Preovulatory Follicle Development Research Articles

Effects of Nesting Material Provision and High-Dose Vitamin C Supplementation during the Peripartum Period on Prepartum Nest-Building Behavior, Farrowing Process, Oxidative Stress Status, Cortisol Levels, and Preovulatory Follicle Development in Hyperprolific Sows.

Hyperprolific sows often experience increased oxidative stress during late gestation and lactation periods, which can adversely affect the farrowing process and overall lactation performance. This study examines the influence of providing a coconut coir mat (CCM; 1 × 1 m) as nesting material, supplementing high-dose vit-C (HVC; 20% vit-C, 10 g/kg feed) as an antioxidant, or both on maternal behavior, the farrowing process, oxidative status, cortisol levels, and preovulatory follicle developments in sows with large litters. In total, 35 sows (Landrace × Yorkshire; litter size 15.43 ± 0.27) were allocated to the following four treatment groups: control (n = 9, basal diet), vit-C (n = 8, basal diet + HVC), mat (n = 10, basal diet + CCM), and mat + vit-C (n = 8, basal diet + HVC + CCM). A post-hoc analysis showed that compared with sows that were not provided CCM, mat and mat + vit-C groups demonstrated increased durations of nest-building behavior during the period from 24 h to 12 h before parturition (p < 0.05 for both), reduced farrowing durations, and decreased intervals from birth to first udder contact (p < 0.01 for both). The mat group exhibited lower advanced oxidation protein product (AOPP) levels during late gestation and lactation periods than the control group (p < 0.05). Sows with HVC supplementation showed longer farrowing durations than those without HVC supplementation (p < 0.0001). The vit-C group had higher salivary cortisol levels on day 1 after farrowing than the other treatment groups (p < 0.05). Furthermore, the follicle diameters on day 3 after weaning in the vit-C group tended to be smaller than those in the control group (p = 0.077). HVC supplementation prolonged farrowing and increased the physiological stress on postpartum, and no advantageous effects on maternal behavior and developmental progression of preovulatory follicles were observed. Hence, alternative solutions beyond nutritional approaches are required to address increased oxidative stress in hyperprolific sows and secure their welfare and reproductive performance. The present results substantiated the positive impact of providing CCM as nesting material for sows with large litters on nest-building behavior and the farrowing process, which could mitigate the deleterious consequences induced by peripartum physiological and oxidative stress.

Comparison of the efficacy of altrenogest versus transdermal progestin patches on estrus synchronization and ovulation in mares.

The adhesive progestin patch was investigated for estrus synchronization in mares because this method is convenient and safe in other species. This study aimed to compare the efficacy of a transdermal progestin patch versus oral altrenogest on estrus synchronization, preovulatory follicle development, and ovulation. Twenty-four broodmares were randomly divided into two groups. In Group 1, mares (n = 12) were fed 0.044 mg/kg altrenogest daily for 14 days. Group 2 (n = 12) mares were treated with adhesive transdermal progestin patches for 14 days. Mares were observed for estrus daily, from day 3 until day 7, after hormone withdrawal. Follicular development was determined by ultrasonography. The preovulatory follicle size and ovulation time after hormone treatments and ovulation rate were determined for both groups. Plasma progesterone levels were measured during the experimental periods and 2 days after hormone termination in both groups. The results revealed that the transdermal progestin patch efficiently controlled follicular growth and estrus synchronization in mares. The percentage of mares exhibiting estrus was similar in the altrenogest (100.00%) and transdermal patch (91.67%) groups. Ovulation rates were equivalent with either altrenogest or progestin patch protocols (91.67% [11/12] vs. 83.33% [10/12]). In addition, the preovulatory follicle size was similar in mares treated with altrenogest and progestin patches. This study revealed that the transdermal progestin patch provides effective estrus synchronization and ovulation, similar to altrenogest treatment. However, the transdermal patch was more convenient with a shorter predictable ovulation time after estrus synchronization and should be considered as an alternative method for mares.

Mechanism of negative modulation of FSH signaling by salt-inducible kinases in rat granulosa cells.

