Temporality of ovarian steroids and LH/FSH pulse profiles encompassing selection of the dominant follicle in heifers†.

The tested hypotheses were (1) LH/FSH pulses and F2 diameter are diminished by P4 and, (2) E2 increases during the transition to deviation and alters LH/FSH pulses. On Day 5 (Day 0 = ovulation), heifers were randomized into an untreated group (HiP4, n = 11), and a prostaglandin analog treated group (NoP4, n = 10). On Day 6, a follicular wave was induced by follicle ablation. Ultrasound and blood collections were performed every 12 h from Days 7 to 11. Blood was collected every 15 min for 10 h on Day 9 (largest follicle expected to be ~7.5 mm). Estradiol was ~75% greater (0.36 ± 0.14 vs 0.63 ± 0.19 pg/mL) in heifers with F1 ≥ 7.2 mm than in heifers with F1 < 7.2 mm. The HiP4 had smaller second largest follicle (F2) diameter, lower estradiol (P = 0.06), LH pulse baseline and peak concentrations (P < 0.007), in addition to half the frequency of LH/FSH pulses (4.1 ± 0.3 vs 9.6 ± 0.7 in 10 h) than the NoP4. Within HiP4, heifers with F1 ≥ 7.2 mm had ~25% fewer (P = 0.03) LH pulses compared to heifers with F1 < 7.2 mm. In contrast, within the NoP4, heifers with F1 ≥ 7.2 mm had ~75% greater LH (P = 0.05) and FSH (P = 0.08) pulse amplitude. We propose that greater F2 diameter at deviation in low P4 is related to greater LH baseline and peak concentrations, and greater frequency of LH/FSH pulses. A greater increase in E2 after F1 reaches ~7.2 mm results in further stimulation of LH/FSH pulse amplitude. Elevated P4 not only diminished frequency of LH/FSH pulses but also converted an E2 increase into a negative feedback effect on LH/FSH pulse frequency leading to smaller F2 at deviation.

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.

Endometrial and luteal responses to a prostaglandin F2alpha pulse: a comparison between heifers and mares†.

In heifers and mares, multiple pulses of prostaglandin F2alpha (PGF) are generally associated with complete luteal regression. Although PGF pulses occur before and during luteolysis, little is known about the role of minor PGF pulses during preluteolysis on subsequent luteal and endometrial PGF production that may initiate luteolysis. Heifers (n = 7/group) and mares (n = 6/group) were treated with a single minor dose of PGF (3.0 and 0.5 mg, respectively) during mid-luteal phase (12 and 10 days postovulation respectively). After treatment, a transient decrease in progesterone (P4) concentrations occurred in heifers between Hours 0 and 2 but at Hour 4 P4 was not different from pretreatment. In mares, P4 was unaltered between Hours 0 and 4. Concentrations of P4 decreased in both species by Hour 24 and complete luteolysis occurred in mares by Hour 48. Luteal and endometrial gene expression were evaluated 4 h posttreatment. In heifers, luteal mRNA abundance of PGF receptor and PGF dehydrogenase was decreased, while PTGS2, PGF transporter, and oxytocin receptor were increased. In the heifer endometrium, receptors for oxytocin, P4, and estradiol were upregulated. In mares, luteal expression of PGF receptor was decreased, while PGF transporter and oxytocin receptor were increased. The decrease in P4 between Hours 4 and 24 and changes in gene expression were consistent with upregulation of endogenous synthesis of PGF. The hypotheses were supported that a single minor PGF treatment upregulates endogenous machinery for PGF synthesis in heifers and mares stimulating endogenous PGF synthesis through distinct regulatory mechanisms in heifers and mares.

Effect of elevating luteinizing hormone action using low doses of human chorionic gonadotropin on double ovulation, follicle dynamics, and circulating follicle-stimulating hormone in lactating dairy cows.

