Development of Y-chromosome-Specific SCAR Markers Conserved in Taurine, Zebu and Bubaline Cattle

Contents Sex pre-selection of bovine offsprings has commercial relevance for cattle breeders and several methods have been used for embryo sex determination. Polymerase chain reaction (PCR) has proven to be a reliable procedure for accomplishing embryo sexing. To date, most of the PCR-specific primers are derived from the few single-copy Y-chromosome-specific gene sequences already identified in bovines. Their detection demands higher amounts of embryonic genomic material or a nested amplification reaction. In order to circumvent this, limitation we searched for new male-specific sequences potentially useful in embryo sexing using random amplified polymorphic DNA (RAPD) analysis. Random amplified polymorphic DNA (RAPD) assay reproducibility problems can be overcome by its conversion into Sequence Characterized Amplified Region (SCAR) markers. In this work, we describe the identification of two bovine male-specific markers (OPC16(323) and OPF10(1168)) by means of RAPD. These markers were successfully converted into SCARs (OPC16(726) and OPF10(984)) using two pairs of specific primers.Furthermore, inverse PCR (iPCR) methodology was successfully applied to elongate OPC16(323) marker in 159% (from 323 to 837 bp). Both markers are shown to be highly conserved (similarity >= 95%) among bovine zebu and taurine cattle; OPC16(323) is also highly similar to a bubaline Y-chromosome-specific sequence. The primers derived from the two Y-chromosome-specific conserved sequences described in this article showed 100% accuracy when used for identifying male and female bovine genomic DNA, thereby proving their potential usefulness for bovine embryo sexing.

Impact of short-term preconceptional exposure to particulate air pollution on treatment outcome in couples undergoing in vitro fertilization and embryo transfer (IVF/ET)

To assess the potential effects of short-term exposure to particulate air pollution during follicular phase on clinical, laboratory, and pregnancy outcomes of women undergoing IVF/ET. Retrospective cohort study of 400 first IVF/ET cycles of women exposed to ambient particulate matter during follicular phase. Particulate matter (PM) was categorized into quartiles (Q(1): a parts per thousand currency sign30.48 A mu g/m(3), Q(2): 30.49-42.00 A mu g/m(3), Q(3): 42.01-56.72 A mu g/m(3), and Q(4): > 56.72 A mu g/m(3)). Clinical, laboratory, or treatment variables were not affected by follicular phase PM exposure periods. Women exposed to Q(4) period during the follicular phase of conception cycles had a higher risk of miscarriage (odds ratio, 5.05; 95% confidence interval: 1.04-25.51) when compared to women exposed to Q(1-3) periods. Our results show an association between brief exposure to high levels of ambient PM during the preconceptional period and early pregnancy loss, although no effect of this exposure on clinical, laboratory, and treatment outcomes was observed.

In vitro fertilization, embryo development, and cell lineage segregation after pre- and/or postnatal exposure of female mice to ambient fine particulate matter

Objective: To evaluate effects of pre- and/or postnatal exposure to ambient fine particulate matter on fertilization, embryo development, and cell lineage segregation in preimplantation blastocysts using the IVF mouse model. Design: Animal model. Setting: Academic institution. Animal(S): Six-week-old, superovulated mice. Intervention(s): Pre- and postnatal exposure to filtered air (FA-FA), filtered-ambient air (FA-AA), or ambient air (AA-AA) in exposure chambers 24 hours a day for 9 weeks. Main Outcome Measure(S): Gestation length, litter size, sex ratio, ovarian response to superovulation, fertilization rate, embryo development, blastocyst and hatching rates, total cell count, and proportion of cell allocation to inner-cell mass (ICM) and trophectoderm (TE). Result(S): Gestation length, litter size and birth weight, live-birth index, and sex ratio were similar among exposure groups. Ovarian response was not affected by the exposure protocol. A multivariate effect for pre- and/or postnatal exposure to ambient fine particulate matter on IVF, embryo development, and blastocyst differential staining was found. Cell counts in ICM and ICM/TE ratios in blastocysts produced in the FA-FA protocol were significantly higher than in blastocysts produced in the FA-AA and AA-AA protocols. No difference in total cell count was observed among groups. Conclusion(S): Our study suggests that exposure to ambient fine particulate matter may negatively affect female reproductive health by disrupting the lineage specification at the blastocyst stage without interfering in early development of the mouse embryo. (Fertil Steril (R) 2009;92:1725-35. (C) 2009 by American Society for Reproductive Medicine.)

