Growth Of Preovulatory Follicles Research Articles

Effects of selection for litter size variability on ovarian folliculogenesis, ovarian cell proliferation, apoptosis, and production of regulatory peptides in rabbits

The present study aimed to identify the novel mechanisms regulating rabbit fecundity. For this purpose, the association between litter size variability, fecundity, ovarian morphology, and markers of proliferation, apoptosis, steroidogenesis, and the presence of regulatory proteins were examined in ovarian cells from two rabbit lines divergently selected for low (LL) and high (HL) variability in litter size throughout sixteen generations. Ovaries and uteri were isolated from multiparous non-lactating female rabbits from each line at sixteen generations of selection. One part of the ovary was subjected to histomorphometric analysis of folliculogenesis. From the rest of ovary, ovarian granulosa cells (OGCs) were isolated, cultured, and cell viability, proliferation (accumulation of PCNA and of cyclin B1, and BrdU-positive cells), and apoptosis (accumulation of caspase 3, bax and DNA fragmentation) were evaluated by the Trypan blue exclusion test and BrdU, quantitative immunocytochemistry, and cell death detection assays. Furthermore, OGCs were subjected to proteomic analysis by using the nano HPLC-Chip-MS/MS method. The release of progesterone and oestradiol was measured by ELISA. The LL had more than one kit per litter than the HL (7.6 kits vs. 6.5 kits, p ≤ 0.05). No differences were found in the diameter of primordial and primary ovarian follicles, theca, and granulosa thickness, but the diameter of oocytes in the primary and secondary follicles was higher in the LL than in the HL (88.49 µm in the LL vs. 77.86 µm in the HL for oocytes of primary follicles, p ≤ 0.05; 122.10 µm in the LL vs. 109.87 µm in the HL for oocytes of secondary follicles, p ≤ 0.05). Preovulatory follicles were presented only in the ovaries of the LL. The LL had higher incorporation of BrdU and reduced accumulation of bax within OGCs (0.92% in the LL vs. 0.44% in the HL for incorporation of BrdU, p ≤ 0.05; 41% in the LL vs. 48% in the HL for accumulation of bax, p ≤ 0.05). Ovarian fragments from the LL produced less progesterone and oestradiol than those of the HL (12 ng/mg tissue/day in the LL vs, 45 ng/mg tissue/day in the HL for progesterone, p ≤ 0.05; 11 ng/mg tissue/day in the LL vs. 30 ng/mg tissue/day in the HL for oestradiol, p ≤ 0.05). Besides, the OGCs from the LL produced a higher number of specific regulatory proteins involved in cell differentiation, proliferation, and adhesion than the HL (50 vs 38, p ≤ 0.05). In conclusion, higher prolificacy in the LL line would be caused by: (1) the selection of growing primordial ovarian follicles; (2) better transformation to preovulatory follicles; (3) increased cytoplasmic maturation of oocytes; (4) increased DNA synthesis and decreased cytoplasmic apoptosis in OGCs; (5) changes in ovarian steroidogenesis; and (6) changes in the number of peptides involved in cell differentiation, proliferation, and adhesion. HIGHLIGHTS Assessing fecundity in two divergently rabbit lines for variability in litter size. Fecundity and its variability are related to the diameter and quality of oocytes. Selection and better growth of preovulatory follicles have been related to fecundity.

Double trouble: Implications and management of twinning in lactating dairy cows

Over the past 4 decades, twinning increased concurrently with milk production in dairy cattle, but recently appears to have plateaued, likely from the adoption of hormonal synchroniza­tion protocols. Approximately 95% of Holstein twins are dizy­gotic, making double ovulations the primary mechanism for twinning. Decreased progesterone (P4) concentrations dur­ing preovulatory follicle growth increases double ovulations. Increased hepatic catabolism of P4 because of increased feed intake associated with increased milk production provides a physiological mechanism for decreased P4 concentrations and increased double ovulations. Cows with unilateral twins have more pregnancy losses than cows with bilateral twins which were similar to cows with singletons. Selective embryo reduction followed by P4 treatment did not increase the risk of pregnancy loss for cows with unilateral twins, but increased the risk of loss for cows with bilateral twins. Twinning will likely increase concurrently with milk production over time but can be decreased using 2 approaches: 1) submitting cows to hormonal synchronization protocols that increase P4 dur­ing growth of the preovulatory follicle before timed AI; and 2) identifying twins by laterality and allowing cows with bilateral twins to continue through gestation with extra assistance at calving, while selective reduction can be attempted for cows with unilateral twins.