Double ovulation and twin pregnancy are undesirable traits in dairy cattle. Based on previous physiological observations, we tested the hypothesis that increased LH action [low-dose human chorionic gonadotropin (hCG)] before the expected time of diameter deviation would change circulating FSH concentrations, maximum size of the second largest (F2) and third largest (F3) follicles, and frequency of multiple ovulations in lactating dairy cows with minimal progesterone (P4) concentrations. In replicate 1, multiparous, nonbred lactating Holstein dairy cows (n = 18) had ovulation synchronized. On d 5 after ovulation, all cows had their corpus luteum regressed and were submitted to follicle (≥3 mm) aspiration 24 h later to induce emergence of a new follicular wave. Cows were then randomized to NoP4 (untreated) and NoP4+hCG (100 IU of hCG every 24 h for 4 d after follicle aspiration). Ultrasound evaluations and blood sample collections were performed every 12 h for 7 d after follicle aspiration. All cows were then treated with 200 µg of GnRH to induce ovulation. In replicate 2, cows (n = 16) were resubmitted to similar procedures (i.e., corpus luteum regression, follicle aspiration, randomization, ultrasound evaluations every 12 h, GnRH 7 d after aspiration). However, cows in replicate 2 received an intravaginal P4 device that had been previously used (∼18 d). Only cows with single (n = 15) and double (n = 16) ovulations were used in the analysis. No significant differences were detected for frequency of double ovulation, follicle sizes, and FSH concentrations across replicates (NoP4 vs. LowP4 and NoP4+hCG vs. LowP4+hCG), so data were combined. Double ovulation was 40% for control cows with no hCG (CONT) and 62.5% with hCG (hCG). Double ovulation increased as the maximum size of F2 increased: <9.5 mm and 9.5-11.5 mm (7.7%) and ≥11.5 mm (94.1%). The hCG group had more cows with F2 > 11.5 (69%) than with 9.5 ≥ F2 ≤ 11.5 (25%) and F2 < 9.5 (6%). In agreement, F2 and F3 maximum size were larger in the hCG group, but FSH concentrations were lower after F1 > 8.5 mm compared with CONT. In contrast, FSH concentrations were greater before deviation (F1 closest value to 8.5 mm) in cows with double ovulations than in those with single ovulations, regardless of hCG treatment. In addition, time from aspiration to deviation was shorter in cows with double rather than single ovulation and in cows treated with hCG as a result of faster F1, F2, and F3 growth rates before diameter deviation. In conclusion, greater FSH and follicle growth before deviation seems to be a primary driver of greater frequency of double ovulation in lactating cows with low circulating P4. Moreover, the increase in follicle growth before deviation and in the maximum size of F2 during hCG treatment suggests that increased LH may also have a role in stimulating double ovulation.

Selection of side of ovulation by intraovarianism in Bos taurus heifers†.

Intraovarianism refers to mechanisms within an ovary that affect the local follicle and luteal dynamics and is well-represented in heifers by greater frequency of ovulation from the right ovary (RO) than the left ovary (LO). On average, the RO has more 6-mm follicles than the LO before one follicle is selected to deviate in diameter and become the future ovulatory follicle. Therefore, the ovulatory follicle is more frequently selected from the RO. More follicles in the RO likely develop before birth as indicated by greater weight of the RO with more 0.3- to 4.8-mm follicles in recently born calves. It has been proposed that differences in intraovarianism between sides are a consequence of differences between sides in the inherent intraovarian angioarchitecture. The frequency of the pair of ovulations at the beginning and end of 900 interovulatory intervals (IOI) was lowest for the left/left (LL) pair (16%) and higher and similar among the RR, LR, and RL pairs (28% each). The lower frequency of LO ovulation was entirely a function of the LL pair as indicated by the lower frequency of the LL than RR pairs without a difference among the RR, LR, and RL pairs. Ovulations from the opposite sides at the beginning and end of an IOI (LR and RL pairs) would not have contributed to a difference in ovulation frequency between LO and RO. In conclusion, the greater frequency of RO (56%) than LO (44%) ovulation was mathematically and functionally a direct consequence of the low frequency of the LL pair of ovulations.

