[A 55-year-old male with Raynaud's phenomenon, cutaneous lesions and focal seizures].
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Humans↗
Biomedical subjects
J De La Fuente
Publications and source records attributed to J De La Fuente.
Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression.
We have previously shown that, while the intrinsic quality of the oocyte is the main factor affecting blastocyst yield during bovine embryo development in vitro, the main factor affecting the quality of the blastocyst is the postfertilization culture conditions. Therefore, any improvement in the quality of blastocysts produced in vitro is likely to derive from the modification of the postfertilization culture conditions. The objective of this study was to examine the effect of the presence or absence of serum and the concentration of BSA during the period of embryo culture in vitro on 1) cleavage rate, 2) the kinetics of embryo development, 3) blastocyst yield, and 4) blastocyst quality, as assessed by cryotolerance and gene expression patterns. The quantification of all gene transcripts was carried out by real-time quantitative reverse transcription-polymerase chain reaction. Bovine blastocysts from four sources were used: 1) in vitro culture in synthetic oviduct fluid (SOF) supplemented with 3 mg/ml BSA and 10% fetal calf serum (FCS), 2) in vitro culture in SOF + 3 mg/ml BSA in the absence of serum, 3) in vitro culture in SOF + 16 mg/ml BSA in the absence of serum, and 4) in vivo blastocysts. There was no difference in overall blastocyst yield at Day 9 between the groups. However, significantly more blastocysts were present by Day 6 in the presence of 10% serum (20.0%) compared with 3 mg/ml BSA (4.6%, P < 0.001) or 16 mg/ml BSA (11.6%, P < 0.01). By Day 7, however, this difference had disappeared. Following vitrification, there was no difference in survival between blastocysts produced in the presence of 16 mg/ml BSA or those produced in the presence of 10% FCS; the survival of both groups was significantly lower than the in vivo controls at all time points and in terms of hatching rate. In contrast, survival of blastocysts produced in SOF + 3 mg/ml BSA in the absence of serum was intermediate, with no difference remaining at 72 h when compared with in vivo embryos. Differences in relative mRNA abundance among the two groups of blastocysts analyzed were found for genes related to apoptosis (Bax), oxidative stress (MnSOD, CuZnSOD, and SOX), communication through gap junctions (Cx31 and Cx43), maternal recognition of pregnancy (IFN-tau), and differentiation and implantation (LIF and LR-beta). The presence of serum during the culture period resulted in a significant increase in the level of expression of MnSOD, SOX, Bax, LIF, and LR-beta. The level of expression of Cx31 and Cu/ZnSOD also tended to be increased, although the difference was not significant. In contrast, the level of expression of Cx43 and IFN-tau was decreased in the presence of serum. In conclusion, using a combination of measures of developmental competence (cleavage and blastocyst rates) and qualitative measures such as cryotolerance and relative mRNA abundance to give a more complete picture of the consequences of modifying medium composition on the embryo, we have shown that conditions of postfertilization culture, in particular, the presence of serum in the medium, can affect the speed of embryo development and the quality of the resulting blastocysts. The reduced cryotolerance of blastocysts generated in the presence of serum is accompanied by deviations in the relative abundance of developmentally important gene transcripts. Omission of serum during the postfertilization culture period can significantly improve the cryotolerance of the blastocysts to a level intermediate between serum-generated blastocysts and those derived in vivo. The challenge now is to try and bridge this gap.
Animals↗
Influence of glucose on the sex ratio of bovine IVM/IVF embryos cultured in vitro.
The effect of glucose in the medium used during in vitro culture on the sex ratio of bovine blastocysts derived from in-vitro-matured and in-vitro-fertilized oocytes was evaluated. Oocytes were matured and inseminated with mixed sperm from three bulls and were cultured in vitro in modified synthetic oviducal fluid medium with 10% fetal calf serum, with or without glucose supplementation. The overall rate of cleaved embryos that developed to expanded blastocyst in the medium without glucose (27.0%) was significantly greater (P < 0.05) than the percentage observed when embryos were cultured in medium with glucose (17.5%). Analysis of variance was performed to analyse the effect of glucose on the proportion of male embryos reaching the blastocyst stage (or arrested at the morula stage) during Days 7 to 10. Regardless of the presence or absence of glucose in the medium, significantly (P < 0.05) more male than female embryos were harvested as expanded blastocysts on Day 7 and on Day 8 of culture. On Days 9 plus 10 of culture, a sex ratio imbalance only occurred in the absence of glucose in the culture medium (P < 0.05). Glucose did not produce any significant effect on the sex ratio of the overall number of expanded blastocysts harvested by Day 10 of in vitro culture. However a significantly greater proportion of females (P < 0.01) were found among those embryos that developed only to the morulae stage after 10 days in vitro. These results show that glucose supplementation of culture media produces a preferential loss of female embryos during culture to the blastocyst stage.