The optimal development of preovulatory follicles needs follicle-stimulating hormone (FSH). Recent findings revealed that salt-inducible kinases (SIKs) inhibit FSH actions in humans and rodents. This report seeks to increase our understanding of the molecular mechanisms controlled by SIKs that participate in the inhibition of FSH actions in primary rat granulosa cells (GCs). The results showed that FSH causes a transient induction of Sik1 mRNA. In contrast, SIK inhibition had no effects on FSH receptor expression. Next, we determined whether SIK inhibition enhances the effect of several sequential direct activators of the FSH signaling pathway. The findings revealed that SIK inhibition stimulates the induction of steroidogenic genes by forskolin, cAMP, protein kinase A (PKA), and cAMP-response element-binding protein (CREB). Strikingly, FSH stimulation of CREB and AKT phosphorylation was not affected by SIK inhibition. Therefore, we analyzed the expression and activation of putative CREB cofactors and demonstrated that GCs express CREB-regulated transcriptional coactivators (CRTC2) and that FSH treatment and SIK inhibition increase the nuclear expression of this factor. We concluded that SIKs target the FSH pathway by affecting factors located between cAMP/PKA and CREB and propose that SIKs control the activity of CRTC2 in ovarian GCs. The findings demonstrate for the first time that SIKs blunt the response of GCs to FSH, cAMP, PKA, and CREB, providing further evidence for a crucial role for SIKs in regulating ovarian function and female fertility.

Estradiol Signaling at the Heart of Folliculogenesis: Its Potential Deregulation in Human Ovarian Pathologies.

Estradiol (E2) is a major hormone controlling women fertility, in particular folliculogenesis. This steroid, which is locally produced by granulosa cells (GC) within ovarian follicles, controls the development and selection of dominant preovulatory follicles. E2 effects rely on a complex set of nuclear and extra-nuclear signal transduction pathways principally triggered by its nuclear receptors, ERα and ERβ. These transcription factors are differentially expressed within follicles, with ERβ being the predominant ER in GC. Several ERβ splice isoforms have been identified and display specific structural features, which greatly complicates the nature of ERβ-mediated E2 signaling. This review aims at providing a concise overview of the main actions of E2 during follicular growth, maturation, and selection in human. It also describes the current understanding of the various roles of ERβ splice isoforms, especially their influence on cell fate. We finally discuss how E2 signaling deregulation could participate in two ovarian pathogeneses characterized by either a follicular arrest, as in polycystic ovary syndrome, or an excess of GC survival and proliferation, leading to granulosa cell tumors. This review emphasizes the need for further research to better understand the molecular basis of E2 signaling throughout folliculogenesis and to improve the efficiency of ovarian-related disease therapies.

The relationship between digital ultrasonographic image echotexture parameters of the uterus and preovulatory follicle development and ovulation in purebred Arabian mares

Objective assessment of ultrasonographic images is only possible using computer-assisted echotexture analysis. This study investigated the relationship between cyclic digital echotexture analysis of ultrasonographic uterine images, and preovulatory follicle development and ovulation in 48 cyclic purebred Arabian mares during the breeding season. When the mares’ ovarian follicle diameters exceeded 30 mm, ultrasonographic examinations were performed and ultrasonographic images recorded with uterine endometrial edema degrees following follicle development until ovulation day. Echotexture measurements, including mean grayness value (MGV), heterogeneity (HET) and contrast (CON) parameters, were performed on the days when ovarian follicle diameters reached 30 mm (Group 1, n = 12), 35 mm (Group 2, n = 12), 40 mm (Group 3, n = 12) and 45 mm (Group 4, n = 12) in images recorded the day before ovulation and on the day of ovulation. MGV was the lowest in Group 2 and the difference from the other groups was statistically significant (P&lt;0.001). CON was significantly higher in Group 2 than Groups 3 and 4 (P&lt;0.01). However, there was no significant difference compared to Group 1 (P&gt;0.05). Variations in HOM (P&lt;0.05), contrast (P&lt;0.05) and MGV (P&lt;0.01) were statistically significant on the day before ovulation and ovulation day. Thus, uterine ultrasonographic echotexture parameters are associated with preovulatory follicle development in mares, which suggests that these parameters can indicate the day of ovulation in purebred Arabian mares.

Effect of PGF2α treatments during early corpus luteum development on circulating progesterone concentrations and ovulation in breeding-age Holstein heifers

