Acute and chronic effects of cortisol on milk yield, the expression of key receptors, and apoptosis of mammary epithelial cells in Saanen goats.

Cortisol (CORT) induces mammary development in late gestation and is fundamental to the differentiation of mammary epithelial cells and lactogenesis. The objective of this study was to investigate the relationship between CORT, insulin, prolactin, growth hormone, and insulin-like growth factor-1 in milk as well as the effect of CORT on the expression of receptors of insulin (INSR), prolactin (PRLR), growth hormone (GHR); we also studied the insulin-like growth factor-1 (IGF1R), glucocorticoid (NR3C1), mineralocorticoid (NR3C2), B-cell lymphoma 2 (BCL2), BCL-2-like protein X (BAX) genes, and the apoptosis rate of mammary epithelial cells of lactating Saanen goats in vivo and in vitro. The following experiments were conducted: (1) comparing hormone release in milk and blood after ACTH or a placebo administration; (2) evaluating the effect of acute CORT increases in mammary gland expression and milk yield in vivo; and (3) evaluating the effect of a chronic increase in CORT concentration in epithelial mammary cell apoptosis in vitro. In vivo, ACTH administration significantly increased CORT release but did not affect insulin, prolactin, growth hormone, and insulin-like growth factor-1 release in plasma and milk versus placebo. The results show also that a low CORT release after ACTH administration increased the expression of GHR and PRLR genes in the mammary tissue. Indeed, CORT release significantly increased the milk yield from goats subjected to ACTH versus goats subjected to the placebo. However, a higher amount of CORT added in vitro upregulated the NR3C1, GHR, PRLR, and BAX genes and downregulated the IGF1R and INSR genes, which could negatively modulate the apoptosis of mammary epithelial cells. Finally, the effect of CORT in vivo after ACTH administration demonstrated the increased expression of the PRLR and GHR genes, which may improve epithelial cell responsiveness and be associated with the positive effect of CORT observed on milk yield at mid-end lactation.

Heat stress affects the expression of key genes in the placenta, placental characteristics, and efficiency of Saanen goats and the survival and growth of their kids.

Heat stress is detrimental during gestation; however, the effects of heat stress on goat placental characteristics and kid survival remain unclear. The objective of this study was to evaluate the effects of heat stress at final gestation on cortisol concentration, placenta characteristics, and the expression of genes related to placenta. Forty-six primiparous and multiparous Saanen goats were subjected to control (CT; under a thermoneutral environment: air temperature between 12°C and 25°C and the relative humidity from 45 to 73%, n = 23) or heat stress (HS; under a climatic chamber: air temperature at 37°C and the relative humidity at 60 to 70% from 0800 to 1600 h, n = 23) from the last 60 d of pregnancy until the first colostrum suckling. The heat challenge imposed on HS goats during the prepartum period increased their rectal temperature, respiratory frequency, and cortisol levels in plasma and amniotic fluid versus CT goats. In the placenta, HS treatment also increased the expression of the HSPA1A gene. Heat-stressed goats also showed significantly lower expression of HSD11B2 and greater expression of MC2R and NR3C1 than CT goats, suggesting that heat stress decreased the effectiveness by which the HSD11B2 enzyme converts cortisol to cortisone and increased placental responsiveness to cortisol. The HS goats took longer to release the placenta with lighter placental cotyledons, and HS goats had a lower ratio between the kid's weight at birth and placenta weight than CT goats. There was no treatment effect on the kids' survival or weights at birth, but the kids from goats subjected to HS presented lesser cortisol concentration and greater mortality rates at weaning than kids from CT goats. Finally, the overexpression of HSPA1A by HS goats suggests a protective response of placenta. However, the heat stress negatively affected the placenta's expulsion length, placental cotyledons number, weight and area, the ratio between kid's weight and placenta weight, and cortisol signaling. Indeed, the upregulation of MC2R and NR3C1 and downregulation of HSD11B2 on placenta caused by heat stress were associated with greater cortisol concentrations in the amniotic fluid of HS goats. Although HS and CT kids had adequate weights and survival rate during the first weeks of life, the heat stress increased the mortality at weaning of HS kids versus CT kids, suggesting that the heat stress effect persists and can change the ability of kids to respond to weaning challenge.

Effect of acute stressors, adrenocorticotropic hormone administration, and cortisol release on milk yield, the expression of key genes, proliferation, and apoptosis in goat mammary epithelial cells.

