A century of research on the uteroovarian pathway for uterine-induced luteolysis in mammals.

Year 2023 is the 100-year anniversary of the discovery in guinea pigs that the lifespan of the corpus luteum (CL) is controlled by the uterus. The CL is the gatekeeper between two fundamental reproductive events - the estrous cycle and pregnancy. Uteroluteal research for the initial 33 years was productive but limited to laboratory species until the inclusion of farm animals in 1956. In the early 1960s, it was found that uterine luteolysin in sows travels unilaterally from a uterine horn to the adjacent CL which likely accounted for the heyday of uteroluteal research in the 1960s-70s. The luteolytic properties of PGF2α were demonstrated in rats in 1969. In 1971, (1) surgical separation of the lengthwise adherence between the uteroovarian vein and ovarian artery interfered with luteolysis in ewes, (2) species with primarily unilateral vs systemic uterine-induced luteolysis have a strong vs an absent or weak unilateral venoarterial transfer pathway, and (3) vascular infusions identified PGF2α as a uterine luteolysin. Vascular and PGF2α studies were beginning to merge. In 1973, a venoarterial pathway was firmly demonstrated in ewes and later in heifers by surgical anastomosis of a uterine vein or ovarian artery from a uterine-intact side to the corresponding vessel on the unilaterally hysterectomized side. More recent studies described how prostaglandins likely transfer through the walls of uterine and ovarian vessels using concentration gradients in sows and a prostaglandins transporter system in cows.

Uteroovarian pathway for embryo-empowered maintenance of the corpus luteum in farm animals.

The first luteal response to pregnancy in farm animals at 12-18 days after ovulation involves maintenance of the corpus luteum (CL) if pregnancy has occurred. In most common farm species, regression of the CL results from production of a luteolysin (PGF2α) by the nongravid uterus, and maintenance of the CL involves the production of an antiluteolysin (PGE2) by the gravid uterus and conceptus. The proximal component of a unilateral pathway from a uterine horn to the adjacent CL for transport of PGF2α and PGE2 is the uterine venous and lymphatic vessels and the distal component is the ovarian artery. The mechanisms for venolymphatic arterial transport of PGF2α and PGE2 from a uterine horn to the adjacent CL ovary and transfer of each prostaglandin through the walls of the uteroovarian vein and ovarian artery occur by similar mechanisms probably as a consequence of similarities in molecular structure between the two prostaglandins. Reported conclusions or interpretations during the first luteal response to pregnancy in sows and ewes are that PGE2 increases in concentration in the uteroovarian vein and ovarian artery and counteracts the negative effect of PGF2α on the CL. In cows, treatment with PGE2 increases circulating progesterone concentrations and prevents spontaneous luteolysis and luteolysis induced by estradiol, an intrauterine device, or PGF2α. The prevailing acceptance that interferon tau is the primary factor for maintaining the CL during early pregnancy in ruminants will likely become tempered by the increasing reports on PGE2.

Luteal tissue blood flow and side effects of horse-recommended luteolytic doses of dinoprost and cloprostenol in donkeys.

This study assessed luteolysis and side effects in jennies receiving standard horse-recommended doses of cloprostenol and dinoprost. Sixteen cycles of eight jennies were randomly assigned in a sequential crossover design to receive dinoprost (5 mg, i.m.) and cloprostenol (0.25 mg, i.m.) at 5-d post-ovulation. B-mode and Doppler ultrasonography were employed to assess luteal tissue size and blood flow before (-15 min and 0h) and after (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, and 48h) administering PGF2α. Immunoreactive progesterone concentrations were assayed at similar timepoints via RIA. Side effects such as sweating, abdominal discomfort, and diarrhea were scored at 15-min-intervals for 1h after PGF2α. Data normality was assessed with the Shapiro-Wilk's test. Luteal tissue size and blood flow were analyzed using PROC-MIXED and post-hoc by Tukey. Non-parametric tests analyzed side effect variables. The luteal blood flow increased overtime by 27% at 45 min and peaked by 49% at 3 h for dinoprost, and conversely, it increased by 14% at 30 min and peaked at 39% at 5h for cloprostenol (P<0.05). Luteal blood flow was reduced by 50%, 25%, and 10% on both groups at 8, 12, and 24h (P<0.05). Immunoreactive progesterone concentrations decreased in 0.5h for dinoprost and 1h for cloprostenol and gradually decreased by 48h (P<0.05). Dinoprost induced greater sudoresis scores, while cloprostenol resulted in greater abdominal discomfort and diarrhea scores (P<0.05). In conclusion, dinoprost and cloprostenol effectively induced luteolysis with distinct side effects; this could guide practitioners' case selection to use one or another PGF2α.