The objective of this study was to test the effect of low circulating concentrations of progesterone (P4) on pre-ovulatory follicle development in heifers as part of an overarching objective to develop a model to understand this phenomenon in dairy cattle without the confounding factors of lactation. Holstein heifers between 12 and 13 mo of age were pre-synchronized to ensure all heifers were on d 6 of the estrous cycle at the start of the Ovsynch program. Only heifers with CL regression and ovulation to the following pre-treatment strategy were used in the study: 0.5 mg cloprostenol (PGF2α), 2 d later, 0.1 mg GnRH, 6 d later, GnRH (G1; 1st GnRH of Ovsynch). Heifers (n = 159) responding to pre-treatment were randomly assigned to 4 groups and completed the Ovsynch program: high P4 control (HPC), low P4 control (LPC; PGF2α 24 h after G1), PG2 (PGF2α 24 and 48 h after G1) and PG3 (PGF2α 24, 48, and 96 h after G1). Only heifers that had ovulation to G1 remained in the study. Blood samples were collected in all heifers on d 7 (n = 157) and in a subset of heifers on d 1, 2, 3, 4 (n = 82) after G1 to measure serum P4. Pre-ovulatory follicle size at G1 (13.0 ± 0.1 mm; P = 0.53) and mean serum P4 24 h after G1 (d 1; 3.62 ± 0.11 ng/mL; P = 0.46) did not differ among treatments. HPC heifers had greater (P < 0.001) mean serum P4 compared to LPC, PG2 and PG3 on d 2, 3, 4, and 7. On d 2, 3 and 4, mean serum P4 of LPC, PG2 and PG3 heifers did not differ (P > 0.10). On d 7, LPC heifers had greater (P < 0.001) serum P4 compared to PG2 and PG3 heifers. Mean ± SEM serum P4 on d 7 after G1 was 8.43 ± 0.39, 2.55 ± 0.36, 1.58 ± 0.20, and 1.21 ± 0.15 ng/mL for HPC, LPC, PG2, and PG3, respectively. Percentage of heifers with P4 < 0.50 ng/mL on d 7 was greater (P < 0.05) for LPC, PG2 and PG3 (27, 32 and 26%, respectively) compared to HPC (0%). A greater (P < 0.05) proportion of heifers ovulated before G2 in the LPC, PG2 and PG3 than in the HPC. For heifers that ovulated after G2, low serum concentrations of P4 in LPC, PG2 and PG3 induced double ovulations in 6/97 heifers after the final GnRH of Ovsynch compared to 0/33 in HPC. In summary, PGF2α treatments during early CL development reduced circulating P4 concentrations 7 d after G1 compared with both HPC and LPC. However, it did not effectively control CL and follicle function to be utilized as a model to test high vs. low serum P4 on fertility parameters in Holstein heifers.

Insulin signaling is an essential regulator of endometrial proliferation and implantation in mice.

Insulin signaling is critical for the development of preovulatory follicles and progression through the antral stage. Using a conditional knockout model that escapes this blockage, we recently described the role of insulin signaling in granulosa cells during the periovulatory window in mice lacking Insr and Igf1r driven by Pgr-Cre. These mice were infertile, exhibiting defects in ovulation, luteinization, steroidogenesis, and early embryo development. Herein, we demonstrate that while these mice exhibit normal uterine receptivity, uterine cell proliferation and decidualization are compromised resulting in complete absence of embryo implantation in uteri lacking both receptors. While the histological organization of double knockout mice appeared normal, the thickness of their endometrium was significantly reduced. This was supported by the reduced proliferation of both epithelial and stromal cells during the preimplantation stages of pregnancy. Expression and localization of the main drivers of uterine proliferation, ESR1 and PGR, was normal in knockouts, suggesting that insulin signaling acts downstream of these two receptors. While AKT/PI3K signaling was unaffected by insulin receptor ablation, activation of p44/42 MAPK was significantly reduced in both single and double knockout uteri at 3.5 dpc. Overall, we conclude that both INSR and IGF1R are necessary for optimal endometrial proliferation and implantation.

CITED4 mediates proliferation, apoptosis and steroidogenesis of Hu sheep granulosa cells in vitro

Being a novel target of luteinizing hormone (LH), the effect of CREB-binding protein/P300-interacting trans-activator with ED-rich tail member 4 (CITED4) gene on the proliferation, apoptosis, and steroidogenesis of ovarian granulosa cells (GCs) in Hu sheep was investigated. The presence of CITED4, CREB-binding protein (CBP), CCAAT/enhancer-binding protein alpha (C/EBPα) and -beta (C/EBPβ) proteins was demonstrated in GCs and luteal cells. CITED4 protein in GCs was induced by LH, and CITED4 overexpression moderately increased GC responses to LH. In contrast, CITED4 knockdown in GCs decreased prostaglandin (PGE2)-induced LH target gene levels. Moreover, PGE2-stimulated CITED4 mRNA expression was blocked by ERK1/2 inhibition (U0126), suggesting that CITED4 is a downstream target of the ERK1/2 pathway in sheep GCs. In contrast to CITED4 knockdown, CITED4 overexpression promoted GC proliferation, inhibited apoptosis, upregulated cell cycle-related genes, and downregulated apoptosis-related genes. Additionally, CITED4 overexpression induced cell cycle transition from S to G2/M phase. No effect was observed with CITED4 knockdown. CITED4 overexpression increased progesterone (P4) production levels and STAR mRNA expression, whereas CITED4 knockdown decreased P4 production and STAR and 3β-HSD mRNA expression levels. Thus, our results suggest that CITED4 is involved in regulating the expression of LH-induced genes and the ERK1/2 pathway and the proliferation, apoptosis, and steroidogenesis in Hu sheep GCs by modulating the expression of related genes. These findings will help understand the role of CITED4 in follicular development and ovulation of pre-ovulatory follicles.