DETERMINATION OF OVULATION TIME IN GAYO MARES BASED ON IMAGE OF PREOVULATORY FOLLICLE GROWTH

This study aimed to obtain the ultrasound image of preovulatory follicle growth in gayo mares to predict the ovulation time. This study used three Gayo mares and one gayo stallion for teaser aged 5-10 years and weighed 200-250 kg. Preovulatory follicle growth was observed using transrectal ultrasonography which was performed when the dominant follicle size was ≥ 3.5 cm. Observations were made for 3 consecutive estrous periods on gayo mares at relatively the same time. The absence of previous follicular appearance and the presence of hypoechoic area in the same ovary indicate that ovulation has occurred. The result revealed that the diameter of preovulatory follicle of gayo mares was 4.02±0.32 to 5.05±0.04 cm. There was no significant follicle growth in the last 48 hours before ovulation and the preovulatory follicle shape altered from spherical to pearshape. Many small follicles (2 cm in diameter) were observed in peripheral area of the ovary indicated the recruitment of follicles which form the first follicular wave. To conclude, the alteration in the size and pearshape of preovulatory follicle and the recruitment of small follicles can be used as a marker to determine the time of ovulation on gayo mares.

A single dose of FSH or hCG during a split-time AI program did not enhance follicular growth or pregnancy per artificial insemination in beef heifers

ABSTRACT The objective of this study was to evaluate the effect of a low dose of hCG or FSH on follicle growth and pregnancy per artificial insemination (AI) in crossbreed beef heifers subjected to a split-time AI program. All heifers (n = 386) were subjected to a 7-d Co-synch protocol and an intravaginal controlled internal drug release (CIDR) device (GnRH and CIDR insertion; 7 days later PGF2α, CIDR removal, and application of a tail head estrus detection patch; 36 hours later AI of heifers detected in estrus, 24 hours later timed AI of remaining heifers). At CIDR removal, heifers were randomly assigned to receive either 150 IU of hCG, 20 mg of FSH, or remained as untreated controls. The diameter of the preovulatory follicle and the pregnancy per AI did not differ among treatments. A smaller (P = 0.035) proportion of heifers in the hCG group were detected in estrus compared with control. Additionally, the incidence of premature ovulation was greater (P < 0.01) among heifers treated with hCG compared with control. In conclusion, using a low dosage of hCG or FSH at the time of device removal did not affect pre-ovulatory follicle growth or the proportion of pregnant heifers.

Effect of insulin-like growth factor system on luteinising angiogenesis.

Preovulatory follicle growth and the luteal transition requires intense angiogenesis. This enables progesterone production to increase sufficiently to support a pregnancy. Inadequate follicular or luteal vascularisation can lead to reduced ovarian function and thus compromise fertility. Insulin-like growth factor 1 (IGF1) and IGF2 regulate multiple ovarian processes and are key links between an animal's reproductive and metabolic status. This study investigated the role that the IGF system plays in regulating luteinising follicular endothelial cell (EC) networks and progesterone production in vitro. Bovine luteinising follicular angiogenesis cultures were treated with 1) LR3-IGF1 (10 or 100ng/ml) under basal and angiogenic-stimulated conditions or 2) IGF1 receptor inhibitor (picropodophyllin (PPP); 1µM) in the presence or absence of LR3-IGF1, IGF2, or combined LR3-IGF1+IGF2 (10ng/ml). EC networks were quantified by von Willebrand factor immunohistochemistry. Progesterone production was analysed by ELISA and cell proliferation was determined by MTT assay. LR3-IGF1 had limited effects on EC growth parameters, whilst PPP (p<0.001) markedly reduced EC growth parameters (by 60-70%). Cell proliferation was slightly increased (by 3-5%) by LR3-IGF1 (p<0.001). LR3-IGF1 had variable effects on progesterone production, whilst PPP reduced progesterone concentration (p<0.001) with or without LR3-IGF1 or IGF2 alone or in combination. IGF1 was detected in cell conditioned media and was increased by LH (50ng/ml) (p<0.001). In conclusion, exogenous IGF1 and IGF2 had minimal effects on luteinising follicular angiogenesis and progesterone production, but the inhibitory effect of the IGFR1 inhibitor (PPP) suggests that IGF1 receptor signalling is critical for the development of EC networks and progesterone production in luteinising follicular cells.