Effects of intravaginal administration of prostaglandin F2α on luteolysis and subsequent estrus in Shiba goats (Capra hircus).

This study examined the effects of intravaginal administration of prostaglandin F2α (PGF2α ) on luteolysis and subsequent estrus in cycling goats. Goats with functional corpus lutea received one of five treatments: 2 mg of PG intramuscularly (IM2 × 1; n = 6), 2 mg of PGF2α intravaginally (IVG2 × 1; n = 7), 4 mg of PGF2α intravaginally (IVG4 × 1; n = 7), and 1 or 2 mg of PGF2α intravaginally 8 h apart (IVG1 × 2 group; n = 6 and IVG2 × 2; n = 8). Blood samples were collected at 24-h intervals from 0 to 7 days after PGF2α administration. Estrus was checked twice daily during the experiment. The proportion of goats with complete luteolysis (reduction of progesterone concentrations to <1 ng/mL until 48 h after treatment) in the IVG2 × 1 group (28.6%) was significantly lower than in the other groups (IM2 × 1; 100%, IVG4 × 1; 57.1%, IVG1 × 2; 87.5%, IVG2 × 2; 100%, respectively). For goats completing luteolysis, there was no significant difference in the onset and duration of estrus among the groups. These results suggest that intravaginal administration of PGF2α can be applied as an alternative to intramuscular administration.

Reduced period from follicular wave emergence to luteolysis generated greater steroidogenic follicles and estrus intensity in dairy cows.

The onset of productive life in dairy cattle, concomitant to parturition, is accompanied by a substantial decrease in fertility in comparison with non-lactating, nulliparous heifers. Follicular growth patterns differ between parous and nulliparous dairy cattle. Nulliparous heifers ovulate follicles with reduced antral age (RAA). This study aimed to exogenously reduce ovulatory follicle age in lactating dairy cows from 7 to 5 days old. Cows (n = 80) had their estrous cycles synchronized with the Double-Ovsynch program. At the final portion of this program, luteolysis was induced at either 5 (RAA) or 7 (Control) days following follicular wave emergence. RAA outcomes were estimated in comparison with Controls. RAA resulted in smaller follicles 2 days post-treatment. Despite lower serum concentrations of 17ß-estradiol before treatment compared with Controls, the rate of increase in this hormone was greater for the RAA treatment. There was no difference in luteolysis rates between treatments. Proestrus (luteolysis onset to estrus onset) was prolonged in RAA cows. Cows with RAA had more intense estruses. Collectively, these results indicate that decreasing the age of the ovulatory follicle may improve the steroidogenic capacity of the dominant follicle and estrus expression intensity in lactating dairy cows.

Ovarian and uterine arteries blood flow velocities waveform, hormones and nitric oxide in relation to ovulation in cows superstimulated with equine chorionic gonadotropin and luteolysis induction 10 and 17 days after ovulation.

To investigate the ovarian responses, ovarian and uterine hemodynamics, circulating ovarian hormones, and nitric oxide (NO) with their relations in superstimulated cows. Eight Holstein Friesian dry cows previously synchronized with CIDR underwent rectal Doppler ultrasound scanning and blood sampling after administrating eCG (1500 I.U) on day 10 of the second ovulation (day -5). Cows were treated with 12.5 mg prostaglandin F2α (PGF2α) on days 10 and 17 after ovulation. Estradiol, progesterone, and NO were measured. Results showed that from ≥ 13 follicles, five follicles ovulated from both ovaries. The ovulated follicles increased antrum colored area and colored area % till day -1. The developed corpora lutea (CLs) attained similar diameter, area, colored area, and colored area % from day 2 till day 15. The peak point of velocity (PSV) of uterine arteries decreased while that of ovarian arteries increased from day -4 to day 0. Both ovarian arteries diameter, resistance index (RI), PSV, end velocity (EDV) and systolic/diastolic ratio (S/D) positively correlated (P < 0.0001), but their pulsatility index (PI) negatively correlated (P < 0.0001). The uterine arteries PI, RI, PSV, EDV, time average velocity (TAMV) and S/D negatively correlated (P < 0.0001) but their diameters positively correlated. Estradiol increased but progesterone decreased from day -5 till day 0. After ovulation, P4 reached maximum values on day 9 and started to decrease till day 19.NO showed one peak on day -3 and another one from day 3 to day 9. Conclusions: Blood flow of ovarian arteries is different from uterine arteries and depended on pre- or post-ovulation.

