Role of estrogen and androgen in maintaining the preovulatory follicle

Summary The effects of Nitromifene citrate (CI 628), an antiestrogen, and Flutamide, an antiandrogen, on the ultrastructure and viability of the preovulatory follicle and granulosa cells were examined both in vivo and in vitro. In vivo administration of either antihormone induced degeneration within the granulosa cells. In some of the affected granulosa cells, the nuclear material was condensed while the cytoplasm and associated organelles were unaltered. In others, the density of the cytoplasm was reduced, the smooth endoplasmic reticulum was dilated but the nucleus remained unaltered. In vitro, either antihormone reduced granulosa-cell viability but the granulosa cells were twenty times more sensitive to CI 628 than to Flutamide. In addition, exposure to CI 628 induced nuclear condensation without affecting the cytoplasm, while Flutamide induced the deterioration of the cytoplasm without altering the nucleus. These observations suggest that: (1) both estrogen and androgens control the viability of the granulosa cells and thereby the follicle, (2) the action of estrogen and androgen is mediated through receptors within the granulosa cells since these antihormones prevent the nuclear uptake of their respective hormone, (3) the granulosa cells of preovulatory follicles appear to be more dependent on estrogen than on androgen, and (4) each steroid appears to have a specific role in maintaining the granulosa cell; estrogens control the integrity of the nucleus while androgens preserve the cytoplasmic organization of the granulosa cell.The authors are indebted to Dr. Neri of Schering AG for donating the Flutamide and to Dr. Westland of Warner-Lambert/Parke-Davis for providing CI-628     

Ultrasonic evaluation of the preovulatory follicle in the mare

Ultrasonically visible characteristics of preovulatory follicles in mares which single ovulated were studied daily for 79 preovulatory periods in 40 mares. The preovulatory follicle became the largest follicle in the ovary from which ovulation later occurred six or more days before ovulation in 65 of 79 (82%) preovulatory periods; the mean was day -7 (range, day -14 to day -4). The increase in mean diameter of the preovulatory follicle was linear (R(2)=99.5%) over day -7 (29.4 +/- 0.8 mm) to day -1 (45.2 +/- 0.5 mm; growth rate, 2.7 mm/day). Follicles which double-ovulated were smaller (P<0.05) on day -1 (36 +/- 1.6 mm; n=12 follicles). Preovulatory follicles exhibited a pronounced change in shape from a spherical to a conical or pear-shaped structure in 84% of the preovulatory periods. Remaining follicles retained a spherical shape. Scores representing thickness of the follicular wall increased (P<0.05) as the interval to ovulation decreased. There was no significant difference among days in mean gray-scale value of the follicular wall or in echogenicity of the follicular fluid. Although diameter and shape of the follicle and thickness of the follicular wall changed during the preovulatory period, no reliable ultrasonically visible predictor of impending ovulation was found.     

Complex interrelationships among CL,preovulatory follicle,number of follicular waves,and right or left ovaries in heifers

The diameter of the dominant follicle (DF) of wave 1 was studied on Days 9 to 17 (Day 0 = ovulation) in a survey of the ipsilateral and contralateral relationships between the location of the DF and CL, and number of follicular waves per interovulatory interval (IOI). For contralateral relationships, regardless of number of waves the diameter of the DF of wave 1 decreased (P < 0.03) between Days 11 and 13 when referenced to the follicle–CL relationship of wave 1 and decreased (P < 0.008) between Days 9 and 11 when referenced to the preovulatory follicle (PF)–CL relationship. For wave 2 in two-wave IOIs, the CL ovary of ipsilateral relationships had more (P < 0.05) follicles that reached at least 6 mm than the non-CL ovary. In three-wave IOIs, frequency of IOIs with the DF in the CL ovary was greater (P < 0.02) for wave 2 than for wave 3. In wave 3, the preovulatory and the largest subordinate follicles were located more frequently (P < 0.005) in the contralateral ovary. Ovulation in two-wave IOIs occurred more frequently (P < 0.0009) from the right ovary. In three-wave IOIs with a contralateral relationship ovulation occurred more frequently (P < 0.003) from the left ovary; a negative intraovarian effect of the CL on location of the PF may account for more ovulations from the left ovary and a reported greater frequency of the contralateral relationship. The hypothesis was supported that the ipsilateral versus contralateral relationship between the PF and CL is affected by the DF–CL relationship during the previous follicular waves and by the number and identity of waves per IOI.     