Intraovarianism. Local mechanisms that affect follicle and luteal dynamics in heifers and women†.

Intraovarianism is represented by greater follicle activity in right ovary (RO) than left ovary (LO) during the two or three follicular waves per interovulatory interval in heifers and the ovulatory wave in women. In each species, selection between the dominant follicle (DF or F1) and future largest subordinate follicle (F2) is manifested by diameter deviation. The RO has more ≥6 mm predeviation follicles in heifers, more 2-10 mm predeviation follicles in women, and greater frequency of ovulation in each species. The RO propensity for ovulation likely develops before birth or at least before sexual maturity as indicated by (1) heavier RO with more follicles in recently born calves and heavier right fetal gonad with more DNA content in humans and (2) greater RO frequency for largest follicle of a wave in sexually immature heifers and for first ovulation of puberty. In heifers, intraovarianism is also expressed by (1) two-way enhancement effect between DF and corpus luteum (CL) when in same ovary, (2) regression of predeviation follicles when adjacent to the regressing CL, (3) more frequent location of F2 during predeviation in ovary that contains F1, (4) greater diameter increase during recovery of subordinate follicles when in RO with the future DF, and (5) effect of an intraovarian pattern on the pattern in the next wave. The nature of these forms of intraovarianism is not known but may be physical rather than from local passage of factors between structures. Intraovarianism should be considered when studying systemic hormonal effects on ovarian dynamics.

Up-regulation of endometrial oxytocin receptor is associated with the timing of luteolysis in heifers with two and three follicular waves†.

Initiation of luteolysis in ruminants is variable due to ill-defined mechanisms. Cycles of two follicular waves are shorter and have earlier luteolysis than three-wave cycles. This study validated a cytobrush technique for evaluating dynamics of endometrial gene expression and associated changes in mRNA with timing of luteolysis, based on circulating progesterone and ultrasound-determined changes in blood flow and volume of corpus luteum (CL). On day 8 (ovulation = day 0), Holstein heifers were randomized into two groups: cytobrush group (n = 9) had an endometrial sample collected every 48 h from day 8 until end of luteolysis (CL blood flow ≤ 20%) and control group was sampled only once either before (day 12; n = 4) or at the end of luteolysis (n = 5). Concentrations of progesterone, CL blood flow, CL volume, and the frequency of two and three-wave cycles were similar between groups. Endometrial mRNA for progesterone receptors and estradiol receptors 1 and 2 was greater on day 8 and decreased thereafter similarly in two and three-wave cycles. Oxytocin receptor mRNA increased earlier in two vs three-wave cycles (day 14 vs 18), and the increase was associated with the onset of luteolysis. In conclusion, the cytobrush technique allowed in vivo collection of multiple endometrial samples during the estrous cycle. Endometrial mRNA expression of steroid receptors did not explain the variability in timing of onset of luteolysis in heifers while the later onset of luteolysis in three-wave cycles was associated with later up-regulation of oxytocin receptor mRNA.

Selection of fewer dominant follicles in Trio carriers given GnRH antagonist and luteinizing hormone action replaced by nonpulsatile human chorionic gonadotropin†.