Review: Luteal prostaglandins: mechanisms regulating luteal survival and demise in ruminants.

The corpus luteum (CL) is critical for establishment and maintenance of pregnancy in all mammals. However, the fate of the CL in ruminants is dependent on the presence of a functional uterus or signals from a developing embryo to modify uterine function to ensure its own survival. The key molecule secreted by the uterus that must be modified is prostaglandin F2alpha (PGF2A). At the same time, there is evidence that mechanisms within the CL may influence the ability of PGF2A to cause luteolysis. This review focuses on prostaglandins and steroidogenic capacity as endogenous modulators of the sensitivity of the CL to exogenous PGF2A. Early luteal development and early pregnancy are two different luteal stages in which sensitivity of the CL to PGF2A renders it incapable, or less capable, respectively, of undergoing luteolysis in response to PGF2A compared to a midcycle CL. An analysis of molecular changes that occur during these two stages provides some novel insight into molecules and pathways worth exploring to explain the regulation of luteolytic capacity in corpora lutea of ruminants.

Review: Maintenance of the ruminant corpus luteum during pregnancy: interferon-tau and beyond.

This manuscript reviews the mechanisms that maintain the corpus luteum (CL) of pregnancy in ruminants. In mammals, ovulation and luteinization of the remaining cells in the CL are due to a surge in Luteinizing Hormone (LH). In cattle, continued secretion of pulses of LH is essential for full development and function of the CL during the estrous cycle (LH pulses), however, the few studies on the CL after d20 of pregnancy do not indicate that LH is essential for maintaining the CL of pregnancy. The first essential step in maintaining the CL of pregnancy in ruminants is overcoming the mechanisms that cause regression of the CL in non-pregnant ruminants (d18-25 in cattle; d13-21 in sheep). These mechanisms have a uterine component involving oxytocin-induced prostaglandin F2α (PGF2A) pulses and a luteal component involving decreased progesterone production and luteal cell death. There is a critical role for embryonic interferon-tau (IFNT) in suppressing the uterine secretion of PGF2A during early pregnancy (d13-21 in sheep; d16-25 in cattle) and preventing luteolysis. There are also effects of IFNT on the expression of interferon-stimulated genes in other tissues including the CL but the physiologic role of these interferon-stimulated genes is not yet clear. After the IFNT period, there is another mechanism that maintains the CL of pregnancy in ruminants since embryonic IFNT is inhibited as attachment occurs and trophoblastic binucleate/giant cells begin secretion of pregnancy-associated glycoproteins. The second mechanism for luteal maintenance has not yet been defined but acts in a local manner (ipsilateral to pregnancy), and remains functional from d25 until just before parturition. The most likely mechanisms mediating later maintenance of the CL of pregnancy are increased uterine blood flow or decreased prostaglandin transporter expression in the utero-ovarian vasculature, preventing PGF2A reaching the CL. Finally, implications of these ideas on pregnancy loss in cattle are explored, highlighting the importance of inappropriate regression of the CL of pregnancy as a mechanism for pregnancy loss in cattle.

Plasma cell-free DNA concentration increases during luteolysis in beef cows.