PROGRAMAS DE INSEMINACIÓN Y TRANSFERENCIA DE EMBRIONES: EFECTO DEL CELO SOBRE LA PREÑEZ Y PÉRDIDAS EMBRIONARIAS Y FETALES EN BOVINOS

The primary objective of implementing embryo transfer in meat operations is to accelerate the rate of genetic progress in the herd. Among the main factors that affect the use of these technologies are related to the nutrition, the management and the synchronization of the estrus. As a result of research conducted in the last 20 years, the use of the receptor has increased through the application of protocols that synchronize ovulation and allow the transfer of embryos without the need for oestrus detection, generally known as fixed-time embryo transfer. (FTET). Although these protocols have functioned well for several years, attention has recently been directed to the effect of estrus expression and estradiol concentrations during pre-ovulatory follicle growth on embryonic growth and pregnancy. The experiments reviewed in this document demonstrate that oestrus expression is positively associated with high pregnancy rates and reduced pregnancy losses in recipients receiving frozen / thawed bovine embryos produced in vitro and in vivo.

Gonadotrophic control of follicle growth in the ewe

Preovulatory follicle growth in the ewe is dependent on FSH although no precise relationship appears to exist between plasma concentrations of FSH and the number of preovulatory follicles which develop or ovulation rate. This may be related to a hitherto unrecognized influence of pulsatile LH on the growth of large follicles. Preovulatory follicle growth is dependent on the presence of basal amounts of LH, but pulsatile LH, while being essential to supply an increase in androgen substrate to the granulosa cells of the follicle, may also play a role in reducing the responsiveness of many large follicles to FSH, in particular during the preovulatory phase when plasma concentrations of FSH are reduced. Thus selection of the preovulatory follicle(s) may involve a previously unrecognized interaction between FSH and pulsatile LH secretion in which pulses of LH act in a negative rather than positive manner.

Preovulatory follicle growth dynamics indicates daily spawning synchronicity in wild-caught Atlantic horse mackerel off Portugal

The Atlantic horse mackerel, Trachurus trachurus, is a commercially important species whose daily spawning dynamics are not yet fully understood. Existence of spawning synchronicity would facilitate application of the daily egg production method for estimating the spawning biomass of the southern stock of the species in the Northeast Atlantic. The intent of this work was to study preovulatory follicle (PREF) growth dynamics in wild Atlantic horse mackerel to determine the existence of daily spawning synchronicity in the population. The size and translucency of hydrated oocytes increased significantly between early morning and late afternoon; this pattern was the same for two study years. This morpho-developmental shift led to the appearance of previously indistinguishable oil drops in ovarian whole mounts which is an easily observed microscopic descriptor of PREF growth. The results clearly suggested that PREF growth was well synchronized between Atlantic horse mackerel that were capable of spawning and provided evidence for the existence of daily spawning synchronicity in the population. Apart from sampling time of day, the size of hydrated oocytes was shown to be affected by body size: a 10 cm difference in maternal length corresponded to a 0.030 mm difference in the diameter of hydrated oocytes. In contrast, the size of early germinal vesicle migration oocytes was correlated neither to sampling time nor to maternal size which suggests that the abrupt increase in PREF size only commences after the onset of the actively spawning sub-phase.

Dynamic remodeling of endometrial extracellular matrix regulates embryo receptivity in cattle.

We aimed to evaluate in the bovine endometrium whether (1) key genes involved in endometrial extracellular matrix (ECM) remodeling are regulated by the endocrine peri-ovulatory milieu; and (2) specific endometrial ECM-related transcriptome can be linked to pregnancy outcome. In Experiment 1, pre-ovulatory follicle growth of cows was manipulated to obtain two groups with specific endocrine peri-ovulatory profiles: the Large Follicle-Large CL group (LF-LCL) served as a paradigm for greater receptivity and fertility and showed greater plasma pre-ovulatory estradiol and post-ovulatory progesterone concentrations when compared to the Small Follicle-Small CL group (SF-SCL). Endometrium was collected on days 4 and 7 of the estrous cycle. Histology revealed a greater abundance of total collagen content in SF-SCL on day 4 endometrium. In Experiment 2, cows were artificially inseminated and, six days later, endometrial biopsies were collected. Cows were retrospectively divided into pregnant and non-pregnant (P vs. NP) groups after diagnosis on day 30. In both experiments, expression of genes related to ECM remodeling in the endometrium was studied by RNAseq and qPCR. Gene ontology analysis showed an inhibition in the expression of ECM-related genes in the high receptivity groups (LF-LCL and P). Specifically, there was down-regulation of TGFB2, ADAMTS2, 5 and 14, TIMP3 and COL1A2, COL3A1, COL7A1 and COL3A3 in the LF-LCL and P groups. In summary, the overlapping set of genes differently expressed in both fertility models: (1) suggests that disregulation of ECM remodeling can impair receptivity and (2) can be used as markers to predict pregnancy outcome in cattle.