Studying selection of multiple dominant follicles (DFs) in monovulatory species can advance our understanding of mechanisms regulating selection of single or multiple DFs. Carriers of the bovine high fecundity Trio allele select multiple DFs, whereas half-sib noncarriers select a single DF. This study compared follicle selection during endogenous gonadotropin pulses versus during ablation of pulses with Acyline (GnRH antagonist) and luteinizing hormone (LH) action replaced with nonpulsatile human chorionic gonadotropin (hCG) treatment in Trio carriers (n = 28) versus noncarriers (n = 32). On Day 1.5 (Day 0 = ovulation), heifers were randomized: (1) Control, untreated; (2) Acyline, two i.m. doses (Days 1.5 and D3) of 3 µg/kg; (3) hCG, single i.m. dose of 50 IU hCG on Day 1.5 followed by daily doses of 100 IU; and (4) Acyline + hCG. Treatments with nonpulsatile hCG were designed to replace LH action in heifers treated with Acyline. Acyline treatment resulted in cessation of follicle growth on Day 3 with smaller (P < 0.0001) maximum follicle diameter in Trio carriers (6.6 ± 0.2 mm) than noncarriers (8.7 ± 0.4 mm). Replacement of LH action (hCG) reestablished follicle diameter deviation and maximum diameter of DFs in both genotypes (8.9 ± 0.3 mm and 13.1 ± 0.5 mm; P < 0.0001). Circulating follicle stimulating hormone (FSH) was greater in Acyline-treated than in controls. Finally, Acyline + hCG decreased (P < 0.0001) the number of DFs from 2.7 ± 0.2 to 1.3 ± 0.2 in Trio carriers, with most heifers having only one DF. This demonstrates the necessity for LH in acquisition of dominance in Trio carriers (~6.5 mm) and noncarriers (~8.5 mm) and provides evidence for a role of GnRH-induced FSH/LH pulses in selection of multiple DFs in Trio carriers and possibly other physiologic situations with increased ovulation rate.

Increased dietary energy alters follicle dynamics and wave patterns in heifers.

Understanding the impacts of nutrition on reproductive physiology in cattle are fundamental to improve reproductive efficiency for animals under different nutritional conditions. Starting on Day 0 (day of ovulation) until next ovulation, Holstein heifers (n = 24) were fed: low energy diet (ad libitum grass hay; LED) and high energy diet (ad libitum grass hay + concentrate supplement; HED). Heifers on HED gained more weight (average daily gain: 0.824 ± 0.07 vs 0.598 ± 0.09 kg/day) and had increased insulin concentrations. The dominant follicle of wave 1 in HED had greater growth rate overall from Days 0 to 8 and on Days 6-7 and 8-9 and started atresia later. The dominant follicle of wave 2 in HED had greater growth rate overall from Day 9 to 18 and on Days 14-15 and 15-16. In two-wave patterns, there was no difference in estradiol or progesterone concentrations but concentrations of FSH were lower in HED on Days 15 and 16. Estradiol concentrations increased earlier in two-wave patterns in association with earlier luteolysis. The frequency of two follicular waves was greater in HED than LED (11/12 vs 6/11; 92.7% vs 54.5%). In conclusion, an acute increase in dietary energy altered not only growth rate of the dominant follicle but also follicular wave pattern in heifers by increasing frequency of two follicular waves. The hypotheses were supported that an acute increase in dietary energy (1) prolongs growth period of dominant follicles and (2) alters follicular wave pattern in heifers.

Necessity for LH in selection and continued growth of the bovine dominant follicle.

Previous research demonstrated that acute treatment with GnRH antagonist, Acyline, allowed follicle growth until ~8.5 mm and no dominant follicle was selected. This study evaluated whether deficient LH was the underlying mechanism for Acyline effects by replacing LH action, using human chorionic gonadotropin (hCG), during Acyline treatment. Holstein heifers (n = 24) during first follicular wave were evaluated by ultrasound and randomized into one of three treatments: Control (saline treatments), Acyline (5 µg/kg Acyline), or Acyline+hCG (Acyline plus 50 IU of hCG at start then 100 IU every 12 h). Pulses of LH were present in Control heifers (9 Pulses/10 h) but not during Acyline treatment. Data were normalized to the transition to diameter deviation (day 0; F1 ~7.5 mm). Diameter deviation of the largest (F1) and the second largest (F2) follicle was not observed in Acyline-treated heifers, whereas control heifers had decreased growth of F2 at F1 ~7.5 mm, indicating deviation. Selection of a single dominant follicle was restored by providing LH activity in Acyline+hCG heifers, as evidenced by F1 and F2 deviation, continued growth of F1, and elevated circulating estradiol. Separation of F1 and F2 occurred 12 h (~7.0 mm) earlier in Acyline+hCG heifers than Controls. Circulating FSH was greater in Acyline than Controls, but lower in Acyline+hCG than Controls after day 1.5. In conclusion, dominant follicle selection and growth after follicle deviation is due to LH action as shown by inhibition of this process during ablation of GnRH-stimulated LH pulses with Acyline and restoration of it after replacement of LH action by hCG treatment.