During cell death, DNA is fragmented and reaches the bloodstream in the form of cell-free DNA (cfDNA). Luteal cells must undergo an apoptotic process during structural luteolysis to begin a new oestrous cycle. We hypothesized that cfDNA concentrations would increase when inducing luteolysis by applying prostaglandin F2α (PGF2α) analog to the cycling cow. Multiparous non-pregnant and non-lactating Angus cows (Bos taurus; n = 15) were synchronized using the 7-day CoSynch + CIDR protocol. Ten days after oestrus was detected, two treatments were applied (PGF2α, n = 10; or Con, n = 5). Twice a day, grey mode and colour Doppler ultrasonography were used to calculate area (CL-A) and luteal blood perfusion (LBP%). Additionally, we collected one blood sample for plasma progesterone (P4) and cfDNA concentrations for four consecutive days. Data analysis was performed using the GLM procedure of SAS. The luteolysis induction was demonstrated by a decrease in P4 concentrations (p ≤ .01) and CL-A (p ≤ .01) in the PGF2α group after 12 h of the PGF2α injection. Reduction of LBP% (p ≤ .01) in the PGF2α group after 36 h of the injection. The concentration of cfDNA showed a significant increase (p = .05) after 48 h of the PGF2α application in the PGF2α group. In conclusion, cfDNA showed a significantly increased concentration after luteolysis induction, which can imply that cfDNA could be used as a luteolysis biomarker in plasma.

Basic fibroblast growth factor induces proliferation and collagen production by fibroblasts derived from the bovine corpus luteum†.

Cyclic regression of the ovarian corpus luteum, the endocrine gland responsible for progesterone production, involves rapid matrix remodeling. Despite fibroblasts in other systems being known for producing and maintaining extracellular matrix, little is known about fibroblasts in the functional or regressing corpus luteum. Vast transcriptomic changes occur in the regressing corpus luteum, among which are reduced levels of vascular endothelial growth factor A (VEGFA) and increased expression of fibroblast growth factor 2 (FGF2) after 4 and 12 h of induced regression, when progesterone is declining and the microvasculature is destabilizing. We hypothesized that FGF2 activates luteal fibroblasts. Analysis of transcriptomic changes during induced luteal regression revealed elevations in markers of fibroblast activation and fibrosis, including fibroblast activation protein (FAP), serpin family E member 1 (SERPINE1), and secreted phosphoprotein 1 (SPP1). To test our hypothesis, we treated bovine luteal fibroblasts with FGF2 to measure downstream signaling, type 1 collagen production, and proliferation. We observed rapid and robust phosphorylation of various signaling pathways involved in proliferation, such as ERK, AKT, and STAT1. From our longer-term treatments, we determined that FGF2 has a concentration-dependent collagen-inducing effect, and that FGF2 acts as a mitogen for luteal fibroblasts. FGF2-induced proliferation was greatly blunted by inhibition of AKT or STAT1 signaling. Our results suggest that luteal fibroblasts are responsive to factors that are released by the regressing bovine corpus luteum, an insight into the contribution of fibroblasts to the microenvironment in the regressing corpus luteum.

Resistin as a new player in the regulation of porcine corpus luteum luteolysis: in vitro effect on proliferation/viability, apoptosis and autophagy.

The formation and luteolysis of the corpus luteum (CL) is strictly controlled by many factors. Imbalance between proliferation and apoptosis processes leads to deficiency of the luteal phase and infertility. Our previous study showed resistin expression in porcine luteal cells and an inhibitory effect on progesterone synthesis. Thus, the aim of the present study was to examine the in vitro effect of resistin on the proliferation/viability, apoptosis and autophagy of porcine luteal cells as well as the involvement of mitogen-activated kinase (MAP3/1), protein kinase B (AKT) and signal transducer and activator of transcription 3 (STAT3) in these processes. Porcine luteal cells were incubated with resistin (0.1-10 ng/mL) for 24-72 h and viability was assessed using the alamarBlue or 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Then, the time-dependent effect of resistin on mRNA and protein expression of proliferating cell nuclear antigen (PCNA), caspase 3, BCL2-like protein 4 (BAX), B-cell lymphoma 2 (BCL2), beclin1, microtubule-associated protein 1A/1B-light chain 3 (LC3) and lysosomal-associated membrane protein 1 (LAMP1) was measured by real-time polymerase chain reaction (PCR) and immunoblotting, respectively. We found that resistin enhanced luteal cell viability with no effect on caspase 3 mRNA and protein, increased the BAX/BCL2 mRNA and protein ratio and significantly stimulated the initiation of autophagy, which promotes the maintenance of CL function rather than its regression. Additionally, using pharmacological inhibitors of MAP3/1 (PD98059), AKT (LY294002) and STAT3 (AG490), we observed that the effect of resistin was reversed to the control level in viability and, by influence, MAP3/1 and STAT3 in autophagy. Taken together, our results suggest that resistin, in addition to its well-known effect on granulosa cell function has direct influence on CL luteolysis and the formation and maintenance of luteal cell function.

