Pre-treatment of cattle sperm and/or oocyte with antibody to lipocalin type prostaglandin D synthase inhibits in vitro fertilization and increases sperm-oocyte binding

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO2 in air) for I It in the following treatments either 500 mu L of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for I h either FM or FM with a L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) a L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) a L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 X 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha. L-PGDS: (1) 26.4 +/- 3.0; (2) 25.6 +/- 3.0; (3) 59.7 +/- 3.0; (4) 56.4 +/- 3.0; and (5) 57.1 +/- 3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P < 0.05) compared with the control: (1) 89.2 +/- 2.0%; (2) 87.5 +/- 2.0%; (3) 19.4 +/- 2.0%; (4) 27.2 +/- 3.1%; and (5) 14.1 +/- 3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization. (c) 2007 Elsevier B.V. All rights reserved.

Apoptotic and developmental effects of bovine Herpesvirus type-5 infection on in vitro-produced bovine embryos

Bovine Herpesvirus type-5 (BoHV-5), which is potentially neuropathogenic, was recently described to be related with reproductive disorders in cows. The objective was to elucidate mechanisms involved in propagation of BoHV-5 in embryonic cells. For this purpose, bovine embryos produced in vitro were assayed for apoptotic markers after experimental infection of oocytes, in vitro fertilization, and development. Host DNA fragmentation was detected with a TUNEL assay, expression of annexin-V was measured with indirect immunofluorescence, and viral DNA was detected with in situ hybridization. Infective BoHV-5 virus was recovered from embryos derived from exposed oocytes after two consecutive passages on Madin-Darby bovine kidney (MDBK) cells. The viral DNA corresponding to US9 gene, localized between nucleotides 126243 to 126493, was detected in situ and amplified. There was no significant difference between the ratio of TUNEL stained nuclei and total cells in good quality blastocysts (0.87 +/- 0.05, mean SD), but there were differences (P < 0.05) between infected (0.18 +/- 0.05) and uninfected blastocysts (0.73 +/- 0.07). The Annexin-V label was more intense in uninfected embryos (0.79 +/- 0.04; P < 0.05). The quality of infected and uninfected embryos was considered equal, with no significant effect on embryonic development. In conclusion, we inferred that BoHV-5 infected bovine oocytes, replicated, and suppressed some apoptotic pathways, without significantly affecting embryonic development. (C) 2010 Elsevier Inc. All rights reserved.

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.

Differences in thermoregulatory ability between slick-haired and wild-type lactating Holstein cows in response to acute heat stress

Animals inheriting the slick hair gene have a short, sleek, and sometimes glossy coat. The objective of the present study was to determine whether slick-haired Holstein cows regulate body temperature more effectively than wild-type Holstein cows when exposed to an acute increase in heat stress. Lactating slick cows (n = 10) and wild-type cows (n = 10) were placed for 10 h in an indoor environment with a solid roof, fans, and evaporative cooling or in an outdoor environment with shade cloth and no fans or evaporative cooling. Cows were exposed to both environments in a single reversal design. Vaginal temperature, respiration rate, surface temperature, and sweating rate were measured at 1200, 1500, 1800, and 2100 h (replicate 1) or 1200 and 1500 h (replicate 2), and blood samples were collected for plasma cortisol concentration. Cows in the outdoor environment had higher vaginal and surface temperatures, respiration rates, and sweating rates than cows in the indoor environment. In both environments, slick-haired cows had lower vaginal temperatures (indoor: 39.0 vs. 39.4 degrees C; outdoor 39.6 vs. 40.2 degrees C; SEM = 0.07) and respiration rate (indoor: 67 vs. 79 breaths/min; outdoor 97 vs. 107 breaths/min; SEM = 5.5) than wild-type cows and greater sweating rates in unclipped areas of skin (indoor: 57 vs. 43 g.h(-1)/m(2); outdoor 82 vs. 61 g.h(-1)/m(2); SEM = 8). Clipping the hair at the site of sweating measurement eliminated the difference between slick-haired and wild-type cows. Results indicate that slick-haired Holstein cows can regulate body temperature more effectively than wild-type cows during heat stress. One reason slick-haired animals are better able to regulate body temperature is increased sweating rate.

Periodontal disease in gestational and type 1 diabetes mellitus pregnant women

Objective: The present study evaluated the relationship between periodontal disease and its clinical variables in Brazilian non-diabetic pregnant women (C), gestational diabetes mellitus (GDM), or type 1 diabetes mellitus (T1DM). Subjects and methods: A periodontal exam was performed in one hundred and sixty-one pregnant women (GDM:80; T1DM:31; C:50) by a single-blinded calibrated examiner who recorded plaque index (PI), gingival index (GI), bleeding index (BI), gingival margin location (GM), probing depth (PD), clinical attachment level (CAL), bleeding on probing (BOP), and tooth mobility index (MI). The medical variables were age, pregestational body mass index (pre-BMI), fasting plasma glucose (FPG), and glycated hemoglobin (HbA(1c)). Results: The GI, GM, PD, CAL, BOP, and MI were significantly higher (P < 0.01) among GDM and T1DM than for C. The PI was higher in GDM and similar between C and T1DM. The Adjusted Final Model for medical variables to evaluate the effects of groups on periodontal parameters confirmed these results. Conclusions: The presence of periodontal disease was significantly higher in Brazilian diabetic pregnancies (GDM and T1DM) when compared to non-diabetic pregnant women (C). The degree of periodontal disease was similar between the GDM and T1DM groups. Age, pregestational BMI, and HbA(1c) were factors related to CAL development in these two types of diabetes mellitus.