Spontaneous switching of future dominance to a smaller follicle: commonality among monovular species.

Selection of a dominant follicle from a wave of follicles is manifested by diameter deviation between future dominant (F1) and largest subordinate (F2) follicles. On day -1 or 0 (day 0 = beginning of deviation), growth rate of F1 continues and growth rate of F2 decreases. Deviation occurs during the decline in the wave-stimulating FSH surge when F1 reaches means of 8.5, 10.5, and 22.5 mm in heifers, women, and mares, respectively. Diameter of F1 at the FSH peak vs at deviation is proportionally similar among these monovular species. In conventional deviation, F1 usually emerges first. In F1,F2-switched deviation, F2 is usually first to emerge and to reach a diameter characteristic of deviation. On day -1 or 0, the larger F2 and the smaller F1 switch so that the formerly larger F2 becomes subordinate and the formerly smaller F1 becomes dominant. In heifers and mares, the profile and prominence of the FSH surge are similar between deviation classes. Surge location relative to deviation differs so that the surge ends earlier in switched deviation. When the larger F2 reaches a diameter characteristic of deviation, FSH concentration is too low for continued growth of F2. The decrease in FSH ceases (heifers) or increases (mares) presumably from a decrease in FSH inhibitors; therefore, F1 continues to grow and becomes dominant. The frequency of switched deviation (e.g., 16 to 37% among species) can be problematic in follicle-selection research. Switching is a natural model for study of the interplay between follicles and FSH.

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.

Gonadotropin-releasing hormone, estradiol, and inhibin regulation of follicle-stimulating hormone and luteinizing hormone surges: implications for follicle emergence and selection in heifers.

Mechanisms regulating gonadotropin surges and gonadotropin requirements for follicle emergence and selection were studied in heifers. Experiment 1 evaluated whether follicular inhibins regulate the preovulatory luteinizing hormone (LH)/follicle-stimulating hormone (FSH) surges elicited by gonadotropin-releasing hormone (GnRH) injection (Hour = 0) and the subsequent periovulatory FSH surge. Treatments included control (n = 6), steroid-depleted bovine follicular fluid (bFF) at Hour -4 (n = 6), and bFF at Hour 6 (n = 6). Gonadotropins in blood were assessed hourly from Hours -6 to 36, and follicle growth tracked by ultrasound. Consistent with inhibin independence, bFF at Hour -4 did not impact the GnRH-induced preovulatory FSH surge, whereas treatment at Hour 6 delayed onset of the periovulatory FSH surge and impeded growth of a new follicular wave. Experiment 2 examined GnRH and estradiol (E2) regulation of the periovulatory FSH surge. Treatment groups were control (n = 8), GnRH-receptor antagonist (GnRHr-ant, n = 8), and E2 + GnRHr-ant (n = 4). GnRHr-ant (acyline) did not reduce the concentrations of FSH during the periovulatory surge and early follicle development (<7.0 mm) was unaffected, although subsequent growth of a dominant follicle (>8.0 mm) was prevented by GnRHr-ant. Addition of E2 delayed both the onset of the periovulatory FSH surge and emergence of a follicular wave. Failure to select a dominant follicle in the GnRHr-ant group was associated with reduced concentrations of LH but not FSH. Maximum diameter of F1 in controls (13.3 ± 0.5 mm) was greater than in both GnRHr-ant (7.7 ± 0.3 mm) and E2 + GnRHr-ant (6.7 ± 0.8 mm) groups. Results indicated that the periovulatory FSH surge stems from removal of negative stimuli (follicular E2 and inhibin), but is independent of GnRH stimulation. Emergence and early growth of follicles (until about 8 mm) requires the periovulatory FSH surge but not LH pulses. However, follicular deviation and late-stage growth of a single dominant follicle requires GnRH-dependent LH pulses.

