Luiz Sérgio de Almeida Camargo

Interrelationships among morphology, echotexture, and function of the bovine corpus luteum during the estrous cycle.

It has been suggested that ultrasound image attributes are a potential indicator of physiological and functional status of the corpus luteum (CL) in several species, including cattle. The aims of this study were to evaluate CL morphological, functional and echotextural characteristics, and also to investigate the hypothesis that those attributes are correlated and change similarly throughout an estrous cycle. Ovaries of crossbred (Bos taurus taurus x Bos taurus indicus) heifers were evaluated using ultrasonography daily throughout an interestrus interval using a B-mode, real-time ultrasound machine equipped with a 5 MHz linear-array rectal transducer, during a natural estrous cycle (Experiment 1; n=12) or during a shortened cycle, with luteolysis induction 10d after estrus (Experiment 2; n=6). Blood samples were collected for assay of plasma progesterone concentrations. Corpora lutea areas were measured and daily images of each CL were videotaped and digitized for computer-assisted analysis using custom-developed software. In Experiment 1, area of luteal tissue increased until a maximum value 10d after estrus (P<0.001), followed by a plateau phase, and then a decline beginning 14 d after estrus. Luteal tissue area was highly correlated to plasma progesterone concentrations (r=0.86; P<0.001). When luteolysis was induced in Experiment 2, loss of CL function (decrease in plasma progesterone concentrations to metestrous values) preceded tissue regression by 48 h (24h compared with 72 h; P<0.001). The mean pixel value of ultrasound images did not change in Experiment 1 (P>0.70), but a day effect on this attribute was observed in Experiment 2 (P=0.052). In contrast, mean pixel value was correlated to plasma progesterone concentrations in Experiment 1 (r=-0.63; P<0.05), but not in Experiment 2 (r=-0.28; P>0.10). In regard to CL heterogeneity, defined as the standard deviation of the mean pixel value of the luteal tissue, a time effect was observed following both natural (Experiment 1; P<0.009) and luteolysis-induced (Experiment 2; P<0.05) estrous cycles (P<0.05). Moreover, this variable was correlated with plasma progesterone concentrations (r=-0.71 and -0.58 in Experiments 1 and 2, respectively; P<0.01), indicating that CL images were more heterogeneous during metestrus and after luteolysis (functional regression). In summary, morphological and echotextural attributes were correlated with CL function and underwent similar changes during the estrous cycle. Luteal tissue heterogeneity, assessed by ultrasonography, is considered a potential indicator of CL functional status, because it is correlated to circulating progesterone concentrations.

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 22-24h 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 in 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.

Gestation length, birth weight and offspring gender ratio of in vitro-produced Gyr (Bos indicus) cattle embryos

In vitro embryo production (IVP) has been suggested to result in a greater proportion of male calves, longer gestation and heavier offspring than artificial insemination in Bos taurus cattle. Despite the increasing use of IVP in tropical countries, its effects upon these traits in Bos indicus have not been conclusively investigated. Gyr is a B. indicus dairy breed with known physiological differences from B. taurus, such as a longer gestation period and lighter offspring. The aim of this study was to evaluate the effect of IVP on gestation length, birth weight and gender ratio in Gyr offspring. Oocytes were recovered from Gyr cows by ovum pick-up and were matured and fertilized with thawed Gyr semen in vitro. Embryos were cultured in CR2aa medium with cumulus cells and 10% fetal calf serum under 5% CO(2) at 38.5 degrees C in air. Seven- to eight-day blastocysts were transferred to synchronized recipients. Data on gestation length and birth weight of calves from in vitro-produced embryos were compared to data obtained from Gyr calves produced by artificial insemination (AI) and natural breeding (NB) during the same period using analysis of variance, and the gender ratio was compared to the expected 1:1 ratio using a chi-square test. IVP increased (P<0.01) the percentage of male offspring (76.9%) compared to the expected 1:1 ratio, while no difference (P>0.05) was observed in the AI and NB groups. Gestation length was similar (P>0.05) between the IVP and AI groups, but IVP-derived offspring were heavier (P<0.05) than AI- and NB-derived ones, mainly for male calves (P<0.05). These data show that in vitro production affects the subsequent development of Gyr embryos, resulting in a skewed sex ratio and increased birth weight.

Developmental competence and expression of the MATER and ZAR1 genes in immature bovine oocytes selected by brilliant cresyl blue.

