Effect of Acute and Cumulative Stress on Gene Expression in Mammary Tissue and Their Interactions with Physiological Responses and Milk Yield in Saanen Goats.

This study addresses the hypothesis that different acute stressors can cumulatively decrease milk yield. In fact, in a time of global warming, the impact of environmental stress and farm management practices on milk production remains unclear. In this context, our objective was to investigate the effect of acute and cumulative stress on gene expression in mammary tissue and their interactions with physiological responses and milk yield in Saanen goats. Thirty lactating goats were subjected to two treatments: (1) control (CT), in which goats were maintained following a habitual routine under comfort conditions; (2) stress (ST), in which the goats were subjected to different types of environmental stress: heat stress, adrenocorticotropic hormone administration, hoof care management, and exposure to rain. These stressors were performed sequentially, with one stress per day on four consecutive lactation days, to evaluate their effect on milk quality and milk yield. Our results showed that compared to CT goats, cumulative stress increased the gene expression of glucocorticoid receptor (GR), interferon-gamma (IFN-γ), superoxide dismutase (SOD), and catalase (CAT) in mammary tissue, which are indicators of cortisol action, inflammatory response, and antioxidant enzymes. Furthermore, the acute challenges imposed on ST goats changed their rectal temperature and respiratory frequency and increased cortisol, glucose, cholesterol, triglycerides, and high-density lipoprotein release in plasma when compared to CT goats. Although these physiological and metabolic responses restore homeostasis, ST goats showed lower milk yield and higher somatic cell count in milk than CT goats. In conclusion, the results confirmed our initial hypothesis that different acute stressors cumulatively decrease the milk yield in Saanen goats.

Effect of experimental stress and cortisol release induced by ACTH administration on expression of key genes related to milk synthesis and apoptosis during mammary involution of Saanen goats.

This research paper addresses the hypothesis that stress, induced by ACTH administration and cortisol release increases somatic cell count (SCC) in mammary secretion, and improves the effectiveness of dry off in goats. We report indicators of milk synthesis and mammary gland involution during dry off. Thirty Saanen goats were subjected to abrupt dry off and treatments: (1) ACTH administration (ACTH) or (2) placebo (Control) on days 1, 3, 6, 9, 12, 15, 30, and 60 of dry off. The expression of target genes in mammary tissue that are related to milk synthesis and cell survival such as insulin-like growth factor 1 receptor (IGF1R), phosphatidylinositol-3-kinase (PIK3CA), protein kinase B (AKT1) and mechanistic target of rapamycin (MTOR), casein (CSN2), lactalbumin (LALBA) and lactoferrin (LF) were evaluated, and plasma cortisol concentration, SCC, leucocyte count, and microbiological analyses in milk and mammary secretions were assessed. ACTH significantly downregulated the expression of IGF1R and upregulated the expression of PIK3CA in mammary tissue, increased lactoferrin concentration and SCC, and changed immune cell levels in mammary secretions compared to Control. Furthermore, ACTH administration increased the percentage of dry goats compared to the Control (73 vs. 46%, respectively). We conclude that the effect of stress via ACTH administration and cortisol release accelerated mammary involution during the early dry-off period.

Long-term heat stress at final gestation: physiological and heat shock responses of Saanen goats.

