Productive Performance, Ovarian Follicular Development, Lipid Peroxidation, Antioxidative Status, and Egg Quality in Laying Hens Fed Diets Supplemented with Salvia officinalis and Origanum majorana Powder Levels

This study aimed to assess the effect of dietary supplementation with different levels of Salvia officinalis and/or Origanum majorana on productive performance, ovarian follicular development, lipid peroxidation, antioxidative status, and egg quality in laying hens. Two hundred and ninety-four 45-week-old Bovans brown hens were allocated into seven groups, with seven replicates of six hens each. The first group was fed with the basal considered as a control (A); the second (B) and third (C) groups were provided with the same control diet further supplemented with 0.5 and 1 kg/ton Salvia officinalis, respectively; the fourth (D) and fifth (E) groups received the control diet further supplemented with 0.5 and 1 kg/ton Origanum majorana, respectively; while the sixth (F) and the seventh (G) groups were offered a diet supplemented with 0.5 kg/ton Salvia officinalis and 0.5 kg/ton Origanum majorana and 1 kg/ton Salvia officinalis and 1 kg/ton Origanum majorana, respectively. No significant effects were observed in the final body weight (BW) and feed intake (FI) of the laying hens. In the diets supplemented with Salvia officinalis and Origanum majorana, the egg weights for groups C, F, and G had significantly higher values only compared to group D. The supplementation of the diets with Salvia officinalis and/or Origanum majorana significantly (p < 0.05) increased the Follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol estrogenic hormone concentration, except for Origanum majorana at both levels with regard to estradiol. The dietary utilization of Salvia officinalis and Origanum majorana did not significantly alter the plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT), total protein, albumin, globulin, and High density lipoprotein (HDL) parameters. Cholesterol, glucose, triglyceride, and Low density lipoprotein (LDL) were decreased (p < 0.05) in the birds fed with Salvia officinalis and/or Origanum majorana supplemented diets. Moreover, at both doses, the dietary supplementation with Salvia officinalis and Origanum majorana decreased (p < 0.05) the yolk cholesterol and liver Malondialdehyde (MDA) levels. In addition, the dietary enrichment with Salvia officinalis and/or Origanum majorana decreased (p < 0.05) the palmitoleic and stearic fatty acids’ egg yolk concentration. In contrast, the yolk linoleic fatty acid concentration was significantly increased by Salvia officinalis and/or Origanum majorana. In conclusion, dietary supplementation with Salvia officinalis and/or Origanum positively affected productive performance, ovarian follicular development, antioxidant activity, hormonal status, and steroidogenesis in Bovans brown laying hens.

Aspects of Ovarian Function in a Line of Sheep with a Novel X-Linked Maternally-Imprinted Gene that is Associated with an Increased Ovulation Rate

<p>Fecundity is a term that refers to the number of offspring produced per female. It combines fertility (i.e. ability to produce offspring) and prolificacy (i.e. number of offspring). Ovulation rate i.e. the number of mature eggs released from the ovaries during one reproductive cycle in sheep, as with other mammals, is controlled by an exchange of hormonal signals between the pituitary gland and the ovary. Genetic mutations affecting ovulation are commonly referred to as the fecundity genes (Fec). The most obvious outcome is the number of offspring produced. There is already evidence of a number of major genes affecting the ovulation rate in sheep, specifically the Booroola, Inverdale, Hanna and more recently the Woodlands gene. The sheep carrying the Woodlands gene arose because the mutation was first recognised on a farm in Woodlands, Southland, New Zealand. Woodlands have a novel, X-linked maternally-imprinted, fecundity trait referred to as FecX2w, where Fec = fecundity, X = X chromosome, 2= 2nd mutation identified on X and W= Woodlands. The studies in this thesis investigated ovarian follicular development in both 4-week old Woodland carrier (W+) and non-carrier (++) lambs and adult ewes and evaluated some aspects of the endocrine interactions between the ovary and pituitary gland. The purpose was to identify potential physiological effects of the FecX2w gene on ovarian function. A confounding issue during these studies was the discovery that a large ovary phenotype (LOP) which was present in many of the W+ but not ++ lambs at 4 weeks of age was in fact a coincidence and not linked to the FecX2w mutation. The key findings from the studies of lambs and/or ewes that were carriers (W+) or non-carriers (++) of the FecX2w gene were: 1. No genotype differences were present either in the numbers of primordial (i.e. Type 1/1a follicles) or developing preantral (i.e. Types 2-4 follicles); 2. Significant genotype differences were present in the numbers of small antral (Type 5) follicles (W+>++; p<0.05); 3. An earlier onset of antral follicular development in W+ vs. ++ ewes with irregularities in morphology between the basement membrane and stroma in the former; 4. No genotype differences in the onset of gene expression during follicular development or in the cell-types expressing GDF9, BMP15, alpha inhibin, beta A inhibin and beta B inhibin, FSHR, ER alpha, or ER beta; 5. No genotype differences in the levels of GDF9 or BMP15 gene expression in oocytes throughout follicular growth; 6. No genotype difference in the diameters that follicles reached in W+ vs. ++ ewes; 7. Some lambs at 4-weeks of age had unusually large ovaries with an exceptional level of antral follicular development that is reminiscent of a polycystic ovarian condition. The underlying cause of this condition is unknown. In conclusion, the physiological characteristics of ovarian follicular development in ewes with the FecX2w gene is different from that in ewes with the Booroola, Inverdale, Hanna or other recently identified mutations.</p>