Control of in vitro maturation of bovine cumulus-oocyte complex by preovulatory granulosa cells

The present study was designed to investigate (1) the influence of the secretions of follicular cells on the in vitro maturation of bovine cumulus-oocyte complexes (COCs) and (2) the origin of the factors controlling the metabolic function of cumulus cells during the preovulatory period. Preovulatory granulosa cells were collected from synchronized heifers either before or 7–9 hr after the luteinizing hormone (LH) surge, and their secretions were recovered after a 3 hr incubation. Follicular fluids (FFs) originating from the same follicles and sera from the same animals were also collected. The effects of FFs, sera, and secretions of granulosa cells on COC metabolism were compared during 24 hr of culture. FF stimulated cumulus expansion, progesterone secretion, and overall protein synthesis by COCs but decreased the amount of a major protein of 28 kDa. The time at which FF was collected influenced both cumulus expansion and protein synthesis by COCs. The effects of FF on COC metabolism were detected at the lowest protein concentration studied (0.073 mg/ml) and could be mimicked with serum, but only at a protein concentration 100-fold higher. The inhibitory effect of FF and serum on the amount of the 28 kDa protein was reproduced with the secretions of granulosa cells, acting at protein concentrations five- and 500-fold lower, respectively. However, the secretions of granulosa cells enhanced slightly cumulus expansion and had no effect on progesterone secretion and overall protein synthesis by COCs. These results suggest that COC metabolism is influenced both by endocrine and by local factors secreted by granulosa cells in response to gonadotropins. The paracrine control of COC metabolism by preovulatory granulosa cells could be exerted not only via intercellular contacts but also via substances secreted in FF. © 1993 Wiley-Liss, Inc.     

Qualitative and quantitative changes in protein synthesis of bovine follicular cells during the preovulatory period.

To understand the mechanisms governing oocyte maturation better, the effects of the gonadotropin surge were studied on follicular cells of bovine preovulatory follicles. For this purpose, qualitative and quantitative changes in protein synthesis by both granulosa cells and cumulus cells were compared relative to the luteinizing hormone (LH) surge and the resumption of meiosis in the oocyte. Follicular cells were collected at different times before and up to 25 hr after the LH surge. For each individual preovulatory follicle, granulosa and cumulus cells were incubated separately for 3 hr with 3H-methionine or with 35S-methionine. Newly synthesized cytosolic proteins from granulosa and cumulus cells and proteins secreted into the medium were analyzed by polyacrylamide gel electrophoresis. The radioactivity was measured by liquid scintillation counting after slicing of the gels or revealed by fluorography. Three major peaks of the newly synthesized proteins, with molecular weights of 76, 56, and 30 kDa, were studied throughout the preovulatory period. After the LH surge, the overall level of protein synthesis increased in granulosa cells. In addition, the pattern of cytosolic proteins in granulosa cells changed, and, in particular, the relative synthesis of the 30 kDa peak decreased. These changes in cytosolic protein synthesis may be due to the action of LH since they could be reproduced in vitro in LH-stimulated granulosa cells. A predominant peak of 56 kDa was secreted by granulosa cells throughout the experimental period. No significant change was observed in proteins synthesized by cumulus cells under the same experimental conditions. The amounts of radioactivity incorporated into the three major proteins secreted by granulosa cells, however, were correlated significantly with the amounts of radioactivity incorporated by similar proteins synthesized by cumulus cells. These results indicate that cumulus cells respond differently from granulosa cells to the gonadotropin surge but not in an independent manner.     

Minor FSH surge,minor follicular wave,and resurgence of preovulatory follicle several days before ovulation in heifers

Blood samples were collected and follicle diameters were determined daily beginning on Day 12 (Day 0 = ovulation) in 35 interovulatory intervals (IOIs) in heifers. A minor follicular wave with maximal diameter (6.0 ± 0.3 mm) on Day −4 was detected in six of seven IOIs that were scanned for follicles 4 mm or greater. The number of IOIs with a CV-identified minor FSH surge toward the end of the IOI was greater (P < 0.03) in two-wave IOIs (10/17) than in three-wave IOIs (4/18). The 17 two-wave IOIs were used for study of the temporal relationships among preovulatory follicle, FSH, LH, and estradiol. Daily growth rate of the preovulatory follicle was maximum on Days −11 to −7, minimum (P < 0.05) on Days −7 to −4, and increased (resurged, P < 0.05) on Days −4 to −3. A transient increase in FSH was maximum on mean Day −4, and the peak of a minor FSH surge occurred on Day −4.5 ± 0.2. Concentration of LH and estradiol increased between Days −5 and −4. Results demonstrated resurgence of the preovulatory follicle apparently for the first time in any species. Resurgence seemed more related temporally to the minor FSH surge than to the LH increase, but further study is needed. Results supported the novel hypotheses that a minor FSH surge near the end of the IOI is temporally associated with (1) the emergence of a minor follicular wave and (2) the resurgence in growth rate of the preovulatory follicle.     