The long exposure to heat negatively changes performance and productivity of animals, particularly when heat stress is associated with gestation. Indeed, little is known about the negative effects of long-term heat stress on the final gestation of dairy goats. In this context, the physiological and cellular responses of Saanen goats submitted to heat stress (37°C from 10:00 to 16:00 h) were investigated from day 60th pre-partum to day 60th post-partum. At final gestation, 46 pregnant Saanen goats were randomly assigned to the treatments: control (CT; thermal neutral conditions) and heat stress (HS; climatic chamber). After partum, all experimental goats were maintained in thermal neutral conditions. The rectal, dorsal, mammary temperatures and respiratory frequency, cortisol release, milk yield, milk quality, and the genes HSP60, HSP70, HSP90, Glucocorticoid receptor and ACTHR. Goats subjected to HS showed significantly (P < 0.05) higher rectal, dorsal, and mammary temperatures and significantly mobilized the increase of respiratory frequency to lose heat as compared to CT goats. The HS challenge significantly increased cortisol release from day 15th pre-partum to day 15th post-partum. CT goats produced more milk than HS from weeks 4 to 10 of lactation (P <0.001), with no difference in milk quality. However, on day 15th post-partum, there was a significant effect of HS treatment on the expression of HSP70 and ACTHR genes as compared to CT treatment, confirming the long-term effect of HS on Saanen goats. In conclusion, the physiological parameters studied increased pre-partum in the hottest hour, and cortisol peaked on day 15 pre-partum for heat-stressed goats. Although on the 15th day post-partum, all goats were in thermal comfort, and the physiological parameters were within the normal range, the concentration of cortisol continued to be significantly higher for goats submitted to thermal stress. Indeed, milk yield was greater for goats subjected to pre-partum thermal comfort. Furthermore, the expression of HSP70 and ACTHR genes on peripheral blood mononuclear cells are interesting biomarkers for studying the long-term effect of heat stress on Saanen goats.

Acute heat stress induces changes in physiological and cellular responses in Saanen goats.

The relationships between rectal temperatures and physiological and cellular responses to heat stress can improve the productivity of Saanen goats in tropical environments. In this context, this study evaluated the physiological responses and gene expression of heat shock proteins (HSP60, 70, and 90) and genes related to apoptosis (Bax, Bcl-2, and p53) of Saanen goats subjected to acute heat stress. Ten health Saanen goats were exposed to solar radiation during 3 consecutive days. The expression of HSP60, HSP70, HSP90, Bax, Bcl-2, and p53 genes in blood leukocytes, rectal and superficial temperatures, respiratory frequency, cortisol, triiodothyronine, and thyroxine was measured at 06:00, 13:00, and 18:00 h. In vitro, blood leukocytes were subjected to 38 °C and 40 °C for 3 h to measure the expression of the same target genes. The temperature humidity index, measured from 12:00 to 15:00, was greater than 80 and black globe temperatures were greater at 40 °C, indicating the intensity of the solar radiation. Although the solar radiation caused acute heat stress, increased cortisol release, and the expression of HSP60 and 70 in dry Saanen goats, the increased respiratory frequency and decreased T4 and T3 restored the homeothermy of the experimental goats. In vitro, the 40 °C increased the expression of p53 (pro-apoptotic protein), Bcl-2 (anti-apoptotic protein), HSP60, HSP70, and HSP90, suggesting that these genes have protective functions. However, further studies are necessary to understand the physiological and cellular responses to heat stress.

Isolamento e cultivo de neurônios e neuroesferas de córtex cerebral aviar / Isolation and culture of neurons and neurospheres from chicken brain cortex

Métodos de cultivo celular são convenientes na realização de análises funcionais de alterações/interações protéicas das células neuronais, auxiliando a decifrar o interactoma de proteínas chaves na neurogênese de doenças do Sistema Nervoso Central. Por esse motivo, culturas de neurônios e neuroesferas isolados do córtex cerebral aviar representam um modelo acessível para o estudo de diversas doenças neurológicas, tal como a epilepsia. A espécie aviar apresenta peculiaridades em seu proteoma neuronal, visto a presença de uma expressão diferenciada de proteínas chaves no metabolismo energético cerebral, algumas destas (VDAC1 e VDAC2) desempenham papel importante na compreensão do mecanismo da epilepsia refratária. A metodologia estabelecida no presente estudo obteve cultivo de neuroeferas, onde as células cresceram tipicamente em aglomerados atingindo, dentro de 7 dias, o diâmetro ideal de 100-200 µm. A diferenciação celular das neuroesferas foi obtida após a aderência destas às placas tratadas com poli-D-lisina, evidenciada pela migração de fibras do interior da neuroesfera. Ao contrário das neuroesferas, os neurônios em cultivo extenderam seus neuritos após 11 dias de isolamento. Tal modelo in vitro pode ser utilizado com sucesso na identificação das variáveis neuroproteômicas, propiciando uma avaliação global das alterações dinâmicas e suas interações protéicas. Tal modelo pode ter aplicações em estudos dos efeitos de indutores da morte celular e bloqueadores de canais de membrana mitocondriais em proteínas chaves do metabolismo energético cerebral.