Cortisol is essential to milk synthesis; however, different acute stressors and the exogenous administration of adrenocorticotropic hormone (ACTH) decrease milk yield. Therefore, the effect of cortisol on milk yield and its influence on the survival of mammary epithelial cells have not been fully elucidated. In this context, the objective of this study was to evaluate the effect of cortisol on the expression of growth hormone receptor (GHR), insulin-like growth factor type 1 (IGF1), insulin-like growth factor type 1 receptor (IGF1R), insulin-like growth factor-binding protein 3 and 5 (IGFBP3 and IGFBP5), BAX, and BCL2 genes on the proliferation and apoptotic rates of mammary epithelial cells, and on milk yield in Saanen goats. In the present study, 3 experiments were conducted: (1) comparing the in vivo effects of first milking, vaccination, vermifugation, preventive hoof trimming, and the administration of ACTH or a placebo on cortisol release in dairy goats; (2) studying the in vivo effects of immediate increases in cortisol on the mammary gland of lactating goats; and (3) studying the in vitro effects of a prolonged increase in cortisol on mammary epithelial cells obtained from lactating goats. Cortisol release by goats increased significantly after ACTH administration compared with that observed after a placebo, and the cortisol profiles after first milking, vaccination, vermifugation, hoof trimming, and ACTH administration were similar. However, there was no effect of the immediate increase in cortisol in vivo on IGF-1 release, milk yield, milk quality, or the apoptosis and proliferation rates, nor was there any effect on the expression of the target genes. Furthermore, no interaction was observed between IGF-1 and cortisol in either the in vivo or in vitro experiments. However, the addition of cortisol in vitro significantly increased the expression of the GHR and IGF1R genes, which stimulate cell proliferation, and the BAX gene, which causes apoptosis. These contrasting results can explain why cortisol did not change the rates of proliferation or apoptosis in epithelial cells. Indeed, cortisol supplementation in vitro did not change the number or apoptotic rate of epithelial cells over the course of 5 d. Finally, further studies must be performed to understand the effect of cortisol on the expression of the GHR, IGF1R, and BAX genes by epithelial cells and the roles of these genes in milk synthesis during early lactation.

Effects of different supplemental soya bean oil levels on the performance of prepubertal Saanen goats: Oestrogen and progesterone release.

The aim of this study was to investigate the effects of different levels of soya bean oil in the total diet on the growth rate, metabolic changes, and oestrogen and progesterone release in Saanen goats. After dietary adaptation, 21 prepubertal goats (weight of 29.12 ± 0.91 kg, 230 days old) were randomly distributed among three diets of D2: inclusion of 2% soya bean oil in the total diet; D3: basal diet - inclusion of 3% soya bean oil in the total diet; and D4: inclusion of 4% soya bean oil in the total diet. The basal diet (D3) was formulated to promote a daily gain of 0.140 kg. The goats were weighed, and their blood samples were collected weekly. Glucose, cholesterol, triglycerides, total protein, urea, non-esterified fatty acids, beta-hydroxybutyrate, oestrogen and progesterone in the plasma were measured. Prepubertal goats that were fed D4 exhibited a significantly lower dry matter intake, urea and cholesterol levels compared with the goats that were fed D2 and D3. Indeed, goats that were fed D4 displayed a significantly lower final weight than goats that were fed D2 and D3. In contrast, the inclusion of soya bean oil in the diet increased the progesterone and oestrogen concentrations, and goats that were fed D4 released a significantly higher concentration of progesterone than those that were fed D2 and D3. Furthermore, the percentage of goats with a progesterone level greater than 1 ng/ml (functional Corpus luteum) was significantly higher among the goats that were fed D3 and D4 than among those that were fed D2. In this study, although the inclusion of 4% soya bean oil in the diet decreased dry matter intake and growth rate, it increased progesterone concentration and the percentage of goats with a functional Corpus luteum, suggesting that the inclusion of soya bean oil accelerated puberty in prepubertal goats.

Gene silencing during development of in vitro-produced female bovine embryos.

