Optimal methionine plus cystine requirements in diets supplemented with L-methionine in starter, grower, and finisher broilers.

Correct supplementation of dietary amino acids, such as methionine (Met) and cystine (Cys), is crucial to support the exponential growth of broilers. Historically, most available recommendations with regard to the optimal amount of Met plus Cys are based on studies wherein DL-Met was used as the Met source. Nowadays, L-Met is available as a registered feed additive, urging the need to establish the optimal L-Met plus Cys supplementation. The objective of this trial was to investigate these optimal L-Met plus Cys requirements of broilers in the starter (0-10 d), grower (11-23 d), and finisher (24-35 d) phase of life separately. A basal diet deficient in L-Met plus Cys was created along with 6 other diets with increasing L-Met concentrations for each phase. Birds were only included in one life phase and fed with a commercial diet before inclusion. The BW, daily weight gain, daily feed intake, and feed conversion ratio (gain-to-feed ratio) were measured for all birds. Slaughter parameters were determined for birds included in the finisher phase. At the end of each study period, significant differences (P < 0.05) were observed in all measured performance parameters. Birds fed with the deficient diets were characterized by a lower performance, whereas from some point, no gain in performance could be observed. Correct supplementation of L-Met plus Cys seemed more crucial in the starter and grower phase, which was characterized by bigger differences in performance between test diets compared with the finisher birds. The optimal L-Met plus Cys requirements were determined using linear broken line and exponential asymptotic models. The linear broken line model showed overall the best fit. The optimal L-Met plus Cys level was found to be 0.69, 0.66, and 0.62% for birds in the starter, grower, and finisher phase, respectively. From this study, it could be concluded that broilers have lower L-Met plus Cys requirements based on L-Met supplementation than the conventional requirements based on DL-Met. Nevertheless, further research is required to confirm these findings.

The optimum valine: lysine ratios on performance and carcass traits of male broilers based on different regression approaches.

Three different regression approaches were applied to determine the optimal digestible (d.) and analyzed Val:Lys ratios for broiler performance and carcass yield. One-day-old male Cobb 500 broilers (n = 960) were assigned to 1 of 8 diets, with 6 pens/diet and 20 birds/pen, for 42 days. The negative control consisted of the basal diet with a d.Val:d.Lys ratio of 0.63 and with 93% of the required d.Lys. The positive control consisted of the basal diet with a d.Val:d.Lys of 0.80, with no reduction in d.Lys content. The other (test) diets contained a range of d.Val:d.Lys ratios, all with 93% of the required d.Lys. Data on feed intake (FI), body weight gain (BWG), and feed conversion ratio (FCR) were submitted to regression analysis, applying quadratic polynomial (QP), exponential asymptotic (EA), and linear response plateau (LRP) models. Since Val did not affect carcass or breast meat yield, no regression was performed. Digestible and analyzed Val:Lys ratios were similar based on the regression models. The intercept between the QP and LRP models was used to determine the optimum Val:Lys ratio. Overall, the ideal d.Val:d.Lys ratio will vary according to the main goal of poultry production, i.e., BWG or FCR. For BWG, the ideal ratio was found to be 0.78 (0 to 12 d), 0.73 (0 to 28 d), and 0.76 (0 to 35 or 0 to 42 d). For FCR, the optimum d.Val:d.Lys was found to be 0.80 (0 to 12 d), 0.75 (0 to 28 d), and 0.78 (0 to 35 or 0 to 42 d). The optimum analyzed Val:Lys ratio was slightly higher. For instance, for BWG the optimum ratio was 0.80 (0 to 12 d), 0.76 (0 to 28 d), and 0.79 (0 to 35 or 0 to 42 d). For FCR, the optimum Val:Lys was 0.81 (0 to 12 d), 0.79 (0 to 28 d), and 0.81 (0 to 35 or 0 to 42 d). Valine did not affect carcass or breast meat yield.

Effects of intramuscular administration of folic acid and vitamin B12 on granulosa cells gene expression in postpartum dairy cows.