Modulation of cutaneous inflammation induced by ticks in contrasting phenotypes of infestation in bovines

Tick saliva contains molecules that are inoculated at the site of attachment on their hosts in order to modulate local immune responses and facilitate a successful blood meal. Bovines express heritable, contrasting phenotypes of infestations with the cattle tick, Rhipicephalus (Boophilus) microplus: breeds of Bos taurus indicus are significantly note resistant than those of Bos taurus taurus. Tick saliva may contain molecules that interfere with adhesion of leukocytes to endothelium and resistant hosts may mount an inflammatory profile that is more efficient to hamper the tick`s blood meal. We show in vitro that adhesion of peripheral blood mononuclear cells to monolayers of cytokine-activated bovine umbilical endothelial cells was significantly inhibited by tick saliva. The inflammatory response to bites of adults of R. microplus mounted by genetically resistant and susceptible bovine hosts managed in the same pasture was investigated in vivo. The inflammatory infiltrates and levels of message coding for adhesion molecules were measured in biopsies of tick-bitten and control skin taken when animals of both breeds were exposed to low and high tick infestations. Histological studies reveal that cutaneous reactions of resistant hosts to bites of adult ticks contained significantly more basophils and eosinophils compared with reactions of the susceptible breed. Expression of the adhesion molecules - intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin - was higher in adult-infested skin of susceptible hosts undergoing low infestations compared to resistant hosts; when host was exposed to high infestations expression of these adhesion molecules was down-regulated in both phenotypes of infestations. Expression of leukocyte adhesion glycoprotein-1 (LFA-1) was higher in skin from susceptible hosts undergoing low or high infestations compared to resistant hosts. Conversely, higher levels of E-selectin, which promotes adhesion of memory T cells, were expressed in skin of resistant animals. This finding may explain the resistant host`s ability to mount more rapid and efficient secondary responses that limit hematophagy and infestations. The expression profiles observed for adhesion molecules indicate that there are differences in the kinetics of the inflammatory reactions mounted by resistant and susceptible hosts and the balance between tick and host is affected by the number of tick bites a host receives. We show that the contrasting phenotypes of infestations seen in bovines infested with R. microplus are correlated with differences in the cellular and molecular composition of inflammatory infiltrates elicited by bites with adult ticks. (C) 2009 Published by Elsevier B.V.

Mycobacterium tuberculosis Culture Filtrate Proteins plus CpG Oligodeoxynucleotides Confer Protection to Mycobacterium bovis BCG-Primed Mice by Inhibiting Interleukin-4 Secretion

Culture filtrate proteins (CFP) are potential targets for tuberculosis vaccine development. We previously showed that despite the high level of gamma interferon (IFN-gamma) production elicited by homologous immunization with CFP plus CpG oligodeoxynucleotides (CFP/CpG), we did not observe protection when these mice were challenged with Mycobacterium tuberculosis. In order to use the IFN-gamma-inducing ability of CFP antigens, in this study we evaluated a prime-boost heterologous immunization based on CFP/CpG to boost Mycobacterium bovis BCG vaccination in order to find an immunization schedule that could induce protection. Heterologous BCG-CFP/CpG immunization provided significant protection against experimental tuberculosis, and this protection was sustained during the late phase of infection and was even better than that conferred by a single BCG immunization. The protection was associated with high levels of antigen-specific IFN-gamma and interleukin-17 (IL-17) and low IL-4 production. The deleterious role of IL-4 was confirmed when IL-4 knockout mice vaccinated with CFP/CpG showed consistent protection similar to that elicited by BCG-CFP/CpG heterologous immunization. These findings show that a single dose of CFP/CpG can represent a new strategy to boost the protection conferred by BCG vaccination. Moreover, different immunological parameters, such as IFN-gamma and IL-17 and tightly regulated IL-4 secretion, seem to contribute to the efficacy of this tuberculosis vaccine.

Cultural Consonance, a 5HT2A Receptor Polymorphism, and Depressive Symptoms: A Longitudinal Study of Gene x Culture Interaction in Urban Brazil