Effect of cloprostenol on luteolysis and comparison of different estrus synchronization protocols in jennies.

Estrus synchronization is necessary for intensive donkey farming. Studies on estrus synchronization in jennies are, however, scarce. We aimed to investigate the susceptibility of the donkey corpus luteum to cloprostenol and design a successful estrus synchronization protocol. Firstly, the effects of different cloprostenol doses and the timing effect of cloprostenol treatment on estrous cycle was investigated. The time from treatment to luteolysis, the ovulation interval, pre-ovulatory diameter, and ovulation rates were compared between groups. Secondly, to identify the best protocol, eight estrus synchronization protocols from three categories were examined. In the first category, jennies in groups A (n = 55) and B (n = 30) received a progesterone releasing intra-vaginal device (JVID®) and cloprostenol treatment. In the second category (group C to F), jennies were pretreated with deslorelin, and then treated with JVID and cloprostenol, including groups C (n = 50), D (n = 50), E (n = 70), and F (n = 65). In the third category, jennies were treated with deslorelin and cloprostenol, including groups G (n = 40) and H (n = 40). Comparisons were made among groups regarding the degree of synchronization, ovulation, and pregnancy rates. Treatment with 0.4 mg cloprostenol on the third day following ovulation minimized the length of the luteal phase and estrous cycle. Synchronization rate varied from 60.0% to 88.6% among groups and was highest in group E. Pregnancy rates did not differ among the eight protocols. In conclusion, cloprostenol effectively induced luteolysis in jennies and a treatment protocol combining deslorelin, cloprostenol, and JVID is efficient for estrus synchronization in donkeys.

Hormonal profile prior to luteolysis modulates the uterine luminal transcriptome in the subsequent cycle in beef cross-bred cows†.

Sex steroid concentrations modulate endometrial function and fertility in cattle. Our objective was to compare the post-estrus luminal transcriptome of cows that were exposed to contrasting concentrations of progesterone (P4) before luteolysis that displayed estrus and ovulated spontaneously. Cross-bred beef cows received either (1) a new CIDR and GnRH (day -9; high progesterone treatment; HP4; n = 16) or (2) a previously used CIDR, PGF2α, and GnRH (low progesterone treatment; LP4; n = 24). All cows received PGF2α at CIDR removal (day -2). Ovarian ultrasonography and blood collections were performed on days -9, -2, -0.5, and 0 (day of observed estrus), and days 4, 7, and 14 for measurement of ovarian structures, P4, and estradiol (E2). Luminal epithelial cells were collected using a cytology brush on days 4, 7, and 14 for RNAseq. On day -2, CL area and concentrations of P4 were greater, while on day -0.5, concentrations of E2 were decreased in HP4. Ovarian structures and hormonal concentrations were similar on days 4, 7, or 14 (P > 0.05). There were enriched pathways in HP4 related to activation and signaling of the innate immune system at day 4, downregulation in the network involved in the extracellular matrix remodeling at day 7, and exacerbated inflammatory response as well as differentiation and activation of macrophages at day 14 (Benjamini-Hochberg P-value ≤ 0.05). In conclusion, manipulation of pre-luteolysis sex steroid concentrations altered the post-estrus luminal transcriptome even though all cows showed estrus and ovulated spontaneously.

Contributions to Mare Reproduction Research by the Ginther Team.