Animals↗
Differential expression of two genes located on the X chromosome between male and female in vitro-produced bovine embryos at the blastocyst stage.
The potentially unbalanced expression at preimplantation developmental stages of X-linked genes might be responsible of the faster development of male than female embryos in vitro. Two genes located on the X chromosome, glucose-6-phosphate dehydrogenase (G6PD) and hypoxanthine phosphoribosyl transferase (HPRT), are involved in controlling the amount of oxygen radicals, and hence they might have influence in embryo development. We have quantified mRNA expression of these two genes, using in vitro fertilized-in vitro cultured male and female bovine embryos. In vitro-produced early blastocysts obtained at days 7 and 8 were collected and biopsied for gender determination, and the remaining embryos were kept in LN(2) until RNA purification. After sex determination, embryos were pooled in groups of 3 males or 3 females, and mRNA was purified. Using a semiquantitative sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we detected G6PD and HPRT mRNA expression at the early blastocyst stage in all bovine embryos analyzed. Moreover, mRNA expression of both genes studied was significantly higher in female embryos than in male embryos. The differential expression of G6PD and HPRT at these early stages confirm that sex differences are evident prior to gonadal differentiation and that preimplantation bovine embryos have sexually dimorphic gene expression at least with respect to G6PD and HPRT transcripts. These differences might be responsible of the faster development in culture of in vitro-produced male bovine that has been reported. Mol. Reprod. Dev. 55:146-151, 2000.
Animals↗
Effect of reduced concentrations of glycerol and various macromolecules on the cryopreservation of mouse and cattle embryos.
The effect of different macromolecules [bovine serum albumin (BSA), Pluronic F-68, (ET surfactant), or sodium hyaluronate (SH)] on postthaw survival of mouse morulae and in vivo- and in vitro-derived bovine blastocysts frozen in 10, 5, or 1% glycerol solutions was investigated. Embryos were equilibrated with cryoprotectant solution at 25 degrees C for 10 min, seeded at -5 degrees C, cooled at 0.5 degrees C/min to -35 degrees C, and plunged into liquid nitrogen. Embryos were thawed in a 35 degrees C water bath, glycerol was removed with 0.6 M sucrose at 25 degrees C for 5 min, and postthaw viability was evaluated after 1, 24, and 48 h in culture. The addition of BSA supplementation improved postthaw survival of mouse morulae frozen in 5% glycerol, but not in 10% glycerol. All three macromolecular supplements were effective in increasing survival of mouse morulae in 5% glycerol but only BSA and SH were effective in increasing postthaw survival of in vivo- and in vitro-derived bovine blastocysts. None of the macromolecular supplements improved postthaw survival of embryos frozen in 1% glycerol.
Animals↗
Immunological control of ticks through vaccination with Boophilus microplus gut antigens.
The control of tick infestations and the transmission of tick-borne diseases remain a challenge for the scientific community. Traditional control methods have been only partially successful. Recently, vaccination with recombinant Boophilus microplus gut antigens has been shown to control tick infestations. Our Bm86-containing vaccine formulation (Gavac) has been effective for the control of artificial infestations of B. annulatus, B. decoloratus, and chemically sensitive and resistant B. microplus strains from Australia, Africa, America, and Iran. Preliminary results with Hyalomma spp. and Rhipicephalus spp. suggest partial cross protection. In field trials, vaccination with Gavac controlled B. microplus and B. annulatus infestations and reduced the transmission of babesiosis, resulting in important savings for the cattle industry. Different degrees of susceptibility to the vaccination with Bm86 and sequence variations in the Bm86 locus have been reported. The Bm95 antigen was isolated from the Argentinean Bm86-resistant B. microplus strain A. A Bm95-based vaccine was used to protect cattle against tick infestations under production conditions with similar results to that obtained with Gavac. The Bm95 antigen from strain A was able to protect against infestations with Bm86-sensitive and Bm86-resistant tick strains, thus suggesting that Bm95 could be a more universal antigen in protecting cattle against infestations by B. microplus strains from different geographical areas. These results clearly demonstrate the advantage and possibilities for the immunological control of ticks.
Animals↗
A simulation study of the effects of acaricides and vaccination on Boophilus cattle-tick populations.
This paper describes a simulation model to evaluate different control strategies for Boophilus microplus. The model combines a dynamic life-history module for tick-population dynamics with other modules for vaccination, sterile-hybrid larval release and use of acaricide dipping vats. The tick life-history module considers the cattle's nutritional level and allows for distribution of ticks by age at all stages of growth. Appropriately, the model was sensitive to host resistance and to host-nutritional status. The validity of the life-history module--as well as that of the vaccination and acaricide dipping--vats modules--was demonstrated by comparing simulated and real data for several geographical locations in Cuba and Brazil. Optimum tick-control strategies for the first year of vaccination were designed and the effect of long-term vaccination on tick population was also studied.