In this study in urban Brazil we examine, as a predictor of depressive symptoms, the interaction between a single nucleotide polymorphism in the 2A receptor in the serotonin system (-1438G/A) and cultural consonance in family life, a measure of the degree to which an individual perceives her family as corresponding to a widely shared cultural model of the prototypical family. A community sample of 144 adults was followed over a 2-year-period. Cultural consonance in family life was assessed by linking individuals` perceptions of their own families with a shared cultural model of the family derived from cultural consensus analysis. The -1438G/A polymorphism in the 2A serotonin receptor was genotyped using a standard protocol for DNA extracted from leukocytes. Covariates included age, sex, socioeconomic status, and stressful life events. Cultural consonance in family life was prospectively associated with depressive symptoms. In addition, the interaction between genotype and cultural consonance in family life was significant. For individuals with the A/A variant of the -1438G/A polymorphism of the 2A receptor gene, the effect of cultural consonance in family life on depressive symptoms over a 2-year-period was larger (beta = -0.533, P < 0.01) than those effects for individuals with either the G/A (beta = -0.280, P < 0.10) or G/G (beta = -0.272, P < 0.05) variants. These results are consistent with a process in which genotype moderates the effects of culturally meaningful social experience on depressive symptoms. Am. J. Hum. Biol. 21:91-97, 2009. (C) 2008 Wiley-Liss, Inc.

Oocyte diameter as a predictor of fertilization and embryo quality in assisted reproduction cycles

Objective: To assess the impact of the mean oocyte diameter (MOD) on occurrence of fertilization and embryo quality in assisted reproduction cycles. Design: Prospective observational study. Setting: Sector of Human Reproduction of the University Hospital, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo (HCFMRP-USP). Patient(s): Thirty-five women undergoing intracytoplasmic sperm injection (ICSI) at the University Hospital of Ribeirao Preto from May to October 2007. Intervention(s): MOD assessment. Main Outcome Measure(s): Occurrence of fertilization and qualitative embryo classification on 2nd and 3rd day after ICSI. Result(s): We divided 160 metaphase II oocytes according to MOD into groups A (MOD below the 25th percentile), B (MOD between 25th and 75th percentile), and C (MOD above the 75th percentile). There was no statistically significant association between MOD and the occurrence of fertilization or the qualitative embryo classification on days 2 and 3. There was no statistically significant difference between groups regarding number of cells or the qualitative embryo classification on days 2 and 3. Conclusion(s): The MOD of mature oocytes does not seem to be related to the occurrence of fertilization or to the developmental quality of human embryos on days 2 and 3 after ICSI. (Fertil Steril(R) 2010;93:621-5. (C)2010 by American Society for Reproductive Medicine.)

Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules

In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24 h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured it, TCM plus FSH and 10% estrous cow serum. After fertilization. presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P < 0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless. there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis. (C) 2009 Elsevier Inc. All rights reserved.

Relationship between first polar body morphology before intracytoplasmic sperm injection and fertilization rate, cleavage rate, and embryo quality

Objectives: To evaluate the influence of the morphology of the first polar body (PB) on intracytoplasmic sperm injection (ICSI) outcomes. Methods: The morphology of the first PB was assessed in 3177 metaphase 11 oocytes and classified as: intact and normal size, fragmented, or enlarged size. The rates of fertilization, cleavage, and embryo quality were evaluated on day 2. Results: The rates of fertilization, cleavage, and formation of good quality embryos resulting from the insemination of oocytes with an enlarged first PB (20.7%, 18.7%, and 5.0%, respectively) were significantly lower than those for oocytes with an intact first PB of normal size (70.8%, 62.5%, and 19%, respectively) or a fragmented first PB (69.7%, 60.5%, and 17.1%, respectively). Rates did not differ significantly between oocytes with an intact first PB of normal size and oocytes with a fragmented first PB (P>0.05). Conclusions: The presence of an enlarged PB is related to poorer rates of fertilization, cleavage, and top quality embryos. However, identification of first PB fragmentation does not seem to interfere with ICSI outcomes. (C) 2008 International Federation ofGynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

Correlation between mastitis occurrence and the count of microorganisms in bulk raw milk of bovine dairy herds in four selective culture media

Milk is the normal secretion of the mammary gland, practically free of colostrum and obtained by the complete milking of oil(? or more healthy animals. Mastitis is all inflammatory process of the mammary gland and it may cause alterations in the milk. The present work aimed to verify whether it is possible, by means of the counts of microorganism in the bulk raw milk ill four selective culture media, to establish a correlation with the occurrence of mastitis and therefore, to monitor this disease in bovine dairy herds. The following selective culture media were Used: KF Streptococcus Agar, Edwards Agar, Baird-Parker Agar, Blood Agar plus potassium tellurite. Spearman`s correlation coefficient was calculated in order to compare the Occurrence of mastitis (percentage) in each herd with respective selective culture media counts of microorganisms in bulk raw milk. Thirty-six possibilities were analysed (Tamis and CMT-positive rates were compared with the log-transformed count in four selective Culture media) and there was a negative correlation between Tamis 3 and the Baird-Parker Agar plate count. The total results of microbiological tests showed that there were three correlations of the counts in selective culture media. Fifty-two possibilities were analysed and there was a negative correlation between no-bacteria I-growth mastitis rates and log(10) of KF Streptoccocus Agar plate Count and there were two positive correlations between coagulase-positive staphylococci and log(10) of Baird-Parker Agar plate count and Blood Agar plus potassium tellurite plate count.