The fertility of dairy cows is challenged during early lactation, and better nutritional strategies need to be developed to address this issue. Combined supplementation of folic acid and vitamin B12 improve energy metabolism in the dairy cow during early lactation. Therefore, the present study was undertaken to explore the effects of this supplement on gene expression in granulosa cells from the dominant follicle during the postpartum period. Multiparous Holstein cows received weekly intramuscular injection of 320 mg of folic acid and 10 mg of vitamin B12 (treated group) beginning 24 (standard deviation=4) d before calving until 56 d after calving, whereas the control group received saline. The urea plasma concentration was significantly decreased during the precalving period, and the concentration of both folate and vitamin B12 were increased in treated animals. Milk production and dry matter intake were not significantly different between the 2 groups. Plasma concentrations of folates and vitamin B12 were increased in treated animals. Daily dry matter intake was not significantly different between the 2 groups before [13.5 kg; standard error (SE)=0.5] and after (23.6 kg; SE=0.9) calving. Average energy-corrected milk tended to be greater in vitamin-treated cows, 39.7 (SE=1.4) and 38.1 (SE=1.3) kg/d for treated and control cows, respectively. After calving, average plasma concentration of ß-hydroxybutyrate tended to be lower in cows injected with the vitamin supplement, 0.47 (SE=0.04) versus 0.55 (SE=0.03) for treated and control cows, respectively. The ovarian follicle ≥12 mm in diameter was collected by ovum pick-up after estrus synchronization. Recovered follicular fluid volumes were greater in the vitamin-treated group. A microarray platform was used to investigate the effect of treatment on gene expression of granulosa cells. Lower expression of genes involved in the cell cycle and higher expression of genes associated with granulosa cell differentiation before ovulation were observed. Selected candidate genes were analyzed by reverse transcription quantitative PCR. Although the effects of intramuscular injections of folic acid and vitamin B12 on lactational performance and metabolic status of animals were limited, ingenuity pathway analysis of gene expression in granulosa cells suggests a stimulation of cell differentiation in vitamin-treated cows, which may be the result of an increase in LH secretion.

Transcriptomic analysis of cyclic AMP response in bovine cumulus cells.

Acquisition of oocyte developmental competence needs to be understood to improve clinical outcomes of assisted reproduction. The stimulation of cumulus cell concentration of cyclic adenosine 3'5'-monophosphate (cAMP) by pharmacological agents during in vitro maturation (IVM) participates in improvement of oocyte quality. However, precise coordination and downstream targets of cAMP signaling in cumulus cells are largely unknown. We have previously demonstrated better embryo development after cAMP stimulation for first 6 h during IVM. Using this model, we investigated cAMP signaling in cumulus cells through in vitro culture of cumulus-oocyte complexes (COCs) in the presence of cAMP raising agents: forskolin, IBMX, and dipyridamole (here called FID treatment). Transcriptomic analysis of cumulus cells indicated that FID-induced differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism, and oocyte competence. Functional genomic analysis revealed that protein kinase-A (PKA), extracellular signal regulated kinases (ERK1/2), and calcium (Ca(2+)) pathways as key regulators of FID signaling. Inhibition of PKA (H89) in FID-supplemented COCs or substitution of FID with calcium ionophore (A23187) demonstrated that FID activated primarily the PKA pathway which inhibited ERK1/2 phosphorylation and was upstream of calcium signaling. Furthermore, inhibition of ERK1/2 phosphorylation by FID supported a regulation by dual specific phosphatase (DUSP1) via PKA. Our findings imply that cAMP (FID) regulates cell metabolism, steroidogenesis, intracellular signaling and cumulus expansion through PKA which modulates these functions through optimization of ERK1/2 phosphorylation and coordination of calcium signaling. These findings have implications for development of new strategies for improving oocyte in vitro maturation leading to better developmental competence.

Characterization of FSH signalling networks in bovine cumulus cells: a perspective on oocyte competence acquisition.