The objective of this work was to evaluate the selection of immature bovine oocytes by brilliant cresyl blue dye (BCB) and expression of transcripts MATER and ZAR1. Cumulus-oocyte complexes (COCs) from slaughterhouse ovaries were exposed to BCB diluted in mDPBS and incubated for 60 min at 38.5 degrees C in humidified air. After exposure those COCs were distributed in two groups, according to their cytoplasm colour: BCB+ (coloured cytoplasm) or BCB- (colourless cytoplasm). The control group was submitted to in vitro maturation (IVM) immediately after morphological selection and holding control group COCs were exposed to mDPBS without BCB but in the same incubation conditions of BCB+ and BCB- group. The COCs of all groups were submitted to IVM, in vitro fertilization (IVF) and in vitro culture (IVC). Cleavage rate (72 h post-insemination) was similar between control (65.3%) and BCB+ (64.4%) groups, but greater than (p < 0.05) holding control (49.8%) and BCB- (51.3%) groups. Blastocyst rate (192 h post-insemination) was not different between BCB+ (18.5%) and control (16.3%) groups, but greater (p < 0.05) than BCB- (8.4%) group. No difference was found for blastocyst rate between holding control group (14.2%), control and BCB+ groups. The relative expression of MATER and ZAR1 genes was evaluated by real-time PCR in immature oocytes collected from the control, holding control, BCB+ and BCB- groups. Despite the relative expression of MATER in holding control, BCB+ and BCB- were down regulated in comparison to control group there was no statistical difference (p > 0.05) in the relative expression of MATER and ZAR1 transcripts among groups. The results indicate that the BCB dye detects immature oocyte populations with different developmental competence, although no improvement in in vitro embryo production using oocytes exposed or not to BCB was observed. Development competence of immature oocytes exposed to BCB does not seem to be associated with variations in the expression of MATER and ZAR1 transcripts.

Osmotic challenge and expression of aquaporin 3 and Na/K ATPase genes in bovine embryos produced in vitro

The aim of this study was to evaluate the effect of culture media and stage of development in the osmotic ability of in vitro-fertilized bovine embryos and the expression of aquaporin 3 (Aqp3) and Na/K ATPase isoform 1 (ATPAse1) genes in embryos (i) with different ability to undergo rehydration and (ii) following vitrification. In experiment 1, in vitro fertilized presumptive zygotes were co-cultured in SOFaac or modified CR2aa medium and embryos at blastocyst and expanded blastocyst stages at day 7 post-insemination were exposed to NaCl hypertonic medium (900 mOsm) for 5 min following 120 min of culture in isotonic medium in order to evaluate dehydration and rehydration, respectively. No difference (P>0.05) on blastocyst rate was found between CR2aa and SOFaac medium but embryos co-cultured in SOFaac medium underwent greater (P<0.05) dehydration. Embryos at expanded blastocyst stage underwent greater dehydration but slower rehydration than embryos at blastocysts stage (P<0.05). In the experiment 2, the amount of Aqp3 and ATPase1 transcripts were quantified in blastocysts with high or low rehydration after exposure to hypertonic medium. No difference (P>0.05) on relative amount of transcripts was found in either genes. In the experiment 3, expanded blastocysts produced in a co-culture system were vitrified, warmed and then cultured for 72 h for analysis of embryo survival and amount of Aqp3 and ATPase1 transcripts. Lower (P<0.05) embryo survival rate was found for vitrified-warmed embryos (57.9%) than for their fresh counterparts (84.6%). There was no difference on expression of ATPase1 gene but lower (P<0.01) amount of Aqp3 transcripts was found in the vitrified-warmed embryos. In conclusion, embryo ability to undergo shrinkage and swelling is influenced by medium used in a co-culture system and by embryo stage. Rehydrating ability of embryos after exposure to NaCl hypertonic medium is not associated with variations on expression of Aqp3 and ATPase1 genes, but the vitrification can alter gene expression of in vitro-fertilized bovine embryos produced in a co-culture system.

Effect of oxygen tension and serum during IVM on developmental competence of bovine oocytes.

With an aim to improve the in vitro production of bovine embryos, the present study investigated the effect of serum and oxygen tension during IVM on oocyte developmental competence. Four experimental groups were evaluated: G1, 10% oestrus cow serum (OCS) with 20% O(2); G2, 0.1% polyvinyl alcohol (PVA) with 20% O(2); G3, 10% OCS with 5% O(2); and G4, 0.1% PVA with 5% O(2). The proportion of MII oocytes, blastocyst rates and total cell number were not affected (P > 0.05) when the OCS was replaced with PVA under 5% O(2), whereas a higher (P < 0.05) blastocyst rate and total cell number were found with OCS compared with PVA under 20% O(2). The apoptosis index was lower in blastocysts from oocytes matured with PVA under 5% O(2) (G4) compared with other groups (G1, G2 and G3), but no differences (P > 0.05) were found in maturation and blastocyst rates. Significant differences were found in the amount of specific transcripts in oocytes matured under different conditions. In conclusion maturation with PVA and 5% O(2) provides an efficient in vitro culture condition for the maturation of bovine oocytes.