Characterization by restriction fragment length polymorphism and sequence analysis of field and vaccine strains of infectious laryngotracheitis virus involved in severe outbreaks

At the end of 2002 and throughout 2003, there was a severe outbreak of infectious laryngotracheitis (ILT) in an intensive production area of commercial hens in the Sao Paulo State of Brazil. ILT virus was isolated from 28 flocks, and 21 isolates were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) using four genes and eight restriction enzymes, and by partial sequencing of the infected cell protein 4 (ICP4) and thymidine kinase (TK) genes. Three groups resulted from the combinations of PCR-RFLP patterns: 19 field isolates formed Group I, and the remaining two isolates together with the chicken embryo origin (CEO) vaccine strains formed Group II. Group III comprised the tissue-culture origin (TCO) vaccine strain by itself. The PCR-RFLP results agreed with the sequencing results of two ICP4 gene fragments. The ICP4 gene sequence analysis showed that the 19 field isolates classified into Group I by RFLP-PCR were identical among themselves, but were different to the TCO and CEO vaccines. The two Group II isolates could not be distinguished from one of the CEO vaccines. The nucleotide and amino acid sequence analyses discriminated between the Brazilian and non-Brazilian isolates, as well as between the TCO and CEO vaccines. Sequence analysis of the TK gene enabled classification of the field isolates (Group I) as virulent and non-vaccine. This work shows that the severe ILT outbreak was caused by a highly virulent, non-vaccine strain.

Differentiation of field isolates and vaccine strains of infectious laryngotracheitis virus by DNA sequencing

Two different regions of the infected cell protein 4 (ICP4) gene of infectious laryngotracheitis virus (ILTV) were amplified and sequenced for characterization of field isolates and tissue culture-origin (TCO) and chicken embryo-origin (CEO) vaccine strains. Phylogenetic analysis of the two regions showed differences in nucleotide and amino acid sequences between field isolates and attenuated vaccines. The PCR-RFLP results were identical to those obtained by DNA sequencing and validated their use to differentiate ILTV strains. The approach using the sequencing of the two fragments of the ICP4 gene showed to be an efficient and practical procedure to differentiate between field isolates and vaccine strains of ILTV. (C) 2009 Elsevier Ltd. All rights reserved.

Bovine embryo transfer recipient synchronisation and management in tropical environments

Numerous studies have shown that it is possible to manipulate follicular and luteal dynamics, thereby eliminating the need for oestrus detection in embryo transfer (ET) programmes. Fixed-time ET (FTET) protocols are based on the use of gonadotrophin-releasing hormone (GnRH) and prostaglandin (PG) F or progesterone/progestogen (P4)-releasing devices and oestradiol. The FTET protocols increases the proportion of recipients transferred, and therefore pregnancy rates, compared with the use of PGF followed by ET 7 days after oestrus. Furthermore, the addition of equine chorionic gonadotrophin (eCG) to the P4 and oestradiol-based FTET protocols results in an even higher proportion of recipients transferred, and thus higher pregnancy rates. The beneficial effect of eCG treatment may be related to increased growth of the dominant follicle and increased plasma P4 concentrations during the subsequent luteal phase. In Bos taurus x Bos indicus recipients, pregnancy rates were positively correlated with the diameter of the corpus luteum (CL) and the number of CL at ET. When repeat-breeder Holstein cows were used as recipients, FTET protocols increased number of recipients transferred and pregnancy rates compared with the traditional PGF-based synchronisation protocols. In conclusion, the use of FTET protocols eliminates the need for the detection of oestrus and results in a greater proportion of recipients transferred and satisfactory pregnancy rates. Thus, FTET optimises the use of recipients, reducing labour and animal handling and facilitating the use of ET.

Evaluation of Trypsin Treatment on the Inactivation of Bovine Herpesvirus Type 1 on In Vitro Produced Pre-implantation Embryos

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 mu l of BoHV-1, Los Angeles strain 10(7.5) TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.

Oviductal Fluid Proteins Associated with the Bovine Zona Pellucida and the Effect on In Vitro Sperm-Egg Binding, Fertilization and Embryo Development

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 x 10(5) frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.