Cardiovascular responses to different stages of restorative dental treatment unaffected by local anaesthetic type

Background: The aim of this study was to evaluate the cardiovascular effects of maxillary infiltration using 2% lidocaine with 1: 100 000 adrenaline, 4% articaine with 1: 200 000 adrenaline, and 4% articaine with 1: 100 000 adrenaline in different stages during restorative dental procedures. Methods: Twenty healthy patients randomly received 1.8 mL of the three local anaesthetics. Systolic blood pressure, average blood pressure, diastolic blood pressure, and heart rate were evaluated by the oscillometric and photoplethysmograph methods in seven stages during the appointment. Results: Statistical analysis by ANOVA and Tukey tests of cardiovascular parameters did not show significant differences between the anaesthetic associations. There were significant differences for the parameters among different clinical stages. Conclusions: The variation of cardiovascular parameters was similar for lidocaine and articaine with both adrenaline concentrations and showed no advantage of one drug over the other. Cardiovascular parameters were influenced by the stages of the dental procedures, which showed the effect of anxiety during restorative dental treatment.

Image quality assessment and medical physics evaluation of different portable dental X-ray units

Introduction: Recently developed portable dental X-ray units increase the mobility of the forensic odontologists and allow more efficient X-ray work in a disaster field, especially when used in combination with digital sensors. This type of machines might also have potential for application in remote areas, military and humanitarian missions, dental care of patients with mobility limitation, as well as imaging in operating rooms. Objective: To evaluate radiographic image quality acquired by three portable X-ray devices in combination with four image receptors and to evaluate their medical physics parameters. Materials and methods: Images of five samples consisting of four teeth and one formalin-fixed mandible were acquired by one conventional wall-mounted X-ray unit, MinRay (R) 60/70 kVp, used as a clinical standard, and three portable dental X-ray devices: AnyRay (R) 60 kVp, Nomad (R) 60 kVp and Rextar (R) 70 kVp, in combination with a phosphor image plate (PSP), a CCD, or a CMOS sensor. Three observers evaluated images for standard image quality besides forensic diagnostic quality on a 4-point rating scale. Furthermore, all machines underwent tests for occupational as well as patient dosimetry. Results: Statistical analysis showed good quality imaging for all system, with the combination of Nomad (R) and PSP yielding the best score. A significant difference in image quality between the combination of the four X-ray devices and four sensors was established (p < 0.05). For patient safety, the exposure rate was determined and exit dose rates for MinRay (R) at 60 kVp, MinRay (R) at 70 kVp, AnyRay (R), Nomad (R) and Rextar (R) were 3.4 mGy/s, 4.5 mGy/s, 13.5 mGy/s, 3.8 mGy/s and 2.6 mGy/s respectively. The kVp of the AnyRay (R) system was the most stable, with a ripple of 3.7%. Short-term variations in the tube output of all the devices were less than 10%. AnyRay (R) presented higher estimated effective dose than other machines. Occupational dosimetry showed doses at the operator`s hand being lowest with protective shielding (Nomad (R): 0.1 mu Gy). It was also low while using remote control (distance > 1 m: Rextar (R) < 0.2 mu Gy, MinRay (R) < 0.1 mu Gy). Conclusions: The present study demonstrated the feasibility of three portable X-ray systems to be used for specific indications, based on acceptable image quality and sufficient accuracy of the machines and following the standard guidelines for radiation hygiene. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Development of the osteoblastic phenotype in human alveolar bone-derived cells grown on a collagen type I-coated titanium surface

The aim of this study was to evaluate the development of the osteoblastic phenotype in human alveolar bone-derived cells grown on collagen type I-coated titanium (Ti) surface (Col-Ti) obtained by plasma deposition acrylic acid grafting compared with machined Ti (M-Ti). Osteoblastic cells were cultured until subconfluence and subcultured on Col-Ti and M-Ti for periods of up to 21 days. Cultures grown on Col-Ti and M-Ti exhibited similar cell morphology. Cell adhesion, total protein content, and alkaline phosphatase (ALP) activity were not affected by Ti surface modification in all evaluated periods. Growth analyses indicated that there were significantly more cells in cultures grown on Col-Ti at day 3. Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteoprotegerin (OPG) mRNA expression of cells subcultured on Col-Ti was higher, whereas collagen type I (COL) was lower compared with M-Ti. Ti surface modification neither affected the osteocalcin (OC), ALP and receptor activator of NF-kappa B ligand (RANKL) mRNA expression nor the calcium content extracted from mineralized matrix. These results demonstrated that Col-Ti favours cell growth during the proliferative phase (day 3) and osteoblastic differentiation, as demonstrated by changes in mRNA expression profile during the matrix mineralization phase (day 14), suggesting that this Ti surface modification may affect the processes of bone healing and remodelling. To cite this article:Assis AF, Beloti MM, Crippa GE, de Oliveira PT, Morra M, Rosa AL. Development of the osteoblastic phenotype in human alveolar bone-derived cells grown on a collagen type I-coated titanium surface.Clin. Oral Impl. Res. 20, 2009; 240-246.doi: 10.1111/j.1600-0501.2008.01641.x.