In early development, female embryos (XX) produce twice the transcripts of X-linked genes compared with male embryos (XY). During the course of development, inactivation of the X chromosome equilibrates gene dosage, making the development of female embryos viable. Moreover, the biotechnologies used for producing embryos in vitro seem to work better with male embryos, making it easier for them to reach the blastocyst stage and allow for complete gestation. We investigated the expression of three X-linked genes that are involved in development, XIST, G6PD, and HPRT, and of the transcript interferon-tau, in male and female bovine blastocysts produced by nuclear transfer (NT) and by in vitro fertilization (IVF). Oocytes that had been matured in vitro were enucleated and reconstructed with somatic cells from adult animals at 18 h post-maturation. After fusion (two pulses of 2.25 kv/cm) and chemical activation (5.0 mM ionomycin for 5 min and 2.0 mM 6-DMAP for 3 h), the oocyte-somatic cell units were cultivated in CR2 with a monolayer of granulosa cells at 38.8 degrees C, in a humidified 5% CO(2) atmosphere. IVF embryos were inseminated, after centrifugation in a Percoll gradient, with 2 x 10(6) sperm/mL TALP medium supplemented with BSA and PHE and cultivated under the same conditions as the cloned embryos. We used real-time PCR to analyze the gene expression of individual blastocysts compared to expression of the housekeeping gene, GAPDH. The gene XIST was expressed in female embryos and not in male embryos produced by IVF, though it was expressed at low levels in male embryos produced by NT. Unlike previous reports, we found lower levels of the transcript of G6PD in females than in males, suggesting double silencing or other mechanisms of control of this gene. Female embryos produced by IVF expressed the HPRT gene at a higher level than female embryos produced by NT, suggesting that gene silencing proceeds faster in NT-produced female embryos due to "inactivation memory" from the nucleus donor. In conclusion, male and female embryos express different levels of X-chromosome genes and failures of these genes that are essential for development could reduce the viability of females. Nuclear transfer can modify this relation, possibly due to epigenetic memory, leading to frequent failures in nuclear reprogramming.

Imprinted gene expression in in vivo- and in vitro-produced bovine embryos and chorio-allantoic membranes.

Cloning by nuclear transfer is often associated with poor results due to abnormal nuclear reprogramming of somatic donor cells and altered gene expression patterns. We investigated the expression patterns of imprinted genes IGF2 and IGF2R in 33- to 36-day bovine embryos and chorio-allantoic membranes derived from in vivo- and in vitro-produced embryos by somatic cell nuclear transfer (SCNT), parthenogenetic activation, and in vitro fertilization (IVF). There was a lower IGF2 expression rate in the SCNT (0.19) and parthenogenetic (0.02) groups when compared to in vivo and IVF embryos (2.01; P < 0.05). In the chorio-allantoic membranes, IGF2 showed a baseline expression pattern (P < 0.05) in parthenotes (0.001) when compared to in vivo, IVF (3.13), and SCNT (0.98) groups. IGF2R was less expressed (P < 0.05) in SCNT chorio-allantoic membranes (0.25) when compared to the in vivo group. The low expression of IGF2 in parthenogenetic embryos and chorio-allantoic membranes confirms its imprinted status in cattle. Alterations in the relative frequency of IGF2 and IGF2R transcripts were observed in SCNT-derived bovine embryos and chorio-allantoic membranes, respectively, supporting the hypothesis that abnormalities in the expression of imprinted genes are causes of the low efficiency of SCNT procedures in this species.

Bos indicus or Bos taurus mitochondrial DNA - comparison of productive and reproductive breeding values in a Guzerat dairy herd.

The observation of bovine mitochondrial DNA (mtDNA) polymorphisms allows the separation of American zebu cattle, according to its maternal lineage ancestry, into two groups: one with Bos indicus mtDNA and other with Bos taurus mtDNA. The aim of the present study was to determine the productive and reproductive differences between these two groups, in a Guzerat dairy herd. The genotyping of a sample of 56 animals allowed the categorization of most of the 3835 animals in the pedigree file. The production file included 3528 calving and 3198 lactation records from 729 cows, born during the years 1947 to 2007. The traits considered were: lactation milk yield (LMY); days in milk (DIM); age at first calving (AFC), and calving interval (CI). Heritabilities and breeding values were estimated using an animal model. The regression of the average breeding values per year of birth indicated the genetic trends of the herd. The heritability coefficients estimated for LMY, DIM, AFC, and CI were 0.42, 0.43, 0.20, and 0.10, respectively. The genetic trends were similar for both groups, pointing to an improvement in the productive and a worsening in the reproductive traits. The two groups differed significantly regarding the average estimated breeding values for LMY, DIM and AFC, in the starting period, until 1970, but no differences were observed in the more recent years, after 1970. The segregation between the groups existed in the starting period, probably because the Bos taurus contributions to the herd had occurred more recently at that moment. The conclusion is that mtDNA has no significant effect on these traits.

Evaluation of polyvinyl alcohol for fatty acid supplementation in adipose tissue explant culture.