Examples of research discoveries and first reports on mare reproduction by the O.J. Ginther team are (1) determined daily circulating concentrations of four hormones during the estrous cycle, (2) showed that mares can be induced to ovulate and superovulate by hormone treatment during both ovulatory and anovulatory seasons, (3) demonstrated that prostaglandin F2α was the luteolysin in mares, (4) described the mare's elaborate hormonal and biochemical mechanism for selecting the ovulatory follicle from a pool of like follicles, (5) developed the method for diagnosing fetal sex by Day 60 using location of the genital tubercle, (6) refuted the dogma that the primary corpus luteum regresses at about one month of pregnancy, (7) demonstrated that the uterus induces luteolysis in nonpregnant mares through a systemic pathway unlike the local uteroovarian venoarterial pathway in ruminants, (8) developed the method for greatly reducing the devastating twinning problem, and (9) discovered intrauterine embryo mobility and fixation and thereby solved several enigmas in mare reproduction. During 56 years on the University of Wisconsin faculty, Ginther was sole author of seven hard cover texts and reference books. He supervised 112 graduate-students, postdoctorates, and research trainees from 17 countries. His team published 680 full-length journal papers that were cited 43,034 times according to Google Scholar. The Institute for Scientific Information ranked him among the top 1% of the world's scientists in all fields. According to a survey in 2012-23 by Expertscape, he published more scientific manuscripts than anyone on ovarian follicles, corpora lutea, and luteolysis.

Role of intraluteal and intrauterine prostaglandin signaling in LH-induced luteolysis in pregnant rats.

Luteal dysfunctions lead to fertility disorders and pregnancy complications. Normal luteal function is regulated by many factors, including luteinizing hormone (LH). The luteotropic roles of LH have been widely investigated but its role in the process of luteolysis has received little attention. LH has been shown to have luteolytic effects during pregnancy in rats and the role of intraluteal prostaglandins (PGs) in LH-mediated luteolysis has been demonstrated by others. However, the status of PG signaling in the uterus during LH-mediated luteolysis remains unexplored. In this study, we utilized the repeated LH administration (4×LH) model for luteolysis induction. We have examined the effect of LH-mediated luteolysis on the expression of genes involved in luteal/uterine PG synthesis, luteal PGF2α signaling, and uterine activation during different stages (mid and late) of pregnancy. Further, we analyzed the effect of overall PG synthesis machinery blockage on LH-mediated luteolysis during late pregnancy. Unlike the midstage of pregnancy, the expression of genes involved in PG synthesis, PGF2α signaling, and uterine activation in late-stage pregnant rats' luteal and uterine tissue increase 4×LH. Since the cAMP/PKA pathway mediates LH-mediated luteolysis, we analyzed the effect of inhibition of endogenous PG synthesis on the cAMP/PKA/CREB pathway, followed by the analysis of the expression of markers of luteolysis. Inhibition of endogenous PG synthesis did not affect the cAMP/PKA/CREB pathway. However, in the absence of endogenous PGs, luteolysis could not be activated to the full extent. Our results suggest that endogenous PGs may contribute to LH-mediated luteolysis, but this dependency on endogenous PGs is pregnancy-stage dependent. These findings advance our understanding of the molecular pathways that regulate luteolysis.

Circumvention of luteolysis reveals parturition pathways in mice dependent upon innate type 2 immunity.

Although mice normally enter labor when their ovaries stop producing progesterone (luteolysis), parturition can also be triggered in this species through uterus-intrinsic pathways potentially analogous to the ones that trigger parturition in humans. Such pathways, however, remain largely undefined in both species. Here, we report that mice deficient in innate type 2 immunity experienced profound parturition delays when manipulated endocrinologically to circumvent luteolysis, thus obliging them to enter labor through uterus-intrinsic pathways. We found that these pathways were in part driven by the alarmin IL-33 produced by uterine interstitial fibroblasts. We also implicated important roles for uterine group 2 innate lymphoid cells, which demonstrated IL-33-dependent activation prior to labor onset, and eosinophils, which displayed evidence of elevated turnover in the prepartum uterus. These findings reveal a role for innate type 2 immunity in controlling the timing of labor onset through a cascade potentially relevant to human parturition.

The Temporal Associations of B-Mode and Power-Doppler Ultrasonography, and Ovarian Steroid Changes of the Periovulatory Follicle and Corpus Luteum During Luteogenesis and Luteolysis in Jennies.