Aspects of Ovarian Function in a Line of Sheep with a Novel X-Linked Maternally-Imprinted Gene that is Associated with an Increased Ovulation Rate

<p>Fecundity is a term that refers to the number of offspring produced per female. It combines fertility (i.e. ability to produce offspring) and prolificacy (i.e. number of offspring). Ovulation rate i.e. the number of mature eggs released from the ovaries during one reproductive cycle in sheep, as with other mammals, is controlled by an exchange of hormonal signals between the pituitary gland and the ovary. Genetic mutations affecting ovulation are commonly referred to as the fecundity genes (Fec). The most obvious outcome is the number of offspring produced. There is already evidence of a number of major genes affecting the ovulation rate in sheep, specifically the Booroola, Inverdale, Hanna and more recently the Woodlands gene. The sheep carrying the Woodlands gene arose because the mutation was first recognised on a farm in Woodlands, Southland, New Zealand. Woodlands have a novel, X-linked maternally-imprinted, fecundity trait referred to as FecX2w, where Fec = fecundity, X = X chromosome, 2= 2nd mutation identified on X and W= Woodlands. The studies in this thesis investigated ovarian follicular development in both 4-week old Woodland carrier (W+) and non-carrier (++) lambs and adult ewes and evaluated some aspects of the endocrine interactions between the ovary and pituitary gland. The purpose was to identify potential physiological effects of the FecX2w gene on ovarian function. A confounding issue during these studies was the discovery that a large ovary phenotype (LOP) which was present in many of the W+ but not ++ lambs at 4 weeks of age was in fact a coincidence and not linked to the FecX2w mutation. The key findings from the studies of lambs and/or ewes that were carriers (W+) or non-carriers (++) of the FecX2w gene were: 1. No genotype differences were present either in the numbers of primordial (i.e. Type 1/1a follicles) or developing preantral (i.e. Types 2-4 follicles); 2. Significant genotype differences were present in the numbers of small antral (Type 5) follicles (W+>++; p<0.05); 3. An earlier onset of antral follicular development in W+ vs. ++ ewes with irregularities in morphology between the basement membrane and stroma in the former; 4. No genotype differences in the onset of gene expression during follicular development or in the cell-types expressing GDF9, BMP15, alpha inhibin, beta A inhibin and beta B inhibin, FSHR, ER alpha, or ER beta; 5. No genotype differences in the levels of GDF9 or BMP15 gene expression in oocytes throughout follicular growth; 6. No genotype difference in the diameters that follicles reached in W+ vs. ++ ewes; 7. Some lambs at 4-weeks of age had unusually large ovaries with an exceptional level of antral follicular development that is reminiscent of a polycystic ovarian condition. The underlying cause of this condition is unknown. In conclusion, the physiological characteristics of ovarian follicular development in ewes with the FecX2w gene is different from that in ewes with the Booroola, Inverdale, Hanna or other recently identified mutations.</p>

