B. C. Carvalho

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.

Follicular populations, recruitment and atresia in the ovaries of different strains of mice

Follicular atresia is a key event in the selection of the ovulatory follicles and occurs during all developmental stages. The aims of the study were to evaluate the follicular population as well as the rates of follicular recruitment and atresia in different strains of mice. Ovaries were obtained from four strains of mice: G1/ Swiss, G2/ F1 Swiss×C57BL/6, G3/ inbred strain C57BL/6, and G4/ F1 C57BL/6×Swiss. All mice used in the study were 60 days old. Ovaries collected from the mice were fixed and processed for histological analysis. The G2 ovaries were also used to examine immunolocalization of active caspase-3. The pimordial follicle population was smaller in G3 mice than in G1, G2 and G4 groups (7 565±1 845 vs. 17 180±3 159, 14 785±3 319 and 13 325±2 685, respectively; p<0.05). The rate of follicular recruitment in G3, however, was higher than in the other groups (29.2% vs. 18.2%, 17.3% and 13.0% in G1, G2 and G4, respectively; p<0.05), resulting in a similar (p>0.05) number of antral follicles among groups. The small follicular pool in G3 mice was also associated with a lower rate of follicular atresia (11.4% vs. 17.2%, 16.7% and 13.6% for G3, G1, G2 and G4, respectively; p<0.05). The number of follicles stained with active caspase-3 was higher (p<0.05) during the final stage of preantral folliculogenesis than in other stages of follicular development suggesting that apoptosis in mice occurs earlier in comparison to large animals. Thus, it was concluded that differences in follicle reservoir among mice strains are compensated by an increased rate of follicular recruitment and a decreased rate of follicular atresia; and atresia occurs in mice mainly at the end of the preantral stage of folliculogenesis.

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.

Trans-10, cis-12 conjugated linoleic acid reduces neutral lipid content and may affect cryotolerance of in vitro- produced crossbred bovine embryos

BackgroundDue to high neutral lipids accumulation in the cytoplasm, in vitro-produced embryos from Bos primigenius indicus and their crosses are more sensitive to chilling and cryopreservation than those from Bos primigenius taurus. The objective of the present study was to evaluate the effects of trans-10, cis- 12 conjugated linoleic acid (CLA) on the development and cryotolerance of crossbred Bos primigenius taurus x Bos primigenius indicus embryos produced in vitro, and cultured in the presence of fetal calf serum. Bovine zygotes (n = 1,692) were randomly assigned to one of the following treatment groups: 1) Control, zygotes cultured in Charles Rosenkrans 2 amino acid (CR2aa) medium (n = 815) or 2) CLA, zygotes cultured in CR2aa medium supplemented with 100 μmol/L of trans- 10, cis-12 CLA (n = 877). Embryo development (cleavage and blastocyst rates evaluated at days 3 and 8 of culture, respectively), lipid content at morula stage (day 5) and blastocyst cryotolerance (re-expansion and hatching rates, evaluated 24 and 72 h post-thawing, respectively) were compared between groups. Additionally, selected mRNA transcripts were measured by Real–Time PCR in blastocyst stage.ResultsThe CLA treatment had no effect on cleavage and blastocyst rates, or on mRNA levels for genes related to cellular stress and apoptosis. On the other hand, abundance of mRNA for the 1-acylglycerol-3-phosphate 0-acyltransferase-encoding gene (AGPAT), which is involved in triglycerides synthesis, and consequently neutral lipid content, were reduced by CLA treatment. A significant increase was observed in the re-expansion rate of embryos cultured with trans-10, cis- 12 CLA when compared to control (56.3 vs. 34.4%, respectively, P = 0.002). However, this difference was not observed in the hatching rate (16.5 vs. 14.0%, respectively, P = 0.62).ConclusionsThe supplementation with trans-10, cis- 12 CLA isomer in culture medium reduced the lipid content of in vitro produced bovine embryos by reducing the gene expression of 1-acylglycerol 3-phosphate 0-acyltransferase (AGPAT) enzyme. However, a possible improvement in embryo cryotolerance in response to CLA, as suggested by increased blastocyst re-expansion rate, was not confirmed by hatching rates.

Indução do estro no pós-parto em vacas primíparas Holandês-Zebu

The experiment was carried out to evaluate the effect of two ranges of body weight and norgestomet treatment on the reproductive parameters of postpartum crossbred Holstein-zebu cows. Sixty four primiparous cows were randomly allocated to four treatments 40 days after calving: group I - cows with body weight ranging from 390 to 458kg and norgestomet treated; group II - cows with body weight ranging from 464 to 562kg and norgestomet treated; group III - cows with body weight ranging from 390 to 458kg (control); and group IV - cows with body weight ranging from 464 to 562kg (control). Progestagen auricular implants were mantained during 10 days and the cows were mated to bulls submitted to breeding soundness evaluation. Animals from treatment II showed estrus earlier than animals of the others treatments (II: 64.4; I: 109.4; III: 143.2 and IV: 105.1 days; P 0.05). The hormonal treatment and the body weight did not affect the total and daily milk yield, and length of lactation (P>0.05). Progestagen treated, heavier and better body condition scored animals had shorter open days, and returned to postpartum ovarian ciclicity faster than lighter animals.