This study aimed to determine the associations between B-mode and Power-doppler ultrasonography and ovarian steroids of the periovulatory follicle and respective corpus luteum (CL) during luteogenesis and luteolysis in jennies. Twenty-four periovulatory follicles/estrus of correspondent one inter-ovulatory interval (n = 12 jennies) were assessed in the study. B-mode ultrasonography and teasing were carried out once day until the detection of a periovulatory follicle (≥28 mm, uterine edema, and signs of estrus). Thereafter, jennies were monitored at 4-hour-intervals by B-mode and Power-doppler ultrasonography. Closer to ovulation, jennies were hourly checked. Each CL was checked daily from luteogenesis to luteolysis. Plasma concentrations of estradiol and progesterone were assessed daily with chemiluminescence immunoassay. Granulosa echogenicity and thickness increased from -36 hour to -1 hour before ovulation in 70% of follicles (P < .05) and were strongly associated with impending ovulation (r = 0.80 and r = 0.70, respectively). The follicular-wall blood flow increased from -72 to -24 hour pre-ovulation, while the estradiol concentration declined from 42 pg/mL by -72 hour to 31.6 pg/mL by 24 hour before ovulation (P < .05). The vascularization of the periovulatory follicle decreased from 62% (-36 hour) to 37% (-1 hour) before ovulation (P < .05). The CL vascularization and progesterone concentration gradually increased, reaching the peak at 11- and 10-day after the ovulation, respectively (P < .05). The CL vascularization started to decline 3 day before luteolysis, while progesterone concentrations started to drop 4 day before luteolysis (P < .05). In conclusion, the structural changes of the periovulatory follicle detected on B-mode and Power-doppler can be used to detect impending ovulation in donkeys; however, Power-doppler, but not B-mode ultrasonography, can be used to assess CL function in jennies.

Processes involved in prostaglandin F2alpha autoamplification in heifers.

In brief: Endometrial and luteal synthesis of prostaglandin F2alpha (PGF2A) occurs before and during luteolysis and is critical for luteal regression. This study demonstrates that PGF2A stimulates further PGF2A synthesis (autoamplification) apparently from the corpus luteum. Abstract: Understanding the endocrine profile of prostaglandin F2alpha (PGF2A) autoamplification is fundamental to comprehend luteal and endometrial responses to PGF2A. On day 10 of postovulation (preluteolysis), heifers (n = 6/group) were treated intrauterine with saline or PGF2A (0.5 mg; hour 0). A third group received flunixin meglumine + PGF (FM+PGF) to prevent endogenous synthesis of PGF2A. Exogenous PGF2A was metabolized at hour 2 as measured by PGF2A metabolite (PGFM). From hours 5 to 48, concentrations of PGFM were greatest in the PGF group, smallest in the FM+PGF, and intermediate in the control suggesting endogenous synthesis of PGF2A only in PGF group. Progesterone (P4) concentrations decreased transiently between hours 0 and 1 in PGF and FM+PGF groups but rebounded to pretreatment concentrations by hours 6 and 4, respectively. No control or FM+PGF heifers underwent luteolysis during the experimental period. Conversely, in the PGF group, one heifer had complete luteolysis (P4 < 1 ng/mL), two heifers had partial luteolysis followed by P4 and CL resurgence by hour 48, and three heifers did not undergo luteolysis. Endogenous PGF2A appears to be of luteal origin due to the lack of pulsatile pattern of PGFM and lack of endometrial upregulation of oxytocin receptor (typical of endometrial synthesis of PGF2A), whereas luteal downregulation of PGF receptor and HPGD indicates a classic luteal response to PGF2A signaling although other specific mechanisms were not investigated. The hypothesis was supported that a single PGF2A treatment simulating the peak of a natural luteolytic pulse and the uteroovarian transport of PGF2A stimulates measurable endogenous PGF2A production.

Current knowledge about the processes of luteolysis and maternal recognition of pregnancy in camelids.