257 Awardee Talk: Pursuit of Novel Factors that Regulate Ovarian Follicular Function

As follicles grow, theca cells (TC) and granulosa cells (GC) must proliferate with minimal differentiation while thecal vascularization increases so that follicles do not prematurely ovulate or luteinize before the oocyte is fully mature. In the early 2000s we used Affymetrix microarrays to discover several unique genes involved in ovarian follicular development. Thrombospondin and fibroblast growth factor (FGF) 2 receptor genes were stimulated by IGF1 in porcine GC. We compared GC gene expression in bovine cystic versus normal follicles and discovered several novel genes including Indian hedgehog protein (IHH), FGF9, brain ribonuclease (BRB), and G protein-coupled receptor 34 (GPR34), leading to identification of roles for these proteins in ovarian follicular development. During the past 10 years, follow-up TC microarray and mechanistic studies have identified FGF9 control of cell cycle proteins, tight junction proteins, and microRNA 221 (MIR221), and that the mitogenic and steroidogenic responses to the major trophic hormones of the ovary (including IGF1, LH and FSH) are altered by overexpression of MIR221 in GC. In addition, we discovered that: 1) FGF9 stimulates GC and TC mitosis while inhibiting steroidogenesis; 2) FGF9 induces E2 transcription factor (E2F)-1, E2F-8 and cyclin D1 (CCND1), and that both IGF1 and vascular endothelial growth factor-A (VEGFA) synergize with FGF9 to further induce E2F8 and CCND1 mRNA; 3) FGF9 induces the nuclear protein UHRF1; and 4) an E2F inhibitor blocks the stimulatory and inhibitory effects of FGF9 on GC proliferation and steroidogenesis, respectively, and down-regulates UHRF1 mRNA and up-regulates VEGFA mRNA. Thus, aberrant production of FGF9 and the factors it induces/inhibits may lead to vascular dysfunction and ovarian disorders such as ovarian cysts. With additional research, knowledge about these newly identified factors may be used to help the livestock industry improve reproductive efficiency via new treatments for estrous synchronization, superovulation and cystic ovaries.

rno-miR-128-3p promotes apoptosis in rat granulosa cells (GCs) induced by norepinephrine through Wilms tumor 1 (WT1)

The mechanism related to ovarian follicular is complex, which has not been fully elucidated. Abundant reports have confirmed that the ovarian function development is closely related to sympathetic innervation. As one of the major neurotransmitters, norepinephrine (NE) is considered an effective regulator of ovarian functions like granulosa cell (GC) apoptosis. However, the mechanism between NE and GC apoptosis in rat is still unclear. In our study, GCs were isolated and cultured in vitro with NE treatment. The apoptosis of GCs was facilitated by NE. Wilms tumor 1 (WT1) was found to be significantly downregulated in GCs after NE treatment, and overexpression of WT1 repressed apoptosis in rat GCs induced by NE. rno-miR-128-3p was found to be significantly enhanced by NE in GCs, and inhibition of rno-miR-128-3p repressed apoptosis in rat GCs induced by NE. Mechanistically, rno-miR-128-3p interacted with WT1 and repressed its expression. In summary, inhibition of rno-miR-128-3p may enhance WT1 expression, and then repress NE-induced apoptosis in rat GCs. Our research may provide a new insight for the improvement of ovarian follicular development.

Beneficial effects of a decreased meal frequency on nutrient utilization, secretion of luteinizing hormones and ovarian follicular development in gilts

Background Replacement gilts are typically fed ad libitum, whereas emerging evidence from human and rodent studies has revealed that time-restricted access to food has health benefits. The objective of this study was to investigate the effect of meal frequency on the metabolic status and ovarian follicular development in gilts. Methods A total of 36 gilts (Landrace × Yorkshire) with an age of 150±3 d and a body weight of 77.6±3.8 kg were randomly allocated into one of three groups (n = 12 in each group), and based on the group allocation, the gilts were fed at a frequency of one meal (T1), two meals (T2), or six meals per day (T6) for 14 consecutive weeks. The effects of the meal frequency on growth preference, nutrient utilization, short-chain fatty acid production by gut microbial, the post-meal dynamics in the metabolic status, reproductive hormone secretions, and ovarian follicular development in the gilts were measured. Results The gilts in the T1 group presented a higher average daily gain (+ 48 g/d, P < 0.05) and a higher body weight (+ 4.9 kg, P < 0.05) than those in the T6 group. The meal frequency had no effect on the apparent digestibility of dry matter, crude protein, ether extract, ash, and gross energy, with the exception that the T1 gilts exhibited a greater NDF digestibility than the T6 gilts (P < 0.05). The nitrogen balance analysis revealed that the T1 gilts presented decreased urine excretion of nitrogen (− 8.17 g/d, P < 0.05) and higher nitrogen retention (+ 9.81 g/d, P < 0.05), and thus exhibited higher nitrogen utilization than the T6 gilts. The time-course dynamics of glucose, α-amino nitrogen, urea, lactate, and insulin levels in serum revealed that the T1 group exhibited higher utilization of nutrients after a meal than the T2 or T6 gilts. The T1 gilts also had a higher acetate content and SCFAs in feces than the T6 gilts (P < 0.05). The age, body weight and backfat thickness of the gilts at first estrous expression were not affected by the meal frequency, but the gilts in the T1 group had higher levels of serum luteinizing hormone on the 18th day of the 3rd estrus cycle and 17β-estradiol, a larger number of growing follicles and corpora lutea, and higher mRNA expression levels of genes related to follicular development on the 19th day of the 3rd estrus cycle. Conclusions The current findings revealed the benefits of a lower meal frequency equal feed intake on nutrient utilization and reproductive function in replacement gilts, and thus provide new insights into the nutritional strategy for replacement gilts, and the dietary pattern for other mammals, such as humans.