Cell culture methods are used for studies of protein interactions in neural cells, helping to detect the interactome of proteins linked to generation of central nervous system diseases. For this reason, neural cells and neurospheres isolated from cortical chicken brain are a current model for studies of neurological diseases, such as epilepsy. Chicken brain has key characteristics on its proteome, with a differential expression of proteins linked to energy metabolism, some of them (VDAC 1 and VDAC 2) play an important role in understanding mechanism of refractory epilepsy. Using the methods described, we found neurospheres, in which cells grow in structures with the ideal diameter of 100-200µm within seven days after isolation. Neurospheres differentiation was obtained after adhesion of these cells to surfaces coated with poly-D-Lysine, detected by migration of fibers inside them. Unlike neurospheres, neurons extended neurites after 11 days of isolation. Here we describe a method to isolate and culture neurons and neurospheres from chicken cerebral cortex. Such "in vitro" model can be utilized on studies of neuronal protein differential expression and interaction. Cultures of isolated neurons represent an accessible model on studies of apoptosis and channel blockers of key proteins linked to brain metabolism.

Nuclear transfer with apoptotic bovine fibroblasts: can programmed cell death be reprogrammed?

Cell death by apoptosis is considered to be irreversible. However, reports have indicated that its reversibility is possible if the cells have not yet reached the "point of no return." In order to add new information about this topic, we used cells at different moments of apoptotic process as nuclear donors in somatic cell nuclear transfer (SCNT) in order to test if programmed cell death can be reversed. Adult bovine fibroblasts were treated with 10 µM of staurosporine (STP) for 3 h and analyzed for phosphatidylserine externalization (Annexin assay) and presence of active caspase-9. Annexin-positive (Anx+) and Caspase-9-positive (Casp-9+) cells were isolated by FACS and immediately transferred into enucleated in vitro matured bovine oocytes. After STP treatment, 89.9% of cells were Anx+ (4.6% in control cells; p<0.01) and 24.9% were Casp-9+ (2.4% in control cells; p<0.01). Fusion and cleavage were not affected by the use apoptotic cells (p>0.05). Also, the use of Anx+ cells did not affect blastocyst production compared to control (26.4% vs. 22.9%, respectively; p>0.05). However, blastocyst formation was affected by the use of Casp-9+ cells (12.3%; p<0.05). These findings contribute to the idea of that apoptosis is reversible only at early stages. Additionally, we hypothesize that the "point of no return" for apoptosis may be located around activation of Caspase-9.

Serum-starved apoptotic fibroblasts reduce blastocyst production but enable development to term after SCNT in cattle.

Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.

A retrospective model of oocyte competence: global mRNA and housekeeping transcripts are not associated with in vitro developmental outcome.

SummaryOocyte developmental competence depends on maternal stores that support development throughout a transcriptionally silent period during early embryogenesis. Previous attempts to investigate transcripts associated with oocyte competence have relied on prospective models, which are mostly based on morphological criteria. Using a retrospective model, we quantitatively compared mRNA among oocytes with different embryo development competence. A cytoplasm biopsy was removed from in vitro matured oocytes to perform comparative analysis of amounts of global polyadenylated (polyA) mRNA and housekeeping gene transcripts. After parthenogenetic activation of biopsied oocytes, presumptive zygotes were cultured individually in vitro and oocytes were classified according to embryo development: (i) blocked before the 8-cell stage; (ii) blocked between the 8-cell and morulae stages; or (iii) developed to the blastocyst stage. Sham-manipulated controls confirmed that biopsies did not alter development outcome. Total polyA mRNA amounts correlate with oocyte diameter but not with the ability to develop to the 8-cell and blastocyst stages. The last was also confirmed by relative quantification of GAPDH, H2A and Hprt1 transcripts. In conclusion, we describe a novel retrospective model to identify putative markers of development competence in single oocytes and demonstrate that global mRNA amounts at the metaphase II stage do not correlate with embryo development in vitro.