A grape seed extract maternal dietary supplementation improves egg quality and reduces ovarian steroidogenesis without affecting fertility parameters in reproductive hens.

In broiler hens, the genetic selection increased susceptibility to metabolic disorders and reproductive dysfunctions. In human ovarian cells, grape seed extracts (GSE) improved steroid production. Here, we investigated the effects of a GSE dietary supplementation on egg production and quality, fertility parameters, Reactive Oxygen Species (ROS) and steroid content in yolk egg associated to plasma adipokines in broiler hens. For this, we designed two in vivo experiments, the first one included three groups of hens: A (control), B and C (supplemented with GSE at 0.5% and 1% of the total diet composition, respectively, since week 4), and the second one used two groups of hens: A (control) and D (supplemented with GSE at 1% of the total diet composition since hatching). We assessed the egg production from 23th to 40th weeks and quality at 33th week. After artificial inseminations, the fertility parameters were calculated. In egg yolk, Reactive Oxygen Species (ROS) level and steroid production were evaluated by Ros-Glo H202 and ELISA assay, respectively. Expression of steroidogenic enzymes and adipokines and their receptors was determined by RT-qPCR in ovarian cells and plasma adipokines (RARRES2, ADIPOQ and NAMPT) were evaluated by specific ELISA assays. The fertility parameters and egg production were unaffected by GSE supplementation whatever the experiment (exp.). However, the rate of double-yolk eggs decreased for all GSE supplemented groups (exp. 1 P <0.01, exp.2, P<0.02). In exp.1, C group eggs were bigger and larger (P<0.0001) and the shell elasticity was higher for both B and C (P<0.0003) as compared to control. In the egg yolk, GSE supplementation in both exp. reduced ROS content and steroidogenesis consistent with a decrease in P450 aromatase and StAR mRNA expression and basal in vitro progesterone secretion in granulosa cells (P<0.001). Interestingly, in both exp. RARRES2 plasma levels were positively correlated while ADIPOQ and NAMPT plasma levels were negatively correlated, with steroids and ROS in yolk (P<0.0001). Taken together, maternal dietary GSE supplementation did not affect egg production and fertility parameters whereas it reduced ROS content and steroidogenesis in yolk egg. Furthermore, it ameliorated egg quality by decreasing the number of double-yolk eggs and by improving the size of normal eggs and the elasticity of the shell. Taken together, our data suggest the possibility of using dietary maternal GSE to improve egg quality.

A grape seed extract maternal dietary supplementation in reproductive hens reduces oxidative stress associated to modulation of plasma and tissue adipokines expression and improves viability of offsprings.

In reproductive hens, a feed restriction is an usual practice to improve metabolic and reproductive disorders. However, it acts a stressor on the animal. In mammals, grape seed extracts (GSE) reduces oxidative stress. However, their effect on endocrine and tissue response need to be deepened in reproductive hens. Here, we evaluated the effects of time and level of GSE dietary supplementation on growth performance, viability, oxidative stress and metabolic parameters in plasma and metabolic tissues in reproductive hens and their offsprings. We designed an in vivo trial using 4 groups of feed restricted hens: A (control), B and C (supplemented with 0.5% and 1% of the total diet composition in GSE since week 4, respectively) and D (supplemented with 1% of GSE since the hatch). In hens from hatch to week 40, GSE supplementation did not affect food intake and fattening whatever the time and dose of supplementation. Body weight was significantly reduced in D group as compared to control. In all hen groups, GSE supplementation decreased plasma oxidative stress index associated to a decrease in the mRNA expression of the NOX4 and 5 oxidant genes in liver and muscle and an increase in SOD mRNA expression. This was also associated to decreased plasma chemerin and increased plasma adiponectin and visfatin levels. Interestingly, maternal GSE supplementation increased the live body weight and viability of chicks at hatching and 10 days of age. This was associated to a decrease in plasma and liver oxidative stress parameters. Taken together, GSE maternal dietary supplementation reduces plasma and tissue oxidative stress associated to modulation of adipokines without affecting fattening in reproductive hens. A 1% GSE maternal dietary supplementation increased offspring viability and reduced oxidative stress suggesting a beneficial transgenerational effect and a potential use to improve the quality of the progeny in reproductive hens.

The kisspeptin analog C6 is a possible alternative to PMSG (pregnant mare serum gonadotropin) for triggering synchronized and fertile ovulations in the Alpine goat.

In temperate regions goat's reproduction is seasonal. To obtain year-round breeding, hormonal treatments are currently applied. These treatments usually combine a progesterone analog with the pregnant mare serum gonadotropin (PMSG). However, their use has significant ethical and environmental drawbacks. Therefore, alternative methods to manage reproduction are needed. The discovery that in mammals the neuropeptide kisspeptin is a major positive regulator of hypothalamo-pituitary gonadal axis offered an attractive alternative strategy to control reproduction. We have previously designed a kisspeptin analog, called C6, which offers pharmacological advantages over endogenous kisspeptin. These include a longer lasting effect and enhanced activity following intramuscular injection. In the present work, we evaluated C6 effect on LH and FSH plasma concentrations in the Alpine goat breed and tested whether C6 could replace PMSG to trigger ovulation. An intramuscular injection of C6 (15 nmol/doe) given 24 hours after the end of progestogen treatment induced a surge-like peak of both LH and FSH. This was followed by an increase of progesterone, a hallmark of ovulation induction and corpus luteus formation. These results were obtained at three different time of the year: during the breeding season, the non-breeding season and at the onset of the breeding season. Furthermore, we compared the efficacy of C6 and PMSG to induce fertile ovulations when these treatments are given at the onset of the breeding season and are followed by artificial insemination. The results of this first attempt were extremely promising with gestation rates of 45% and 64% for C6 and PMSG respectively. Pending optimization of the treatment procedure in order to improve efficacy, kisspeptin analogs could be the long sought-after alternative to PMSG.