Comparison of fertility following use of one versus two intravaginal progesterone inserts in dairy cows without a CL during a synchronization protocol before timed AI or timed embryo transfer

The objective was to evaluate the effect of increased progesterone (P4) during preovulatory follicle growth during timed AI (TAI) or timed embryo transfer (TET) protocols. Lactating dairy cows with no CL and low circulating P4 (≤1.0 ng/mL) were submitted to a protocol using one or two intravaginal P4 implants (controlled intravaginal releasing device [CIDRs]), and were bred to TAI or TET. The low P4 cows for this experiment were identified on nine farms, four utilized TAI (n = 326 of 1160 cows examined), and five utilized TET (n = 445 of 1396). All cows were synchronized by insertion of P4 implant(s) (CIDR[s]) at start of protocol (Day −11) and simultaneous treatment with 2 mg of E2-benzoate. After 7 days, cows were treated with PGF (Day −4) and 2 days later treated with 1.0-mg E2-cypionate and CIDR(s) were removed (Day −2). Cows received TAI on Day 0 or TET on Day 7. Cows were randomly assigned to receive either one or two CIDRs on Day −11 until Day −2 (1CIDR vs. 2CIDR). Presence of CL was determined by ultrasound on Day −11 and Day 7 after protocol (to determine ovulation to protocol), P4 concentrations were determined on a subset of cows (Day −11, Day −4, Day 7), and ovulatory follicle diameter was evaluated on Day 0. Pregnancy success (P/AI or P/ET) was evaluated on Days 32 and 60. The 2CIDR treatment increased circulating P4 by Day −4 (1.77 ± 0.23 vs. 2.18 ± 0.24 ng/mL) but had no effect on ovulation at the end of protocol (83.6 vs. 82.6%) or ovulatory follicle diameter (15.6 ± 0.3 vs. 15.3 ± 0.3 mm). If only cows that ovulated to the protocol were included, 1CIDR tended to have lower P/AI than 2CIDR by Day 32 (42.8 vs. 52.6%; P = 0.10) and Day 60 (37.7 vs. 48.1%; P = 0.08) but there was no effect on pregnancy loss. There was an interaction (P = 0.05) between ovulatory follicle diameter and CIDR treatment on P/AI (Day 60). In cows ovulating larger follicles (≥14 mm), 2CIDR treatment increased P/AI compared with 1CIDR (53.3 vs. 34.9%; P = 0.02) but not in cows ovulating small follicles (<14 mm). There was no effect of treatment on P/ET on Day 32 (30.0% vs. 32.0%) or Day 60 (24.7% vs. 25.6%). Thus, these results add evidence to the concept that increased circulating P4 during preovulatory follicle development may improve P/AI, most likely due to improved oocyte quality in cows that ovulate larger follicles, since improvement was only in cows ovulating larger follicles and no effect of preovulatory P4 was observed in cows that received ET.

Developmental programming: rescuing disruptions in preovulatory follicle growth and steroidogenesis from prenatal testosterone disruption.

BackgroundPrenatal testosterone (T) excess from days 30-90 of gestation disrupts gonadotropin surge and ovarian follicular dynamics and induces insulin resistance and functional hyperandrogenism in sheep. T treatment from days 60-90 of gestation produces a milder phenotype, albeit with reduced fecundity. Using this milder phenotype, the aim of this study was to understand the relative postnatal contributions of androgen and insulin in mediating the prenatal T induced disruptions in ovarian follicular dynamics.MethodsFour experimental groups were generated: 1) control (vehicle treatment), 2) prenatal T-treated (100 mg i.m. administration of T propionate twice weekly from days 60-90 of gestation), 3) prenatal T plus postnatal anti-androgen treated (daily oral dose of 15 mg/kg/day of flutamide beginning at 8 weeks of age) and 4) prenatal T and postnatal insulin sensitizer-treated (daily oral dose of 8 mg/day rosiglitazone beginning at 8 weeks of age). Follicular response to a controlled ovarian stimulation protocol was tested during their third breeding season. Main outcome measures included the determination of number and size of ovarian follicles and intrafollicular concentrations of steroids.ResultsAt the end of the controlled ovarian stimulation, the number of follicles approaching ovulatory size (≥6 mm) were ~35 % lower in prenatal T-treated (6.5 ± 1.8) compared to controls (9.8 ± 2.0). Postnatal anti-androgen (10.3 ± 1.9), but not insulin sensitizer (5.0 ± 0.9), treatment prevented this decrease. Preovulatory sized follicles in the T group had lower intrafollicular T, androstenedione, and progesterone compared to that of the control group. Intrafollicular steroid disruption was partially reversed solely by postnatal insulin sensitizer treatment.ConclusionsThese results demonstrate that the final preovulatory follicular growth and intrafollicular steroid milieu is impaired in prenatal T-treated females. The findings are consistent with the lower fertility rate reported earlier in these females. The finding that final follicle growth was fully rescued by postnatal anti-androgen treatment and intrafollicular steroid milieu partially by insulin sensitizer treatment suggest that both androgenic and insulin pathway disruptions contribute to the compromised follicular phenotype of prenatal T-treated females.

