R. F. Gonçalves

Lipid characterization of embryo zones by silica plate laser desorption ionization mass spectrometry imaging (SP-LDI-MSI)

Lipid pathways play important biological roles in mammalian embryology, directing early developmental pathways to differentiation. Phospholipids and triglycerides, among others, are the main composing lipids of zona pellucida in several embryo species. Lipid analysis in embryos by mass spectrometry usually requires sample preparation and/or matrix application. This novel approach using silica plate laser desorption/ionization mass spectrometry imaging (SP-LDI-MSI) allows direct single-cell imaging and embryo region discrimination with no matrix coating. Its application is herein described for two- and eight-cell embryos. Lipid biomarkers for blastomere and intact zona pellucida are reported and corroborated by both fragmentation reactions (MS/MS) and images. Results obtained in this work are understood to be of great use for further developments on in vitro bovine fertilization. Since much of the processes can be monitored by characteristic biomarkers, it is now possible to precisely identify cell division errors during early embryo stages, as well as evaluate pre-implantation conditions.

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 microl 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.

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% CO(2) in air) for 1h in the following treatments either 500 microL 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 1h either FM or FM with alpha 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) alpha L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) alpha 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.

A Metabolomic Overview of Follicular Fluid in Cows

Follicular fluid (FF) protects the oocyte against proteolysis and extrusion during ovulation, providing an appropriate microenvironment that favors proper embryonic development; thereby, FF plays a key role in embryo quality. Being directly related to cattle breeding, studying FF is extremely important in livestock science to measure cattle fertility. This may eventually help to assess the quality of both meat and milk, products widely consumed worldwide. There is an important commercial interest in the evaluation and characterization of compounds present in the FF of livestock that present greater likelihood of pregnancy. Mass spectrometry is a great ally for this type of analysis and can provide quick and efficient screening for molecular markers in biological samples. The present study demonstrated the potential of high-resolution mass spectrometry in analyzing FF samples from two distinct groups of Nellore cows (Bos indicus): high and low fertility, as determined by the number of oocytes produced. We were able to delineate markers of interest for each group, which may ultimately be related to biochemical pathways that lead to higher or lower reproductive performance.

In vitro interaction of bovine herpesvirus 1 with uterine tube epithelial cells and oocytes

The aims of this study were to assess in vitro if bovine oocytes and oviductal epithelial cells from slaughterhouses for in vitro fertilization use may be infected with bovine herpesvirus 1; to analyze whether the treatment with trypsin according to the International Embryo Transfer Society guideline is efficient to inactivate the bovine herpesvirus 1; to morphologically study the virus-oocyte interaction through optical microscopy. In this study, Madin Darby Bovine Kidney (MDBK) cells that were co-cultured with oocytes matured in vitro and exposed to bovine herpesvirus 1 showed a cytopathic effect. The nested polymerase chain reaction for the supernatant was positive for the bovine herpesvirus 1, thus suggesting that the cytopathic effect observed in the MDBK monolayer was seen due to virus replication and not because of any culture toxicity. It was also observed cytopathic effect and positive nested polymerase chain reaction in MDBK cells co-cultured with in vitro maturated oocytes free of virus, but that were co-cultured in uterine epithelial cells pre-infected with bovine herpesvirus 1 and washed or not with trypsin, demonstrating an oocyte contamination by the virus. When trypsin-washing efficacy was evaluated, we could observe that the trypsin treatment was not able to eliminate the bovine herpesvirus 1 of the oocytes, and it was not observed any morphological difference in the infected oocytes.

203 Pretreatment of bovine sperm or oocyte with antibody to lipocalin-type prostaglandin d synthase inhibits fertilization

Lipocalin-type prostaglandin D synthase (L-PGDS) has been identified in cow uterine tube fluid (UTF), and as fertility-associated protein in the Holstein bull seminal plasma, but its function is unclear. A previous study demonstrated that L-PGDS is associated with the bovine zona pellucida, and that antibody incubated with UTF decreased embryo development in vitro. This study was conducted to determine whether IVF of bovine oocytes would be affected by pretreating either the sperm or oocytes, or both, with L-PGDS antibody. In vitro-matured bovine oocytes were incubated for 1 h in IVF TALP medium supplemented with penicillamine, hypotaurine, epinephrine, and heparin containing (a) no antibody, or (b) a rabbit polyclonal antibody against recombinant bovine L-PGDS (α L-PGDS; 1:2000). Frozen–thawed spermatozoa were washed by a 45:90% layered Percoll gradient centrifugation and incubated for 1 h in IVF TALP with (a) no antibody, or (b) α L-PGDS. For this study we had 4 different treatments: (1) no antibody (control), (2) α L-PGDS at fertilization time, (3) α L-PGDS-treated oocytes, or (4) α L-PGDS-treated sperm. Oocytes were inseminated with 10 × 104 washed spermatozoa in 4-well culture dishes. After 18 h (39°C, 5% CO2 in air), oocytes were vortexed to remove cumulus cells and accessory spermatozoa, and fixed in 3.7% paraformaldehyde and 10% Triton X–100 for 1 h. Oocytes were washed and transferred to a solution with PBS, 0.3% BSA, and 1% Triton for 1 h, stained with Hoechst 33342, and observed in the presence of 2 pronuclei in the cytoplasm. There were 4 replicates of 200 to 250 oocytes for fertilization assays. Data were analyzed by SAS. Addition of α L-PGDS with sperm, oocytes, or both significantly decreased fertilization (P < 0.05) compared with the control: (1) 89.2 ± 2.0%; (2) 19.4 ± 2.0%; (3) 27.2 ± 3.1%; or (4) 14.1 ± 3.4%. These studies demonstrated that a rabbit polyclonal antibody against recombinant bovine L-PGDS reacts with both oocytes and spermatozoa, resulting in inhibition of fertilization in vitro, and has a possible role in bovine fertilization. This study was supported by FAPESP #2007/00363-5 and #2006/06008-0, Brazil.