Caracterização de seqüelas subseqüentes à punção folicular em bovinos

Objetivaram-se avaliar lesoes no trato genital em animais utilizados como doadores de oocitos. Foram examinadas vacas da raca Gir (n=20) que participaram de diferentes experimentos envolvendo a tecnica de puncao folicular. Ao termino de cada periodo experimental, os animais foram examinados por palpacao retal, ultra-sonografia e vaginoscopia. Um grupo de vacas (n=8) foi selecionado para a caracterizacao histopatologica das lesoes ovarianas. Os ovarios foram retirados cirurgicamente, fixados e processados, e as lâminas obtidas coradas e analisadas. Foram avaliados animais submetidos a entre nove e 42 sessoes de puncao, e que produziram entre 10 e 719 oocitos. Observou-se uma baixa prevalencia (5,26%) de processos inflamatorios na mucosa do fundo de saco vaginal. A ocorrencia de aderencias apresentou uma correlacao positiva com o numero de sessoes de puncao a que o animal foi submetido (r=0,31; P 0,05). Tendencia oposta foi observada em relacao a fibrose ovariana, que apresentou uma alta correlacao com o numero de oocitos recuperados (r=0,53; P<0,05). A avaliacao histopatologica demonstrou a presenca de tecido cicatricial, infiltrados de celulas inflamatorias e presenca de tecido luteal disperso no estroma ovariano.

Post-biopsy bovine embryo viability and whole genome amplification in preimplantation genetic diagnosis.

OBJECTIVE To evaluate the effect of the biopsy of 8-cell to 16-cell bovine embryos on their subsequent development and the effect of whole genome amplification (WGA) on removed blastomeres. DESIGN Randomized study. SETTING Molecular genetics and animal reproduction laboratories. PATIENT(S) Cow ovaries obtained from slaughterhouses. INTERVENTION(S) The ovaries were punctured, and the oocytes were matured and fertilized in vitro. On the fourth day after fertilization, 8-cell to 16-cell bovine embryos were biopsied, one quarter of each embryo being removed. The blastomeres were submitted to WGA followed by polymerase chain reaction (PCR). The embryos were returned to culture for evaluation of their development. MAIN OUTCOME MEASURE(S) Subsequent rate of blastocyst development, embryo cell number, WGA efficiency, and sex determination. RESULT(S) A total of 92 embryos were submitted to biopsy. The blastocyst production was 53.3%, with 44.9% of hatching rate. These results were similar to those of the control group (66.0% and 42.6%) of 103 embryos. Overall, no impact was detected on embryo quality in blastocyst cell number between the two groups. Removed blastomeres were submitted to WGA, resulting in 98.2% of efficiency. However, only 59% of the samples were sexed by PCR. CONCLUSION(S) Biopsy of 8-cell to 16-cell bovine embryos did not affect their subsequent development. WGA was successful in removed blastomeres.

Effect of crotamine, a cell-penetrating peptide, on blastocyst production and gene expression of in vitro fertilized bovine embryos.

The present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 μM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 μM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 μM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 μM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 μM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1-10 μM for 6-24 h.

Efficient delivery of DNA into bovine preimplantation embryos by multiwall carbon nanotubes.

The pellucid zone (PZ) is a protective embryonic cells barrier against chemical, physical or biological substances. This put, usual transfection methods are not efficient for mammal oocytes and embryos as they are exclusively for somatic cells. Carbon nanotubes have emerged as a new method for gene delivery, and they can be an alternative for embryos transfection, however its ability to cross the PZ and mediated gene transfer is unknown. Our data confirm that multiwall carbon nanotubes (MWNTs) can cross the PZ and delivery of pDNA into in vitro-fertilized bovine embryos. The degeneration rate and the expression of genes associated to cell viability were not affected in embryos exposed to MWNTs. Those embryos, however, had lower cell number and higher apoptotic cell index, but this did not impair the embryonic development. This study shows the potential utility of the MWNT for the development of new method for delivery of DNA into bovine embryos.