Vascular endothelial growth factor and angiopoietins during hen ovarian follicle development

Growth and maturation of ovarian follicles in the hen (Gallus gallus) requires a network of blood vessels that increases in complexity during development. The present studies investigate expression of vascular endothelial growth factor A (VEGF), angiopoietin1 (ANGPT1) and ANGPT2 mRNAs together with their associated receptors (VEGFR and TIE2, respectively) during maturation. Elevated expression of VEGF and its receptors is associated with healthy, compared to atretic, follicles. Levels of VEGF significantly increase, while antagonistic ANGPT2 decrease, in granulosa cells (GC) at follicle selection. By comparison, levels of VEGF, VEGFR1, VEGFR2, ANGPT1, ANGPT2 and TIE2 within the theca layer do not change (P>0.05) relative to developmental stages surrounding follicle selection (6–8mm versus 9–12mm follicles). Prior to selection, treatment with transforming growth factor β1 (TGFβ1) significantly increases levels of VEGF in undifferentiated GC from prehierarchal (6–8mm) follicles and actively differentiating GC from selected (9–12 and F4) follicles. Moreover, subsequent to selection follicle stimulating hormone (FSH) increases VEGF expression in GC from 9 to 12mm follicles, and eventually luteinizing hormone (LH) promotes VEGF expression in GC from more mature preovulatory follicles. It is concluded that prior to follicle selection VEGF expression is regulated by autocrine and paracrine actions of TGFβ1 (but not FSH), and that a comparatively limited extent of vasculature is sufficient to maintain prehierarchal follicles in a viable and undifferentiated state. At follicle selection, FSH- and subsequently LH-induced VEGF production within the GC layer enhance angiogenesis within the theca layer, which facilitates the rapid growth of preovulatory follicles via enhanced incorporation of yellow yolk.

Studying the Functions of TGF-β Signaling in the Ovary.

In mammals, ovulation is a multistep physiological process that includes preovulatory follicle growth, oocyte meiotic maturation, cumulus-oocyte complex (COC) expansion, follicle rupture, and luteinization. TGF-β signaling pathway has multiple functions in mammalian ovary, as its complexity in ovarian function has been demonstrated by mouse models with knockouts of TGF-β receptors and SMADs. We describe the protocol that we use to study functions of TGF-β signaling pathway in follicle development and ovulation. Because total knockout of TGF-β pathway components often causes embryonic lethality, which prevents further investigation of these genes in ovarian functions, people have generated ovarian cell type-specific knockout mouse strains for TGF-β signaling pathway genes. These mouse models are also described.

106 THE RECEPTIVE BEEF COW ENDOMETRIUM: POTENTIAL KEY FEATURES FAVOURING THE COMMUNICATION BETWEEN EMBRYONIC AND MATERNAL TISSUES