Animals↗
Relationship between sex ratio and time of insemination according to both time of ovulation and maturational state of oocyte.
The aim of this study was to explore how some reproductive methodologies may affect the sex ratio. We first confirmed the association between the maturation stage of bovine oocytes at the time of in vitro fertilisation (IVF) and the sex ratio of in vitro-derived embryos. Secondly, we studied whether the time of insemination, prior to or after ovulation, could alter the sex ratio in sheep. In the first experiment, bovine oocytes were matured in vitro for 16 h; then oocytes were either fertilised in vitro immediately after extrusion of the first polar body or IVF was delayed for 8 h. The proportion of cleaving embryos and their development to the 8-cell stage was enhanced with delayed insemination. Moreover, delaying IVF produced a male-to-female sex ratio of 1.67:1.00, which was significantly different from the expected 1:1 ratio (p < 0.05), whereas more female embryos were produced when oocytes were fertilised in vitro immediately after polar body extrusion (sex ratio of 1.00:0.67; p < 0.05). In the second experiment, 380 ewes were inseminated at different times before or after ovulation, producing 537 lambs. Significant differences in the sex ratio were obtained when we compared the sex of the offspring of ewes inseminated during the 5 h preceding ovulation (more females) with those inseminated during the 5 h after ovulation (more males). Our results suggest that the differential ability of X- or Y-bearing spermatozoa to fertilise oocytes depending either on time of insemination or oocyte maturation state, may be due, at least partially, to 'intrinsic' differences in the physiological activity of X- or Y-bearing spermatozoa before fertilisation.
Animals↗
Altered frontostriatal relationship in unmedicated schizophrenic patients.
Positron emission tomography with [18F]-fluoro-2-deoxy-D-glucose as tracer was used to investigate frontal glucose metabolism in 15 unmedicated schizophrenic patients and 15 healthy subjects under resting conditions. Although no difference in absolute frontal cerebral metabolic rates of glucose (CMRglu) were found between schizophrenic patients and control subjects, relative measures significantly differentiated the two groups. Whole frontal metabolism and frontocaudate ratio were significantly decreased in both hemispheres in the patients. The results confirm the existence of hypofrontality in unmedicated schizophrenia and indicate disturbances in metabolic relationships between the frontal cortex and the striatum in this disorder.
Adult↗
Detection of Bm86 antigen in different strains of Boophilus microplus and effectiveness of immunization with recombinant Bm86.
The control of tick populations by using conventional strategies poses several problems, including the appearance of organophosphate resistant strains, among others. The possibility of using alternative strategies such as vaccination with tick antigens has been suggested by several authors. One particular antigen (Bm86) has been described and shown to be able to induce a protective immunity against the cattle tick Boophilus microplus. In this paper we demonstrate by means of immunohistochemical staining that this antigen is conserved among several strains of this species. These results correlate with those showing that animals vaccinated with a preparation of recombinant Bm86 were protected against challenge with the four different strains tested, including one resistant to organophosphates. These results favour the immunization with recombinant Bm86 for the control of the cattle tick B. microplus.
Animals↗
Does "mycobacterioascites" exist?
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Adenosine Deaminase↗
Effect of post-thaw cell rehydration at 4 degrees C on survival of frozen and vitrified IVP-derived bovine embryos.
In vitro-produced bovine embryos (IVP) were either frozen in 10% glycerol in a phosphate-buffered saline solution (PBS) using conventional slow freezing or vitrified in 25% glycerol and 25% ethylene glycol in PBS. The results of viability and hatching rates were compared between frozen and vitrified embryos after thawing and dilution using one of three different protocols: (A) a three-step dilution procedure, (B) a one-step dilution procedure or (C) a procedure in which embryos were kept in situ inside the straw at 4 degrees C for 10 min during a one-step dilution procedure. No significant differences in embryo survival were found among protocols A, B and C for frozen embryos and between protocols A and B for vitrified embryos. Viability and hatching rates of vitrified embryos thawed and diluted by protocol C (73 and 62%) were significantly enhanced (P < 0.05) in comparison to those obtained with protocol A (55 and 41.6%) or protocol B (54.5 and 35.3%). These results indicate that for vitrified IVP bovine embryos, direct in-straw rehydration at 4 degrees C for 10 min improves embryo survival and it could be a practical procedure for use under field conditions where there is sometimes a longer interval between thawing and transfer.
Animals↗