Brain mapping of the gonadotropin-inhibitory hormone-related peptide 2 with a novel antibody suggests a connection with emotional reactivity in the Japanese quail (Coturnix japonica, Temminck & Schlegel, 1849).

Gonadotropin-inhibitory hormone (GnIH) is a neuropeptide first discovered in the quail brain that is involved in the control of reproductive physiology and behaviors, and stress response. GnIH gene encodes a second peptide, GnIH-related peptide-2 (RP2), the distribution and function of which remain unknown. We therefore studied GnIH-RP2 distribution by immunohistochemistry using a novel antibody capable of discriminating between GnIH and GnIH-RP2. The overall distribution of GnIH-RP2 is similar to that of GnIH. The vast majority of labeled neurons is located in the paraventricular nucleus (PVN) of the hypothalamus. Labeling of fibers is conspicuous in the diencephalon, but present also in the mesencephalon and telencephalon. Several regions involved in the control of reproduction and stress response (the PVN, septum, bed nucleus of the stria terminalis and nucleus commissura pallii) showed a dense network of immunolabeled fibers. To investigate the potential function of GnIH-RP2 we compared its expression in two quail lines genetically selected for divergence in their emotional reactivity. A quantitative analysis in the above-mentioned brain regions showed that the density of fibers was similar in the two lines. However, the number of GnIH-RP2 labeled neurons was higher in the median portion of the PVN in birds with higher emotional reactivity. These results point to a possible involvement of GnRH-RP2 in modulating stress response and/or emotional reactivity.

The Sole DEAD-Box RNA Helicase of the Gastric Pathogen Helicobacter pylori Is Essential for Colonization.

Present in every kingdom of life, generally in multiple copies, DEAD-box RNA helicases are specialized enzymes that unwind RNA secondary structures. They play major roles in mRNA decay, ribosome biogenesis, and adaptation to cold temperatures. Most bacteria have multiple DEAD-box helicases that present both specialized and partially redundant functions. By using phylogenomics, we revealed that the Helicobacter genus, including the major gastric pathogen H. pylori, is among the exceptions, as it encodes a sole DEAD-box RNA helicase. In H. pylori, this helicase, designated RhpA, forms a minimal RNA degradosome together with the essential RNase, RNase J, a major player in the control of RNA decay. Here, we used H. pylori as a model organism with a sole DEAD-box helicase and investigated the role of this helicase in H. pylori physiology, ribosome assembly, and during in vivo colonization. Our data showed that RhpA is dispensable for growth at 37°C but crucial at 33°C, suggesting an essential role of the helicase in cold adaptation. Moreover, we found that a ΔrhpA mutant was impaired in motility and deficient in colonization of the mouse model. RhpA is involved in the maturation of 16S rRNA at 37°C and is associated with translating ribosomes. At 33°C, RhpA is, in addition, recruited to individual ribosomal subunits. Finally, via its role in the RNA degradosome, RhpA directs the regulation of the expression of its partner, RNase J. RhpA is thus a multifunctional enzyme that, in H. pylori, plays a central role in gene regulation and in the control of virulence.IMPORTANCE We present the results of our study on the role of RhpA, the sole DEAD-box RNA helicase encoded by the major gastric pathogen Helicobacter pylori We observed that all the Helicobacter species possess such a sole helicase, in contrast to most free-living bacteria. RhpA is not essential for growth of H. pylori under normal conditions. However, deletion of rhpA leads to a motility defect and to total inhibition of the ability of H. pylori to colonize a mouse model. We also demonstrated that this helicase encompasses most of the functions of its specialized orthologs described so far. We found that RhpA is a key element of the bacterial adaptation to colder temperatures and plays a minor role in ribosome biogenesis. Finally, RhpA regulates transcription of the rnj gene encoding RNase J, its essential partner in the minimal H. pylori RNA degradosome, and thus plays a crucial role in the control of RNA decay.

Targeting of Helicobacter pylori thymidylate synthase ThyX by non-mitotoxic hydroxy-naphthoquinones.

ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the functionally analogous human enzyme, thus providing means for selective inhibition of bacterial growth. To identify novel compounds with anti-bacterial activity against the human pathogenic bacterium Helicobacter pylori, based on our earlier biochemical and structural analyses, we designed a series of eighteen 2-hydroxy-1,4-naphthoquinones (2-OH-1,4-NQs) that target HpThyX. Our lead-like molecules markedly inhibited the NADPH oxidation and 2'-deoxythymidine-5'-monophosphate-forming activities of HpThyX enzyme in vitro, with inhibitory constants in the low nanomolar range. The identification of non-cytotoxic and non-mitotoxic 2-OH-1,4-NQ inhibitors permitted testing their in vivo efficacy in a mouse model for H. pylori infections. Despite the widely assumed toxicity of naphthoquinones (NQs), we identified tight-binding ThyX inhibitors that were tolerated in mice and can be associated with a modest effect in reducing the number of colonizing bacteria. Our results thus provide proof-of-concept that targeting ThyX enzymes is a highly feasible strategy for the development of therapies against H. pylori and a high number of other ThyX-dependent pathogenic bacteria. We also demonstrate that chemical reactivity of NQs does not prevent their exploitation as anti-microbial compounds, particularly when mitotoxicity screening is used to prioritize these compounds for further experimentation.