A receptive state of the endometrium is crucial for proper communication with the pre-implantation embryo and, thereby, a perquisite for a successful pregnancy. Both reduction of transmembrane mucin and initiation of apoptotic events have been proposed as key features prompting embryo/endometrial cell interaction and facilitating maternal receptivity of the murine and human embryo. In beef cows, however, the signature for receptivity needs to be elucidated. Therefore, we aimed to characterise “receptive” versus “refractory” endometrial tissues with the focus on (1) transcription profiles related to mucin, apoptosis and proliferative pathways, and 2) phenotypic features; that is, epithelial transmembrane mucin and apoptotic cell rates (ACR). Using a bovine model, preovulatory follicle growth was manipulated to produce 2 groups: cows with larger preovulatory follicle, longer proestrus, and higher receptivity (LF-LCL) versus cows with smaller preovulatory follicle, shorter proestrus, and lower receptivity (SF-SCL). Seven days post-induced ovulation, endometrium was collected. Transcriptome profiles of endometrial tissue (n = 3/group) were determined using Illumina RNA sequencing analyses. In addition, paraffin embedded endometrial samples (n = 6/group) were stained with Alcian Blue for semiquantitative analyses of mucin staining intensity and treated with antibodies against activated Casp3 to determine ACR. RNA-seq data showed that cell surface MUC1 gene expression was drastically reduced (fold-change –3.33) in LF-LCL tissue (P &lt; 0.05). In contrast, the LF-LCL endometrial tissue displayed an up-regulated expression of genes involved in apoptosis pathways, such as Casp9, CRADD, DAPL1 (fold-change 1.80, 2.10, and 3.60, respectively; P &lt; 0.05). Moreover, the expression of genes related to proliferation, such as MYC and NOV, was significantly down-regulated in LF-LCL endometrial tissue (fold-change –2.2 and –6.3, respectively; P &lt; 0.01). Histology of endometrial samples revealed that the signal for transmembrane, anti-adhesive, mucin at the LF-LCL epithelium was consistently low, whereas the SF-SCL tissue group displayed variable amounts of transmembrane mucin at the apical epithelial boarder. Immunohistochemistry showed that gland cells from LF-LCL endometrial tissue displayed a significantly higher ACR compared with gland cells of the SF-SCL tissue (29.6 ± 1.52% v. 11.9 ± 1.26%; P &lt; 0.05). In the surface epithelium and the stromal tissue, Casp3 positive cells were rare and ACR was similar between groups. In conclusion, a down-regulated expression of the transmembrane mucin might indicate a receptive condition of the endometrium. Furthermore, the prominent apoptotic characteristics in the LF-LCL tissue suggest that the receptive endometrium must surpass the proliferative status in order to differentiate to more specialised functions, such as embryotrophy.

Exposure to the endocrine disruptor di(2-ethylhexyl)phthalate affects female reproductive features by altering pulsatile LH secretion

The patterns of growth of preovulatory follicles and corpora lutea, as well as plasma concentrations of estradiol, progesterone and LH were evaluated in sheep exposed to DEHP. There were not found effects on the preovulatory follicular dynamics nor on the ovulatory efficiency between DEHP-exposed and control sheep. However, plasma estradiol concentration was significantly higher in the ewes exposed to DEHP than in the control females (P<0.001). Afterwards, DEHP-exposed ewes had significantly higher plasma progesterone concentration from Day 2 of the luteal phase (P<0.05), although there were no differences between groups in the macro- and microscopic features of the corpora lutea. Analysis of mean basal concentrations of LH showed lower values in DEHP-exposed than in control sheep (0.3±01ng/mL vs. 0.7±0.1; P<0.05). Moreover, the frequency of LH pulses was 0.3pulses/4h, with amplitude of 0.6ng/mL, in the control group; on the other hand, the pulsatile secretion of LH in the DEHP-exposed group was undetectable during the period of sampling.

Comparison of Different Treatments for Oestrous Induction in Seasonally Anovulatory Mares

The aim of this study was to evaluate the effects of different treatments for induction and synchronization of oestrus and ovulation in seasonally anovulatory mares. Fifteen mares formed the control group (C), while 26 mares were randomly assigned to three treatment groups. Group T1 (n=11) were treated with oral altrenogest (0.044mg/kg; Regumate(®) ) during 11days. Group T2 (n=7) was intravaginally treated with 1.38g of progesterone (CIDR(®) ) for 11days. In group T3 (n=8), mares were also treated with CIDR(®) , but only for 8days. All mares received PGF2α 1day after finishing the treatment. Sonographic evaluation of follicles, pre-ovulatory follicle size and ovulation time was recorded. Progesterone and leptin levels were analysed. Results show that pre-ovulatory follicles were developed after the treatment in 88.5% of mares. However, the pre-ovulatory follicle growth was dispersal, and sometimes it was detected when treatment was not finished. While in mares treated with intravaginal device, the follicle was soon detected (1.5±1.2days and 2.3±2.0days in T2 and T3 groups, respectively), in T1 group, the pre-ovulatory follicle was detected slightly later (3.9±1.6days). The interval from the end of treatment to ovulation did not show significant differences between groups (T1=13.1±2.5days; T2=11.0±3.6days; T3=13.8±4.3days). The pregnancy rate was 47.4%, similar to the rate observed in group C (46.7%; p>0.05). Initial leptin concentrations were significantly higher in mares, which restart their ovarian activity after treatments, suggesting a role in the reproduction mechanisms in mares. It could be concluded that the used treatments may be effective for oestrous induction in mares during the late phase of the seasonally anovulatory period. Furthermore, they cannot synchronize oestrus, and then, it is necessary to know the reproductive status of mares when these treatments are used for oestrous synchronization.

