 80%) each with two replicates, using six different wool single jersey knits (mean fibre diameter 19.5–27.0 µm). As it was difficult to achieve exactly some of the extreme combinations of temperature and RH, some combinations were repeated, providing a total of 23 different assessment conditions. Data were analysed using restricted maximum likelihood mixed model analysis. The best fixed model included RH, RH2, temperature and the interaction of temperature and RH, accounting for 95% of the variation in Wool ComfortMeter readings. Wool ComfortMeter values were almost constant at 55–60% RH. Generally, the Wool ComfortMeter value reduced with increasing RH > 60% at temperatures of 25°C and 28.5°C as the regain of the fabric increased. However, at 20°C little change was detected as RH was increased from 50 to 80% as there were only small changes in fabric regain. The observed effects were in a good agreement with existing knowledge on the effect of regain on the mechanical properties of wool fibre. Wool ComfortMeter is best operated under standard conditions for textile testing of 65% RH and 20°C.