Cultures of adipose tissue explants are a valuable tool for studying the intracellular mechanisms involving hormones and nutrients. However, testing how fatty acids affect cells requires a carrier molecule; bovine serum albumin (BSA) has been used for this purpose. However, contaminants can alter the cellular response. Our objectives were to: 1) test BSA as a fatty acid carrier and 2) evaluate polyvinyl alcohol (PVA) as a replacement for BSA. Adipose tissue explants from nine pigs were cultured in medium 199 for 4, 12, 24, and 48 h, with the following treatments: control, PVA (100 mM PVA added) and PVA + pGH (100 mM PVA plus 0.1 mg/mL porcine growth hormone). After each culture period, explants were collected and assayed for lipogenesis. After 48 h in culture, explants were assayed for lipolysis. A preliminary study with different commercial sources and high concentrations showed that BSA affected lipogenic rates. On the other hand, there were no effects of PVA on lipid synthesis, while pGH (positive control) reduced glucose incorporation into lipids (P < 0.01) when compared to both control and PVA (P < 0.05). There was no difference between control and PVA for lipolysis rates. However, pGH increased lipolysis when compared to control (P < 0.01) and PVA (P < 0.05). We demonstrated that BSA can alter lipogenesis, which precludes its use as a carrier molecule. On the other hand, addition of PVA had no effect on lipolysis or lipogenesis. We suggest the use of PVA instead of BSA for adding bioactive fatty acids to cultures of adipose tissue.

Evaluation of polyvinyl alcohol for fatty acid supplementation in adipose tissue explant culture

Cultures of adipose tissue explants are a valuable tool for studying the intracellular mechanisms involving hormones and nutrients. However, testing how fatty acids affect cells requires a carrier molecule; bovine serum albumin (BSA) has been used for this purpose. However, contaminants can alter the cellular response. Our objectives were to: 1) test BSA as a fatty acid carrier and 2) evaluate polyvinyl alcohol (PVA) as a replacement for BSA. Adipose tissue explants from nine pigs were cultured in medium 199 for 4, 12, 24, and 48 h, with the following treatments: control, PVA (100 mM PVA added) and PVA + pGH (100 mM PVA plus 0.1 mg/mL porcine growth hormone). After each culture period, explants were collected and assayed for lipogenesis. After 48 h in culture, explants were assayed for lipolysis. A preliminary study with different commercial sources and high concentrations showed that BSA affected lipogenic rates. On the other hand, there were no effects of PVA on lipid synthesis, while pGH (positive control) reduced glucose incorporation into lipids (P < 0.01) when compared to both control and PVA (P < 0.05). There was no difference between control and PVA for lipolysis rates. However, pGH increased lipolysis when compared to control (P < 0.01) and PVA (P < 0.05). We demonstrated that BSA can alter lipogenesis, which precludes its use as a carrier molecule. On the other hand, addition of PVA had no effect on lipolysis or lipogenesis. We suggest the use of PVA instead of BSA for adding bioactive fatty acids to cultures of adipose tissue

Development and optimization of a fluorescent differential display PCR system for studying bovine embryo development in vitro

Differential display is a widely used methodology to identify genes that are differentially expressed in biological samples. We developed a new protocol for the amplification and recovery of differentially expressed genes from extremely small initial amounts of RNA (10 to 25 pg mRNA) from preimplantation bovine embryos. The cDNAs generated with an anchor primer, associated with a universal sequence, were amplified with an arbitrary primer and a single fluorescently labeled primer. Amplification products were easily visualized with a fluorescence scanner, without the need for radioisotopes. Nineteen isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the recognition and identification of gene transcripts involved in bovine embryonic physiology.

Genome activation and developmental block in bovine embryos.

The ultimate goal of in vitro embryo culture systems is to perfectly mimic the condition of oocyte maturation, fertilization and embryo development. These systems are far more complex than standard in vitro cell culture because of the various environments through which the gametes and embryos pass during in vivo development. Improvement of the medium and other culture conditions has allowed for full development of a percentage of the fertilized oocytes but the great majority of bovine zygotes stop developing within a few cell cycles after initiating cleavage. This developmental block arises in the bovine embryo at the eight-cell-stage and is likely correlated with the cytoplasmic quality of the oocyte. Oocytes harbor all mRNAs and proteins needed to reach the fourth or fifth cell cycle, however, embryos that fail to transcribe their own genome fail to further develop. In this article, we review some of the advances in developmental block knowledge and describe a possible role of active embryo transcription that drives incompetent embryos to block and death.