Effects of synchronization treatments on ovarian follicular dynamics, corpus luteum growth, and circulating steroid hormone concentrations in lactating dairy cows

Lactating dairy cows (n=57) ≥45 d postpartum at first service were enrolled in a randomized complete block design study to evaluate treatments to synchronize estrus and ovulation. At 10 d before artificial insemination (AI), animals were randomly assigned to 1 of 3 treatments: (1) d −10 GnRH (GnRH1; 10μg of buserelin, i.m.) and controlled internal drug release insert [CIDR, 1.38g of progesterone (P4)]; d −3 PGF2α (PGF; 25mg of dinoprost, i.m.); d −2 CIDR out; and AI at observed estrus (CIDR_OBS); (2) same as CIDR_OBS, but GnRH (GnRH2) 36h after CIDR out and timed AI (TAI) 18h later (CIDR_TAI); or (3) same as CIDR_TAI, but no CIDR (Ovsynch). Transrectal ultrasound was used to assess follicle size before ovulation and on d 4, 8, and 15 after the presumptive day of estrus (d 0) to measure the corpus luteum (CL). Blood samples were collected to determine concentrations of estradiol (E2; d −10, −9, −3, −2, −1, and 0) and P4 (d −10, −9, −2, −1, 0, 1, 4, 6, 8, 11, and 15). No treatment differences were observed in either circulating concentrations of P4 or the ovulatory response to GnRH1 at the onset of synchronization treatments. Circulating concentrations of P4 were greater for CIDR_OBS and CIDR_TAI compared with Ovsynch at 24h after CIDR insertion (5.34 and 4.98 vs. 1.75 ng/mL) and immediately before CIDR removal (1.65 and 1.48 vs. 0.40 ng/mL). Peak circulating concentrations of E2 were greater for CIDR_OBS compared with Ovsynch (3.85 vs. 2.39pg/mL), but CIDR_TAI (2.82pg/mL) did not differ from either CIDR_OBS or Ovsynch. The interval from PGF injection to peak circulating E2 did not differ between CIDR_TAI and Ovsynch (52.1 vs. 49.8h). Both CIDR_TAI and Ovsynch, however, had shorter intervals from PGF injection to peak circulating E2 concentrations compared with CIDR_OBS (67.8h). The diameter of the dominant follicle before ovulation was greater for CIDR_OBS compared with Ovsynch (18.5 vs. 16.0mm) but CIDR_TAI (17.1mm) did not differ from either of the other treatments. The mean interval from PGF to ovulation was longer for CIDR_OBS (100.0h) compared with CIDR_TAI and Ovsynch (84.4 and 83.2h, respectively). Use of CIDR_OBS resulted in increased preovulatory follicle size and greater circulating concentrations of E2 due to a longer period of preovulatory follicle growth. Progesterone supplementation during synchronization and GnRH on the day before TAI affected ovulatory follicle size, and periovulatory circulating concentrations of P4 and E2. No differences, however, in postovulatory P4 or luteal volume profiles were observed.

Pre-ovulatory LH/hCG surge decreases C-type natriuretic peptide secretion by ovarian granulosa cells to promote meiotic resumption of pre-ovulatory oocytes

In mammalian follicles, oocytes are arrested at the diplotene stage of prophase I until meiotic resumption following the LH surge. Recently, C-type natriuretic peptide (CNP), encoded by natriuretic peptide precursor type C (NPPC) was found to suppress mouse oocyte maturation by promoting cyclic guanosine 5'-monophospate (cGMP) production in cumulus cells. However, regulation of NPPC/CNP expression during the pre-ovulatory period and their regulation by the LH surge have not been investigated. Based on genome-wide analysis of DNA microarray data sets using samples from periovulatory ovaries, we found increases in NPPC transcripts in granulosa cells during pre-ovulatory follicle growth in mice and a rapid decline induced by the pre-ovulatory LH/hCG stimulation. Treatment of pre-ovulatory animals with hCG decreased ovarian CNP content. In isolated ovarian cells, NPPC mRNA was predominantly expressed in mural granulosa cells exhibiting similar regulation following gonadotrophin treatment. In cultured mouse pre-ovulatory follicles, meiosis resumption in oocytes by hCG treatment was accompanied by decreases in NPPC transcript levels. In cultured mouse cumulus cell-oocyte complexes, CNP treatment inhibited the resumption of meiosis with increases in cGMP levels in both cumulus cells and oocytes. In human ovaries, CNP levels in ovarian follicular fluid were also decreased following treatment of patients with an ovulatory dose of hCG. Our findings demonstrate gonadotrophins regulation of NPPC/CNP expression in mouse and human ovaries and confirm the role of CNP as a potent paracrine oocyte maturation inhibitor.