Active immunization against AMH reveals its inhibitory role in the development of pre-ovulatory follicles in Zhedong White geese

The aim of this study was to investigate the effects of active immunization against recombinant Anti-Müllerian hormone (AMH) protein on the ovarian follicular development, egg production, and molecular regulatory mechanisms in broody-prone Zhedong White geese. For this, a recombinant goose AMH protein was expressed using a prokaryotic expression system. Fifty incubating geese from the same genetic background were selected and equally divided into two groups. The immunization group was actively immunized against the recombinant goose AMH protein, whereas the control group was immunized against bovine serum albumin (BSA). Immunization against AMH accelerated ovarian follicular development and increased clutch sizes by one to two eggs in two consecutive laying-incubation cycles. Furthermore, immunization against AMH upregulated the mRNA transcription levels of the FSH-beta gene in the pituitary gland, and FSHR, 3beta-HSD, and Smad4 genes in the granulosa layer of pre-ovulatory follicles; however, immunization downregulated the expression of the OCLN gene in the granulosa layer of pre-ovulatory follicles, and Smad5 and Smad9 genes in the granulosa layer of SYFs. These results suggest that AMH might hinder ovarian follicular development by decreasing both pituitary FSH secretion as well as ovarian follicular sensitivity to FSH. The latter molecular mechanism could be fulfilled by regulating Smad5 or Smad9 signals in SYFs, as well as the FSHR and Smad4 signals that affect progesterone synthesis and yolk deposition in the pre-ovulatory follicles.

Relationship of the conception rate and the side (left or right) of preovulatory follicle location at artificial insemination in dairy heifers.

In this study, we examined the locational effect (left or right ovary) of the preovulatory follicle (PF) on fertility in dairy heifers. In total, 1,111 artificial inseminations (AI) were analyzed. At AI, PF locations were examined using rectal palpation, and heifers were divided into two groups on their PF locations: (i) the PF located in the left ovary (L-PF); and (ii)the PF located in the right ovary (R-PF). Pregnancy was diagnosed by rectal palpation 60days after AI. The conception rate was 50.7% in all heifers. Conception rate was significantly higher in the L-PF (60.1%) than in the R-PF (46.2%). The conception rate was significantly lower by sexed semen (48.6%) than conventional semen (59.1%). Conception rates divided by the semen type (sexed: n=896, conventional: n=215) were significantly higher in the L-PF than in the R-PF for both semen types (sexed; L-PF vs. R-PF: 57.3% vs. 44.4%, conventional; L-PF vs. R-PF: 72.3% vs. 53.3%). In addition, season, age, AI number, and the number of re-inseminations at the same estrus did not affect conception rates. In summary, PF development in the left ovary was associated with increased conception rates in dairy heifers.

The polycystic ovary syndrome-associated gene Yap1 is regulated by gonadotropins and sex steroid hormones in hyperandrogenism-induced oligo-ovulation in mouse.