Follicle Selection in Mares as a Model for Illustrating the Many Hormonal and Biochemical Interactions That Drive a Single Physiological Mechanism.

The mechanism for selection of the future dominant or ovulatory follicle in mares involves a relatively abrupt separation in growth rates between the future dominant follicle and several subordinate follicles and is termed diameter deviation. The event is used to illustrate that a coordinated complex of many follicular, hormonal, and biochemical factors interact and interbalance during a single physiological mechanism. For example, a positive effect of follicle stimulating hormone (FSH) on development of all follicles during the growing phase can later involve a positive effect of luteinizing hormone (LH) but apparently only on the future dominant follicle. In turn, the developing and future dominant follicle produces estradiol which at appropriate times and degrees reduces FSH concentrations to accommodate follicle functions at certain levels of FSH. Meanwhile, the estradiol prevents LH from increasing from a useful to an adverse concentration. These interactions enmesh with the production and roles of other factors (e.g., inhibin, insulin-like growth factor) during follicle selection. The wide array of morphological, hormonal, and biochemical activities occur in harmony even when in the same tissue and often at the same time.

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.

Gross and ultrasonic morphology of the equine conceptus on days 10 to 40.

The gross and ultrasonic equine embryo morphology are described with emphasis on specific days after ovulation. Included are labeled colored photographs and detailed descriptions of the embryo proper (future fetus and foal) and of the entire embryonic vesicle on Days 21, 24, 30, 35/36, and 40. A few related aspects are included for the early fetus on Days 45 and 50. Regression lines for growth in the length of the embryo proper and diameter of the embryonic vesicle along with the mean days of the morphological event are included. Ultrasonograms of the embryonic vesicle are shown and discussed from Days 10 to 45. Major morphological changes in the embryo proper include: (1) appearance of forelimb and hindlimb buds, (2) appearance of the pontine flexure, (3) appearance of the genital tubercle, (4) closure of the pontine flexure, and (5) tapering of limbs toward the midline with hoof-shaped tips. Major changes in the embryonic vesicle are: (1) vascularization of mesoderm, (2) appearance of sinus terminalis, (3) emergence of allantoic sac, (4) formation of embryonic circulatory system, (5) formation and maturation of chorionic girdle, and (6) transition from yolk sac to allantoic sac.

Dynamics of circulating progesterone concentrations before and during luteolysis: a comparison between cattle and horses.

The profile of circulating progesterone concentration is more dynamic in cattle than in horses. Greater prominence of progesterone fluctuations in cattle than in horses reflect periodic interplay in cattle between pulses of a luteotropin (luteinizing hormone; LH) and pulses of a luteolysin (prostaglandin F2alpha; PGF2alpha). A dose of PGF2alpha that induces complete regression of a mature corpus luteum with a single treatment in cattle or horses is an overdose. The overdose effects on the progesterone profile in cattle are an immediate nonphysiological increase taking place over about 30 min, a decrease to below the original concentration, a dose-dependent rebound 2 h after treatment, and a progressive decrease until the end of luteolysis. An overdose of PGF2alpha in horses results in a similar nonphysiological increase in progesterone followed by complete luteolysis; a rebound does not occur. An overdose of PGF2alpha and apparent lack of awareness of the rebound phenomenon has led to faulty interpretations on the nature of spontaneous luteolysis. A transient progesterone suppression and a transient rebound occur within the hours of a natural PGF2alpha pulse in cattle but not in horses. Progesterone rebounds are from the combined effects of an LH pulse and the descending portion of a PGF2alpha pulse. A complete transitional progesterone rebound occurs at the end of preluteolysis and the beginning of luteolysis and returns progesterone to its original concentration. It is proposed that luteolysis does not begin in cattle until after the transitional rebound. During luteolysis, rebounds are incomplete and gradually wane. A partial rebound during luteolysis in cattle is associated with a concomitant increase in luteal blood flow. A similar increase in luteal blood flow does not occur in mares.