The effects of increasing amounts of milk replacer powder added to whole milk on mammary gland measurements using ultrasound in dairy heifers

The aim of this study was to determine the effect of increasing total solids (TS) in the liquid diet during the preweaning phase on mammary gland development in dairy heifers. The increase in TS was obtained with the addition of milk replacer powder to whole milk. Crossbreed Holstein-Gyr heifers (n = 60) were distributed in 4 treatments with different TS concentrations: 13.5% (n = 15), 16.1% (n = 15), 18.2% (n = 15), and 20.4% (n = 15). The liquid diets were provided from 5 to 55 d of age. From 56 to 59 d of age, the total amount of liquid feed was reduced by half. Heifers were weaned at 60 d and monitored until 90 d of age. Ultrasound mammary gland evaluations were performed weekly between 5 to 11 wk of age, using a B-mode ultrasound equipped with microconvex transducer at a frequency of 6 MHz. In those same weeks, the manual palpation of mammary parenchyma (PAR) was performed. Increased TS concentration of liquid diet during the preweaning period increased body weight of heifers, but did not alter PAR growth and the deposition of adipose tissue in the mammary gland evaluated by ultrasonography. The oval-shaped structure of PAR was altered after 2 mo of age. In the evaluated period, PAR growth was isometric with respect to the body growth rate. Palpation scoring of PAR had a strong correlation with the ultrasound evaluation of the PAR.

Effect of increasing amounts of milk replacer powder added to whole milk on postweaning performance, reproduction, glucose metabolism, and mammary fat pad in dairy heifers

The aim of this study was to evaluate the effects of increasing the total solids (TS) content of liquid feed, by adding increasing amounts of milk replacer powder to whole milk, on age at puberty and postweaning performance, glucose metabolism, and mammary fat pad of dairy heifers. Crossbreed Holstein × Gyr heifers (n = 58) were distributed into 4 different treatments during the preweaning period. Treatments consisted of liquid feeds with TS content of 12.5 (actual TS = 13.5 ± 0.53%; n = 15), 15.0 (actual TS = 16.1 ± 0.03%; n = 15), 17.5 (actual TS = 18.2 ± 0.14%; n = 13), or 20.0% (actual TS = 20.4 ± 0.24%; n = 15), which were provided up to 59 d old. From 60 to 89 d old, the animals received the same starter offered during the preweaning period in addition to corn silage ad libitum starting at 70 d old. From 90 to 104 d old, the animals were adapted to a total mixed ration. At 105 d old, the animals were distributed in 4 paddocks equipped with electronic feed and water bins and were fed the same total mixed ration ad libitum. A period of adaptation to the electronic feed bins occurred from 105 to 119 d old. Feed and water intake and body weight and body frame development were assessed until puberty. Mammary gland evaluations were performed monthly by ultrasonography from 120 d of age until puberty onset. Puberty onset was determined as plasma progesterone concentration greater than 1 ng/mL in 2 consecutive samples collected 7 d apart. The date of puberty onset was recorded as the collection day of the first of these samples. A glucose tolerance test was performed at 280 d of age. The increased TS content of the liquid feed fed during the preweaning period did not affect dry matter intake, performance, age at puberty, glucose metabolism, or mammary gland fat pad at later stages of rearing process. Conversely, hip width and heart girth increased linearly up to 150 and 240 d of age, respectively, as a result of the increased TS concentration of the liquid feed during the preweaning period.

Teofilina como agente capacitante do sêmen bovino.

RESUMO Objetivou-se avaliar a teofilina como agente capacitante substituto ou associado a heparina sobre a reacao acrossomica dos espermatozoides e o desenvolvimento de embrioes produzidos in vitro. O experimento foi realizado com quatro touros e tres tratamentos, totalizando 12 grupos experimentais. O semen dos touros foi avaliado nos tratamentos descritos a seguir: tratamento 1 (HEP): heparina ? 10µg/mL; tratamento 2 (TEO): teofilina ? 5mM; tratamento 3 (HEP + TEO): heparina (10µg/mL) + teofilina (5mM), por zero, seis, 12 e 18 horas, corados com trypan blue/Giemsa para avaliacao da reacao acrossomica. Para a producao dos embrioes, os agentes capacitantes foram adicionados aos meios de fertilizacao. Na analise espermatica, a taxa de reacao acrossomica verdadeira foi maior (P 0,05) entre touros na analise de reacao acrossomica nem na PIVE. A utilizacao da teofilina foi tao eficiente quanto a da heparina na inducao da reacao acrossomica, no entanto resultou em menores taxas de producao embrionaria.

Carbon Nanotubes as a DNA Delivery Agent for Generation of Genetically Modified Mammals Embryos

Several research centers and pharmaceutical corporations routinely use genetically modified animals (GMAs) in the development of new drugs, in the identification of new drug targets and to test drugs’ efficacy and safety. The most usual methods to produce GMAs are pronuclear microinjection, somatic cell nuclear transfer, retroviral vectors, and recently, embryonic stem cell transgenesis. These methods make use of DNA vectors and present several limitations. Recently, nanomaterials have been applied as an alternative vector for delivery of exogenous DNA into mammalian cells. This chapter addresses the use of carbon nanotubes (CNTs) as a DNA delivery agent for the generation of genetically modified mammals embryos. CNTs can be easily bound to DNA by non-covalent attachement. The DNA strand spontaneously wraps around the carbon nanotubes and DNA molecules can be encapsulated within or around them. The process of interaction of DNA/RNA with CNT favors their protection from degradation by cytoplasmic nucleases, increasing the integration of the transgene into cell nucleus. Thus, the use of CNTs can be far simpler and less laborious when compared to other techniques to produce GMAs.