What is the physiological function of Yes-associated protein-1 (Yap1), a susceptibility gene for polycystic ovary syndrome (PCOS), in ovarian granulosa cells (GCs)? Physiologically, steroid sex hormones stimulate follicle growth by activating YAP1; however, the preovulatory inhibition of YAP1 activity in GCs is a prerequisite of LH actions. PCOS is a common gynecologic and endocrine disease with multiple short and long-term consequences. Many PCOS patients suffer anovulation caused by hyperandrogenism, but its etiology remains unclear. To study the effect of acute hyperandrogenism on ovulation, we injected pregnant mare serum gonadotrophin (PMSG)-primed (44 h) pubertal mice with dihydrotestosterone (DHT), the major biologically active form of androgen, in a superovulation assay. We investigated if YAP1 is regulated by testosterone and if it is potentially involved in follicle development and ovulation. Cultured primary GCs were subjected to Yap1 depletion by RNA interference and Yap1 overexpression by adenoviral infections. Female mice at postnatal day (PD)-21~23 were analyzed to avoid the complexity of ovarian functions associated with estrous cycles and endogenous surges of gonadotropins. Immature mice were injected intraperitoneally with five IU PMSG to stimulate preovulatory follicle development followed 44 h later with five IU hCG to stimulate ovulation. For DHT treatments, female mice at PD23 were injected intraperitoneally with five IU PMSG followed 44 h later with five IU hCG alone (as control) or five IU hCG plus 100 μg DHT, which was dissolved in 0.1 ml DMSO. Methods of gene expression detection used include immunohistochemistry, immunofluorescence, Western blotting and quantitative PCR. More than three biological and technical replicates were included in each experiments. we provide novel evidence in a mouse model that YAP1 is required for proliferation of ovarian GCs, but is down-regulated by LH through the extracellular-regulated kinase-1/2 (ERK1/2) cascade. Acute hyperandrogenism blocks LH actions and causes oligo-ovulation by activating YAP1. N/A. Results shown were obtained only in mouse, and need to be further confirmed in human samples. These findings not only elucidated the role of YAP1 in maintaining normal ovarian functions, but also link the YAP1 deregulation to the pathogenesis of PCOS. This study is funded by the National Key Research and Development Program of China (2016YFC1000600 and 2017YFSF1001500) and National Natural Science Foundation of China (31528016, 31371449 and 31671558). The authors have no competing interests.

Comparison of 2 protocols to increase circulating progesterone concentration before timed artificial insemination in lactating dairy cows with or without elevated body temperature

Two treatments designed to increase circulating progesterone concentration (P4) during preovulatory follicle development were compared. One treatment used 2 intravaginal P4 implants (controlled internal drug-releasing inserts; CIDR) and the other used a GnRH treatment at beginning of the protocol. Lactating Holstein cows that had been diagnosed as nonpregnant were randomly assigned to receive timed artificial insemination (TAI) following 1 of 2 treatments (n = 1,638 breedings): (1) GnRH: CIDR+ 2 mg of estradiol (E2) benzoate + 100 µg of GnRH on d -11, PGF2α on d -4, CIDR withdrawal + 1.0 mg of E2-cypionate + PGF2α) on d -2, and TAI on d 0; or (2) 2CIDR: 2 CIDR + 2 mg of E2-benzoate on d -11, 1 CIDR withdrawn + PGF2α on d -4, second CIDR withdrawn + 1.0 mg of E2-cypionate + PGF2α on d -2, and TAI on d 0. Milk yield was measured daily between d 0 and d 7. Rectal temperature was measured using a digital thermometer at d 0 and 7, and elevated body temperature was defined as an average rectal temperature ≥39.1°C. Pregnancy diagnoses were performed on d 32 and 60 after TAI. We detected no effect of treatments on pregnancy per AI or pregnancy loss regardless of elevated body temperature, body condition score, parity, milk yield, or presence or absence of a corpus luteum (CL) on d -11 or d -4. Pregnancy per AI at 60 d was reduced [elevated body temperature = 22.8% (162/709), no elevated body temperature 34.1% (279/817)] and pregnancy loss tended to increase [elevated body temperature = 20.2% (41/203), no elevated body temperature 14.4% (47/326)] in cows with elevated body temperature. Various physiological measurements associated with greater fertility were also reduced in cows with elevated body temperature, such as percentage of cows with a CL at PGF2α (decreased 7.9%), ovulatory follicle diameter (decreased 0.51 mm), expression of estrus (decreased 5.1%), and ovulation near TAI (decreased 2.8%) compared with cows without elevated body temperature. A greater proportion of cows (30.2%) had a CL at PGF2α in the GnRH treatment [74.1% (570/763)] than in the 2CIDR treatment [56.9% (434/763)]; however, circulating P4 concentration was greater at the time of PGF2α treatment (d -4) for cows 2CIDR (4.26 ± 0.13 ng/mL) than in cows in GnRH (3.99 ± 0.14 ng/mL). Thus, these 2 protocols yield similar fertility results that might be due to somewhat different physiological alterations. Treatment with GnRH increased the proportion of cows with a CL at PGF2α; however, the 2CIDR protocol increased circulating P4 under all circumstances.

Clomiphene Citrate Treatment Cycle Outcomes of Polycystic Ovary Syndrome Patients Based on Basal High Sensitive C-Reactive Protein Levels: A Cross-Sectional Study.