Influence of synthetic lamprey GnRH-III on gonadotropin release and steroid hormone levels in gilts

Based on the supposition that lamprey GnRH-III (lGnRH-III) elicits FSH releasing activity in swine, synthetic lGnRH-III (peforelin, Maprelin® XP10) was used in puberal estrus synchronized gilts. The secretion of reproductive hormones FSH, LH, estradiol and progesterone was analyzed, and follicle growth and ovulation recorded. Altogether, 24 German Landrace gilts were treated after an 18-day long synchronization of the estrus cycle with Regumate® as follows: 48 h after the last Regumate® feeding they received im either 150 μg Maprelin® XP10 (lGnRH-III, group Maprelin, n = 6), 50 μg Gonavet Veyx® (GnRH-I agonist, group GnRH, n = 6), 850 IE Pregmagon® (eCG, group eCG, n = 6) or saline (group Control, n = 6). Additionally, in eight gilts the concentrations of FSH and LH were analyzed after treatment with 150 μg Maprelin® XP10 (n = 3), 50 μg Gonavet Veyx® (n = 3) or saline (n = 2) at mid-cycle (day 10 of the estrus cycle). Blood samples were collected via implanted jugular vein catheters. Ovarian features were judged endoscopically at the end of the Regumate® feeding and on days 5 and 6 after treatment. Maprelin® XP10 had no effect on FSH release in gilts; neither at the pre-ovulatory period or at mid-cycle. Furthermore, LH levels were unaffected. In contrast, GnRH-I agonist stimulates FSH release, however less compared to LH secretion. LH secretion was induced by GnRH-I both during the follicular phase and at mid-cycle. Equine CG did not stimulate the release of pituitary hormones FSH and LH due to its direct action on the ovary. Increased estradiol concentrations during days 2 to 5 after Regumate® in all treatment groups indicated pre-ovulatory follicle growth in gilts. Equine CG stimulated a higher (P < 0.01) number of ovulatory follicles compared to the other treatment groups. All together, 83 to 100 % of gilts ovulated by day 6 post treatment. In summary, results of our study on reproductive hormone secretion do not provide evidence that synthetic lGnRH-III (Maprelin® XP10) selectively releases FSH in estrus synchronized gilts.

Age-dependent role of steroids in the regulation of growth of the hen follicular wall

BackgroundThe ovaries are the primary targets of senescence effects in mammalian and avian species. In the present study, relationships between reproductive aging, sex steroids and the growth pattern of the pre-ovulatory follicle wall were investigated using young hens with long clutch (YLC), old hens with long clutch (OLC), old hens with short clutch (OSC), and old hens with interrupted long clutch (OILC).MethodsExperiment 1: Hens were sacrificed 1.5 and 14.5 h after ovulation. Experiment 2: YLC and OILC hens were sacrificed 3.5 h after treatments with LH and/or aminoglutethimide (AG), an inhibitor of steroid synthesis. Volumes of pre-ovulatory follicles (F1-F5) and plasma concentrations of ovarian steroids were determined. Experiment 3: Granulosa and theca cells from F3 follicles of OSC and/or YLC hens were exposed in vitro to estradiol-17beta (E2), testosterone (T) and LH and the proliferative activity of the cells was examined using CellTiter 96 Aqueous One Solution Assay.ResultsIn YLC and OLC groups, the total volume of F1-F5 follicles rose between 1.5 and 14.5 h after ovulation (P < 0.01), negatively correlating with the plasma level of E2 (P < 0.01). There was no growth of pre-ovulatory follicles in the middle of the ovulatory cycle in the OSC group, with a positive correlation being present between E2 and the follicular volume (P < 0.05). In young hens, AG caused a rise in the total follicular volume. This rise was associated with a fall in E2 (r = -0.54, P < 0.05). E2 enhanced proliferation of granulosa cells from YLC and OSC groups. The proliferative activity of granulosa and theca cells of YLC hens depended on the interaction between T and LH (P < 0.01).ConclusionsThese data indicate for the first time that the growth pattern of pre-ovulatory follicles during the ovulatory cycle changes in the course of reproductive aging. E2 seems to play a dual role in this adjustment; it stimulates the growth of the follicular wall in reproductive aged hens, whereas it may inhibit this process in young birds. T and LH are apparently involved in the growth regulation during the pre-ovulatory surge in young hens.