Tribbles Pseudokinase 2 (TRIB2) Regulates Expression of Binding Partners in Bovine Granulosa Cells

Members of the Tribbles (TRIB) family of pseudokinases are critical components of intracellular signal transduction pathways in physiological and pathological processes. TRIBs, including TRIB2, have been previously shown as signaling mediators and scaffolding proteins regulating numerous cellular events such as proliferation, differentiation and cell death through protein stability and activity. However, the signaling network associated with TRIB2 and its binding partners in granulosa cells during ovarian follicular development is not fully defined. We previously reported that TRIB2 is differentially expressed in growing dominant follicles while downregulated in ovulatory follicles following the luteinizing hormone (LH) surge or human chorionic gonadotropin (hCG) injection. In the present study, we used the yeast two-hybrid screening system and in vitro coimmunoprecipitation assays to identify and confirm TRIB2 interactions in granulosa cells (GCs) of dominant ovarian follicles (DFs), which yielded individual candidate binding partners including calmodulin 1 (CALM1), inhibin subunit beta A (INHBA), inositol polyphosphate phosphatase-like 1 (INPPL1), 5′-nucleotidase ecto (NT5E), stearoyl-CoA desaturase (SCD), succinate dehydrogenase complex iron sulfur subunit B (SDHB) and Ras-associated protein 14 (RAB14). Further analyses showed that all TRIB2 binding partners are expressed in GCs of dominant follicles but are differentially regulated throughout the different stages of follicular development. CRISPR/Cas9-driven inhibition along with pQE-driven overexpression of TRIB2 showed that TRIB2 differently regulates expression of binding partners, which reveals the importance of TRIB2 in the control of gene expression linked to various biological processes such as proliferation, differentiation, cell migration, apoptosis, calcium signaling and metabolism. These data provide a larger view of potential TRIB2-regulated signal transduction pathways in GCs and provide strong evidence that TRIB2 may act as a regulator of target genes during ovarian follicular development.

Lnc-GULP1–2:1 affects granulosa cell proliferation by regulating COL3A1 expression and localization

Backgrounds Long non-coding RNA is a novel group of non-protein coding transcripts over 200 nt in length. Recent studies have found that they are widely involved in many pathological and physiological processes. In our previous study, we found that lnc-GULP1–2:1 was significantly down-regulated in the ovarian cortical tissue of patients with primary ovarian insufficiency and predicted that lnc-GULP1–2:1 has a regulatory effect on COL3A1. Results In this study, we found that lnc-GULP1–2:1 was mainly localized in the cytoplasm of luteinized granulosa cells. The expression of lnc-GULP1–2:1 was lower in patients with diminished ovarian reserve but substantially elevated in patients with polycystic ovary syndrome. Overexpression of lnc-GULP1–2:1 in KGN cells significantly inhibited cell proliferation, likely through cell cycle related genes CCND2 and p16. Moreover, lnc-GULP1–2:1 expression was positively correlated with the level of COL3A in luteinized granulosa cells from patients with different ovarian functions as well as in multiple cell lines. Overexpression of lnc-GULP1–2:1 in KGN cells promoted the expression of COL3A1 and its translocation into the nucleus. Consistently, silencing COL3A1 in KGN cells also significantly inhibited cell proliferation. Conclusions Lnc-GULP1–2:1 affects the proliferation of granulosa cells by regulating the expression and localization of COL3A1 protein, and may participate in the regulation of ovarian follicle development. This study will provide new insight into molecular mechanisms underlying ovarian follicular development, which will help generate novel diagnostic and therapeutic strategies for diseases related to ovarian follicular development disorders.