Polycystic ovary syndrome (PCOS) is highly associated with an ovulatory infertility, features of the metabolic syndrome, including obesity, insulin resistance and dyslipidemia. Serum concentrations of high sensitive C-reactive protein (hs-CRP) were significantly higher in obese than in non-obese PCOS patients at baseline, suggesting a relationship between elevated hs-CRP levels and obesity. The aim of this study was to evaluate whether cycle day 3 hs-CRP levels before clomiphene citrate (CC) treatment would predict cycle outcomes in women with PCOS. This cross-sectional study was conducted among 84 infertile women with PCOS who were treated with CC at Zekai Tahir Burak Women's Health Education and Research Hospital, Ankara, Turkey, between January 2014 and January 2015. Based on the exclusion criteria, cycle outcomes of remaining 66 infertile women with PCOS treated with CC were analyzed. The hs-CRP levels and insulin resistance indexes were evaluated on day 3 of the CC treatment cycle. The primary outcome measures were number of preovulatory follicles measuring≥17 mm and pregnancy rates. The mean ± SD age of the patients was 24.0 ± 3.8 years (range 18-36). The mean ± SD body mass index (BMI) of the patients was 25.7 ± 4.9 (range 17-43). Fifty patients developed dominant follicle (75%) and 5 patients established clinical pregnancy during the study (clinical pregnancy rate: 7%). The mean ± SD baseline hs-CRP, fasting insulin and Homeostasis Model Assessment-Insulin Resistance (HOMA-IR) values of the patients with and without dominant follicle generation during treatment cycle were 6.42 ± 7.05 and 4.41 ± 2.95 (P=0.27), 11.61 ± 6.94 and 10.95 ± 5.65 (P=0.73), 2.68 ± 1.79 and 2.41 ± 1.30 (P=0.58), respectively. The mean ± SD baseline hs-CRP, fasting insulin and HOMA-IR values of the patients with and without clinical pregnancy establishment following treatment cycle were 6.30 ± 2.56 and 5.90 ± 6.57 (P=0.89), 11.60 ± 7.54 and 11.44 ± 6.61 (P=0.95), 2.42 ± 1.51 and 2.63 ± 1.70 (P=0.79), respectively. In this study, we did not observe a predictive value of cycle day 3 hs-CRP levels on preovulatory follicle development and pregnancy rates among infertile PCOS patients treated with CC. Also, no relationship between HOMA-IR values and dominant follicle generation or clinical pregnancy establishment was demonstrated in our study, confirming the previous studies emphasizing the neutral effect of metformin utilization before and/or during ovulation induction to pregnancy rates.

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.

Does Clinical Treatment with Phenylbutazone and Meloxicam in the Pre-ovulatory Period Influence the Ovulation Rate in Mares?

The presence of anovulatory haemorrhagic follicles during the oestrous cycle of mares causes financial impacts, slowing conception and increasing the number of services per pregnancy. Non-steroidal anti-inflammatory drugs (NSAIDs) such as meloxicam and phenylbutazone are used in the treatment of several disorders in mares, and these drugs can impair the formation of prostaglandins (PGs) and consequently interfere with reproductive activity. This study aimed to evaluate the effects of treatment with NSAIDs on the development of pre-ovulatory follicles in mares. In total, 11 mares were studied over three consecutive oestrous cycles, and gynaecological and ultrasound examinations were performed every 12 h. When 32-mm-diameter follicles were detected, 1 mg of deslorelin was administered to induce ovulation. The first cycle was used as a control, and the mares received only a dose of deslorelin. In the subsequent cycles, in addition to receiving the same dose of deslorelin, each mare was treated with NSAIDs. In the second cycle, 4.4 mg/kg of phenylbutazone was administered, and in the third cycle, 0.6 mg/kg of meloxicam was administered once a day until ovulation or the beginning of follicular haemorrhage. All of the mares ovulated between 36 and 48 h after the induction in the control cycle. In the meloxicam cycle, 10 mares (92%) did not ovulate, while in the phenylbutazone cycle, nine mares (83%) did not ovulate. In both treatments, intrafollicular hyperechoic spots indicative of haemorrhagic follicles were observed on ultrasound. Thus, our results suggested that treatment with meloxicam and phenylbutazone at therapeutic doses induced intrafollicular haemorrhage and luteinization of anovulatory follicles.