Understanding the mechanisms regulating oocyte developmental competence is essential to enhance the clinical efficiency of assisted reproduction. FSH orchestrates the acquisition of oocyte competence, both in vivo and in vitro. Multiple pathways are implicated in FSH signalling; however, their precise coordination remains unresolved. A robust system to investigate FSH signalling is oocyte in vitro maturation (IVM) and we have previously demonstrated better bovine embryo development after FSH addition for the first 6 h during IVM. Using this model, we investigated FSH signalling in cumulus through transcriptomic and pharmacological tools. We demonstrate modulation of cumulus transcriptome by FSH mainly through protein kinase A (PKA) and epidermal growth factor (EGF) pathways. Differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism and oocyte competence. FSH required rouse-sarcoma oncogene (SRC) for EGF receptor transactivation. PKA and EGF pathway crosstalk was investigated using extracellular signal-regulated kinases (ERK1/2) phosphorylation as the functional end-point. FSH enhanced ERK1/2 activation by the EGF pathway with a simultaneous diminution through PKA. More specifically, FSH increased dual specific phosphatase (DUSP1) transcripts via PKA although DUSP1 protein did not change since EGF was required to prevent degradation. Our findings implicate FSH in PKA and EGF pathway activation, which interact to maintain appropriate levels of ERK1/2 phosphorylation and eventually cumulus expansion, metabolism and steroidogenesis. Moreover, considering the implication of the EGF pathway in GDF9 and BMP15 actions, our findings suggest that FSH may have a role in modulation of the cumulus response to oocyte-secreted factors. This information has implications for improvement of IVM and hence oocyte developmental competence.

Retrotransposon expression as a defining event of genome reprogramming in fertilized and cloned bovine embryos.

Genome reprogramming is the ability of a nucleus to modify its epigenetic characteristics and gene expression pattern when placed in a new environment. Low efficiency of mammalian cloning is attributed to the incomplete and aberrant nature of genome reprogramming after somatic cell nuclear transfer (SCNT) in oocytes. To date, the aspects of genome reprogramming critical for full-term development after SCNT remain poorly understood. To identify the key elements of this process, changes in gene expression during maternal-to-embryonic transition in normal bovine embryos and changes in gene expression between donor cells and SCNT embryos were compared using a new cDNA array dedicated to embryonic genome transcriptional activation in the bovine. Three groups of transcripts were mostly affected during somatic reprogramming: endogenous terminal repeat (LTR) retrotransposons and mitochondrial transcripts were up-regulated, while genes encoding ribosomal proteins were downregulated. These unexpected data demonstrate specific categories of transcripts most sensitive to somatic reprogramming and likely affecting viability of SCNT embryos. Importantly, massive transcriptional activation of LTR retrotransposons resulted in similar levels of their transcripts in SCNT and fertilized embryos. Taken together, these results open a new avenue in the quest to understand nuclear reprogramming driven by oocyte cytoplasm.

Apoptosis in fresh and cryopreserved buffalo sperm.

The objectives of present study were (a) validation of annexin V/PI assay for estimation of sperm apoptosis in buffalo (Experiment 1) and (b) determining the effect of stages of cryopreservation on sperm apoptosis and its correlation with sperm motility and plasma membrane integrity (Experiment 2). In Experiment 1, different levels of apoptosis were artificially induced in buffalo semen (100x10(6)sperm/aliquot) through graded doses of camptothecin (5, 10 and 20microM/aliquot). Higher concentrations of camptothecin (10 and 20microM) successfully (P<0.05) induced apoptosis as compared to the lower (5microM) dose and/or control. In Experiment 2, semen samples (n=9, three pooled semen samples from each of the three buffalo bulls separately) were cryopreserved using vapor freezing. The mean percentage of apoptotic, necrotic and viable sperm did not differ between fresh and before freezing stages. However, freezing and thawing increased (P<0.05) the percentage of apoptotic sperm (25.4+/-0.6 vs. 36.5+/-1.9) while decreased (P<0.05) the necrotic (35.1+/-1.2 vs. 29.7+/-0.7) and viable sperm (37.2+/-1.3 vs. 32.8+/-1.9, (P<0.07). Likewise, the mean percent motility and plasma membrane integrity decreased (P<0.05) (64+/-2.1 vs. 49.4+/-1.3) and (79.6+/-0.5 vs. 38.7+/-0.3) respectively, at post thaw compared to other stages. Coefficient of correlation, combined at all stages for each variable revealed that sperm apoptosis was inversely correlated with sperm motility and plasma membrane integrity. It is concluded that (a) the annexin V/PI assay can be used as a tool to determine the buffalo semen apoptosis and (b) freezing and thawing induces apoptosis in buffalo sperm.