Plasma clearance and half-life of prostaglandin F2alpha: a comparison between mares and heifers.

Horses are about five times more sensitive to the luteolytic effect of prostaglandin F2alpha (PGF) than cattle, as indicated by a recommended clinical dose of 5 mg in horses and 25 mg in cattle. Novel evaluations of the PGF plasma disappearance curves were made in mares and in heifers, and the two species were compared. Mares and heifers (n = 5) of similar body weight were injected (Min 0) intravenously with PGF (5 mg per animal). Blood was sampled every 10 sec until Min 3, every 30 sec until Min 5, every 10 min until Min 60, and every 30 min until Min 240. The mean PGF concentration was greater (P < 0.05) in mares than in heifers at Min 1 through Min 60 and at Mins 180 and 240. The mean time to maximum PGF concentration was not different between mares (42.0 ± 8.6 sec) and heifers (35.0 ± 2.9 sec). The apparent plasma clearance, distribution half-life, elimination half-life, and maximum plasma PGF concentration were 3.3 ± 0.5 L h(-1) kg(-1), 94.2 ± 15.9 sec, 25.9 ± 5.0 min, and 249.1 ± 36.8 ng/ml, respectively, in mares and 15.4 ± 2.3 L h(-1) kg(-1), 29.2 ± 3.9 sec, 9.0 ± 0.9 min, and 51.4 ± 22.6 ng/ml, respectively, in heifers. Plasma clearance was about five times less (P < 0.0005), maximum plasma PGF concentration was five times greater (P < 0.002), and the distribution half-life and elimination half-life were about three times longer (P < 0.005) in mares than in heifers. The fivefold greater plasma clearance of PGF in heifers than in mares corresponds to the recommended fivefold greater clinical dose of PGF in cattle and supported the hypothesis that the metabolic clearance of PGF is slower in mares than heifers.

The Dynamic Equine Embryo from Postfixation (Day 17) to the End of the Embryo Stage (Day 40).

After the cessation of equine embryo mobility (fixation) on mean Day 16, the embryonic vesicle is rotated or oriented so that the pole with the embryo proper is opposite to the mesometrial attachment. Orientation involves massage of the vesicle by contractions of the turgid uterine horn and greater thickening of the vesicle at the pole with the embryo proper. Thickening of the dorsal endometrium (encroachment) especially on each side of the mesometrial attachment accounts for a guitar-pick shape of the vesicle when viewed in cross section of the uterine horn. On Days 21-40, the allantoic sac expands, and the relative size of the yolk sac diminishes highlighted by carrying of the embryo proper to the dorsal aspect of the embryonic vesicle. There, the blood vessels from the embryonic vesicle meet at the mesometrial attachment to become the beginning of the umbilical cord. At the end of the embryo stage and beginning of the fetal stage (Day 40), the umbilical cord lengthens in association with the descent of the fetus to the bottom of the allantoic sac. After unilateral fixation of twins, a natural embryo reduction process frequently (∼85%) eliminates one of the embryos. The twins are massaged by the uterine contractions, and a critical proportion of the thicker wall of the doomed embryonic vesicle is forced into the thinner wall of the survivor. Natural embryo reduction does not occur for bilateral twins and reduction requires intervention from the theriogenologist.