ACTH administration during formation of preovulatory follicles impairs steroidogenesis and angiogenesis in association with ovulation failure in lactating cows

Ovulation failure, follicular persistence, and formation of follicular cysts are known to impair dairy cow fertility. Although the underlying mechanism is not entirely clear, stress-induced alteration in adrenal hormone secretion can cause these ovarian pathologies. Six synchronized lactating cows were scanned daily by ultrasound, and plasma samples were taken throughout the estrous cycle. Treatment cows (n = 3) were administered with ACTH analog every 12 h from day 15 to day 21 of the cycle to induce formation of follicular cysts. Ovaries were collected at the slaughterhouse on day 23 of the cycle before appearance of follicular pathologies. Control cows (n = 3) were administered placebo, resynchronized, and administered PGF2α on day 6 of the new cycle to induce development of a preovulatory follicle. Follicular fluid was aspirated from the preovulatory follicles of each group to determine their steroid milieu. Slices were taken from the follicular wall for total messenger (m) RNA isolation and semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Administration of ACTH increased (P < 0.02) plasma cortisol concentration and reduced (P < 0.01) milk production. Androstenedione and estradiol concentrations in the follicular fluids were lower (P < 0.05) in ACTH-treated follicles than those in controls. The mRNA expression of luteinizing hormone receptor, 3β-hydroxysteroid dehydrogenase, cytochrome P450 aromatase (P450arom), and cytochrome P450 17α-hydroxylase (P450c17) were lower (P < 0.02) in the ACTH-treated vs control cows. On the other hand, the expression of steroidogenic acute regulatory protein and cytochrome P450 side-chain cleavage did not differ between groups. In addition, mRNA expression of vascular endothelial growth factor (VEGF)120 and VEGF164 was higher (P < 0.01) in control than in ACTH-treated follicles, but that for angiopoietin-1 and 2 did not differ between groups. Findings indicated that ACTH administration throughout preovulatory follicle development alters follicular steroidogenesis in association with impaired angiogenesis. Such alterations might explain, in part, the mechanism underlying ovulation failure and the formation of persistent or cystic follicles under stress.

Effect of adding a gonadotropin-releasing-hormone treatment at the beginning and a second prostaglandin F2α treatment at the end of an estradiol-based protocol for timed artificial insemination in lactating dairy cows during cool or hot seasons of the year

Our hypothesis was that fertility could be increased in a timed artificial insemination (TAI) protocol based on estradiol (E2) and progesterone (P4) by combining GnRH with E2-benzoate at the start of the protocol to increase circulating P4 during preovulatory follicle development and by using 2 prostaglandin F2α (PGF) treatments at the end to decrease P4 near TAI. Lactating Holstein cows (n=1,808) were randomly assigned during the cool or hot season of the year to receive TAI (d 0) following 1 of 3 treatments: (1) control: controlled internal drug-release insert + 2mg of E2-benzoate on d −11, PGF on d −4, controlled internal drug-release insert withdrawal + 1.0mg of E2-cypionate on d −2, and TAI on d 0; (2) 2PGF: identical to control protocol with addition of a second PGF treatment on d −2; (3) GnRH: identical to 2PGF protocol with addition of a 100-μg GnRH treatment on d −11. Pregnancy diagnoses were performed on d 32 and 60 after TAI. Season had major effects on many reproductive measures, with cool season greater than hot season in percentage of cows with corpus luteum (CL) at PGF (62.9 vs. 56.2%), ovulatory follicle diameter (15.7 vs. 14.8mm), expression of estrus (86.7 vs. 79.9%), ovulation following the protocol (89.7 vs. 84.3%), and pregnancies per artificial insemination (P/AI; 45.4 vs. 21.4%). The GnRH protocol increased percentage of cows with CL (control=56.9%; 2PGF=55.8%; GnRH=70.5%) and P4 at PGF (control=3.28±0.22; 2PGF=3.35±0.22; GnRH=3.70±0.21ng/mL), compared with control and 2PGF protocols. The GnRH protocol increased P/AI at the pregnancy diagnosis at 32d [37.3% (219/595)] and 60d [31% (179/595)] after TAI, compared with control [30.0% (177/604); 25.1% (145/604)], with intermediate results with 2PGF protocol [33.2% (196/609); 28.0% (164/609)]. The positive effects of GnRH treatment on P/AI were only detected during the cool season (GnRH=50.9%; 2PGF=44.2%; control=41.0%) and not during the hot season. In addition, the effect of GnRH was only observed in cows with low P4 (<3ng/mL) at the start of the protocol and not in cows that began the protocol with high P4. Furthermore, presence of CL at PGF interacted with follicle diameter such that cows with a CL at PGF had greater P/AI if they ovulated larger rather than smaller follicles near TAI. Thus, fertility to TAI can be improved by inducing ovulation at the beginning of an E2/P4-based protocol using GnRH treatment, particularly during the cool season of the year and in cows with low P4 at the start of the protocol.

Fertility of a high-altitude sheep model is compromised by deficiencies in both preovulatory follicle development and plasma LH availability.