Camelids have many unique reproductive features that considerably differ from those of other domestic species. Females are induced ovulators with subsequent development of a corpus luteum (CL) with a short lifespan. Plasma progesterone concentration starts to increase on day 4, peaks on day 8-9 and, in non-pregnant animals, basal concentration is reached around day 10-11 post-induction of ovulation. Luteolytic pulses of prostaglandin F2α (PGF2α ) are firstly detected on day 7 or 8 (approximately on day 5-6 after ovulation), with maximal luteolytic peaks observed between days 9 and 11 post-mating, in coincidence with a high endometrial expression of cyclooxygenase 2, a limiting enzyme in prostaglandins synthesis. Unlike other species, oxytocin seems not to be involved in the luteolytic process in these species. The CL is the main source of progesterone secretion, and its function is required to support pregnancy. Despite constant research efforts, aspects of reproduction and maternal recognition of pregnancy in camelids remain not fully understood. A transient decrease and subsequent recovery in plasma progesterone concentration are observed after day 9 post-mating in pregnant animals in association with a pulsatile release of PGF2α and a transitory decrease in CL vascularization. Thus, embryo recognition should occur between days 8 and 12 post-mating. In camels, conceptus tissues exhibit aromatizing activity with the capacity to synthesize large amounts of oestradiol. Similarly, llama blastocysts secrete oestradiol-17ß during the preimplantation stage, with a higher production during the elongation period. An increase in the endometrial expression of oestrogen receptor α is also observed on day 12 post-mating. All these evidences suggest that oestrogen could be the signal released by the embryo at the time of its recognition in camelids. Besides, nearly 98% of pregnancies are carried out in the left horn. A decrease in the endometrial expression of mucin 1 and 16 genes has been reported, suggesting that these changes are crucial for successful embryo implantation; however, no differences have been observed between horns. Thus, maternal recognition of pregnancy in camelids is a particularly complex process that must occur in a concise time to allow the rescue of the CL and embryo survival.

Effect of dose and frequency of prostaglandin F2α treatments during a 7-day Ovsynch protocol with an intravaginal progesterone releasing device on luteal regression and pregnancy outcomes in lactating Holstein cows.

Our objective was to evaluate the effect of 3 different Ovsynch protocols on progesterone (P4) and pregnancies per artificial insemination (P/AI), where all cows received a P4 releasing intravaginal device (PRID) from d 0 until d 8. We hypothesized that (1) both modified PGF2α treatments lead to decreased P4 at the second GnRH treatment (G2), resulting in greater P/AI, (2) the treatment effect is influenced by the presence of a corpus luteum (CL) at the beginning of the protocol, and (3) potential vaginal discharge caused by the PRID does not have a negative influence on fertility. Lactating Holstein cows (n = 1,056) were randomly assigned to 1 of 3 treatment groups on a weekly basis (n = 356; control: d 0, 100 µg of GnRH + PRID; d 7, 25 mg of dinoprost; d 8, PRID removal; d 9, 100 µg of GnRH). Cows in the second group (n = 353) received an Ovsynch protocol with a double dose of PGF2α (DoubleDose: d 0, 100 µg of GnRH + PRID; d 7, 50 mg of dinoprost; d 8, PRID removal; d 9, 100 µg of GnRH). Cows in the third group (n = 347) received an Ovsynch protocol with a second PGF2α treatment 24 h after the first one (2PGF: d 0, 100 µg of GnRH + PRID; d 7, 25 of mg dinoprost; d 8, 25 mg of dinoprost and PRID removal; d 9, 100 µg of GnRH). All cows had their ovaries scanned to determine the presence of a CL at the beginning of the Ovsynch protocol. Vaginal discharge score (VS) was evaluated at PRID removal. All cows received timed artificial insemination approximately 16 h after G2. Pregnancy diagnosis was performed via transrectal ultrasonography (d 38 ± 3 after timed artificial insemination) and rechecked on d 80 ± 7 after timed artificial insemination. Blood samples were collected on d 0, 7, and 9 of the protocol to determine P4 concentrations. Treatment affected P4 at G2. Progesterone was lower for 2PGF and DoubleDose cows compared with cows in the control group (control 0.35 ± 0.02 ng/mL; DoubleDose 0.29 ± 0.02 ng/mL; 2PGF 0.30 ± 0.02 ng/mL). Overall, P/AI did not differ among treatments. We found, however, an interaction between treatment and CL at the first GnRH treatment. Cows lacking a CL at the first GnRH treatment in the 2PGF group had greater P/AI (47.9%) compared with the same type of cows in the DoubleDose group (32.7%). We observed an effect of VS on P4 concentration at d 7. We found an increase in P4 with greater VS. Vaginal discharge score at PRID removal tended to have a positive effect on P/AI at d 38 (VS0: 36.5%; VS1: 41.3%; VS2: 49.7%). In conclusion, the addition of a second PGF treatment on d 7 and 8 of a 7-d Ovsynch protocol increased luteal regression and decreased mean P4 at G2. Cows treated with PGF2α 2 times 24 h apart showed greater P/AI, compared with cows treated with an increased dose of PGF2α.