At high altitude, hypoxia and/or oxidative stress may compromise fertility. This study tested the relative effect of short- or long-term exposure to high-altitude hypobaric hypoxia and oxidative stress in sheep on preovulatory follicle dynamics and gonadotrophin secretion. Thus, growth dynamics, stereidogenic function and competence to ovulate of preovulatory follicles, as well as FSH and LH availability throughout the entire oestrous cycle, were compared among sheep native from low and high altitude, and sheep newcomers to high altitude. The results indicates that short-term exposure in sheep newcomers to high altitude has a deleterious effect on both the ovarian function (affecting preovulatory follicular development) and the pituitary function (diminishing plasma LH availability). On the other hand, there were no detected differences in the preovulatory follicular development in sheep adapted to high altitude for generations and, conversely, LH secretion was increased, which suggests an adaptive mechanism. The treatment with antioxidant agents during a relative short period for the time of folliculogenesis (approximately 1month and a half) changed substantially the development of preovulatory follicles in short-term exposed sheep to similar patterns than in sheep native and living to both high and low altitude. These results highlight the role of oxidative stress in the detriment of the reproductive function in individuals recently exposed to high-altitude hypoxic environment.

Progesterone improves the maturation of male-induced preovulatory follicles in anoestrous ewes.

The first ovulation induced by male effect in sheep during seasonal anoestrus usually results in the development of a short cycle that can be avoided by progesterone priming before ram introduction. In elucidating the involvement of the hypothalamic-pituitary-gonadal axis in the occurrence of short cycles, the effects of progesterone and the time of anoestrus on the development of male-induced preovulatory follicles were investigated in anoestrous ewes using morphological, endocrine and molecular approaches. Ewes were primed with progesterone for 2 (CIDR2) or 12 days (CIDR12) and untreated ewes used as controls during early (April) and late (June) anoestrus. The duration of follicular growth and the lifespan of the male-induced preovulatory follicles were prolonged by ∼1.6 days in CIDR12 ewes compared with the controls. These changes were accompanied by a delay in the preovulatory LH and FSH surges and ovulation. Intra-follicular oestradiol concentration and mRNA levels of LHCGR and STAR in the granulosa and theca cells of the preovulatory follicles were higher in CIDR12 ewes than the control ewes. The expression of mRNA levels of CYP11A1 and CYP17A1 also increased in theca cells of CIDR12 ewes. CIDR2 ewes gave intermediate results. Moreover, ewes ovulated earlier in June than in April, without changes in the duration of follicular growth, but these effects were unrelated to the lifespan of corpus luteum. Our results give the first evidence supporting the positive effect of progesterone priming on the completion of growth and maturation of preovulatory follicles induced by male effect in seasonal anoestrous ewes, thereby preventing short cycles.

Effect of single-chain ovine gonadotropins with dual activity on ovarian function in sheep.

We examined the half-life and biological activity of two single-chain proteins that combined portions of ovine FSH and LH. We proposed the hypothesis that these chimeric proteins would display LH and FSH activities and would promote follicle maturation in ewes. Estrus activity was synchronized using progestogen-impregnated vaginal pessaries. To negate the impact of endogenous LH and FSH, animals received serum-containing antibodies against GNRH 1 day before pessary removal (PR). At PR sheep (five animals per group) received a single injection (10 IU/kg, i.v.) of either the ovine-based (oFcLcα) gonadotropin analog, an ovine-based analog containing oLHβ truncated at the carboxyl terminus (oFcL(ΔT)cα), or a human-based gonadotropin analog (hFcLcα). Control animals received a comparable amount of gonadotropin-free protein. Ovulation was induced 3 days after PR using human chorionic gonadotropin (1000 IU, i.v.). Ovaries were collected 11 days after PR. Neither estradiol (E2) or progesterone (P4) production, development of preovulatory follicles or corpora lutea (CL) were noted in control animals receiving gonadotropin-free protein. Significant increase in the synthesis of E2 and P4 was noted in sheep receiving the dually active gonadotropin analogs. The number of CLs present 11 days after PR was significantly increased in sheep receiving the chimeric glycoproteins compared with control animals. The magnitude of the secretory and ovarian responses did not differ between hFcLcα and oFcLcα or between oFcLcα and oFcL(ΔT)cα. Immunoactivity of LH and FSH was low in control animals, but was significantly elevated in sheep receiving the gonadotropin analogs. In conclusion, ovine-based gonadotropin analogs are functionally active in sheep and a single injection is adequate to induce the development of multiple ovulatory follicles.