Resumption of follicle growth in gilts after ovarian autografting

The aims of the study were to evaluate autografting of porcine ovarian tissue in terms of establishment of a blood supply, follicle survival and development, commencement of oestrous cycles and endocrine patterns in this polyovular species. Experiment 1, a preliminary study on four gilts, showed that ovarian tissue slices survived the grafting procedure and re-vascularised. In Experiment 2, a further six pre-pubertal gilts had both ovaries surgically removed and two thin cortical slices of each ovary were immediately reattached to each of the ovarian pedicles. Blood samples were taken at surgery and then weekly. Two gilts were slaughtered 2 weeks after surgery and ovarian tissue recovered. The remaining four gilts underwent daily checks for behavioural oestrus until slaughter 24 weeks after surgery. All four gilts showed standing heat at least once prior to slaughter. Plasma LH and FSH concentrations increased significantly (P<0.01) by 3 days after surgery, then fell gradually, but did not return to pre-surgery levels. Progesterone concentrations showed some evidence of cyclicity in all animals. In the grafted tissue, re-vascularisation of the tissue was apparent by 2 weeks post-grafting, although no preantral or antral follicles were observed. The tissue recovered after 24 weeks contained healthy preantral and antral follicles, luteal tissue and some large cystic follicles. It is unclear whether these cysts were the result of ovarian or hypothalamic/pituitary disturbance. In conclusion, the results of this study have shown that follicle growth and resumption of cyclicity can be achieved following ovarian autografting in pigs and indicate that this will be a useful model for investigating the mechanisms that control the early stages of follicular growth and ultimately ovulation rate in this multiovular species.     

Remodeling of extracellular matrix at ovulation of the bovine ovarian follicle

Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1-10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n = 6) and or with 25 mg porcine LH (Day 11, Group 2, n = 8 or Day 10, Group 3, n = 8) and ovariectomy on Day 12 (12-14 hr post LH in Group 2, 38-40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains beta2 and gamma1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV alpha1 and perlecan were present prior to ovulation; after ovulation collagen type IV alpha1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV alpha1, laminins beta2 and gamma1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times.     

Hair follicle characteristics and fibre production in South American camelids

Hair follicle and fibre characteristics of Peruvian alpaca and llama and Bolivian llama were analysed in three experimental studies. The first experiment was designed to determine the age at which all the secondary follicles reach maturity, as well as to compare the skin follicular structure and activity among these different types of Peruvian camelids. It is concluded that the South American camelids investigated in this study gained a complete and mature skin follicle apparatus at an early age, and hence producers should practise an early first shearing. A second Peruvian experiment investigated comparative fibre cuticular structure on twenty Peruvian domestic camelids comprising huacaya, suri and llama (woolly) 'chacos' genotypes. The results showed that the number of cuticular scales per 100 μm fibre length proved to be strongly affected by both the fleece type and the fibre diameter. The suri fleece was clearly differentiated from those of both huacaya and llama by possessing the highest percentage of fibres with a number of scales less than eight, the lowest percentage of fibres with more than nine scales, along with the lowest percentage of fibres with a diameter of more than 35 μm. It is concluded that, with the exception of the scale height, the cuticular parameters investigated in this study can be utilised in textile fibre analyses for distinguishing among these three types of fleece, as well as in selection projects designed to produce homogeneous fibres from Peruvian domestic camelids. A further study was conducted to determine the age at which the hair follicles in Bolivian llamas reach maturity as well as for comparing the skin follicular structure and activity between the two distinct genotypes. Thirty-one llama kids were chosen. They were born between January and April 1998 and were of different sex and of 'Q'aras' (or Carguera) or 'T'amphullis' type. Skin biopsies were taken from the right mid-costal region at 2, 4, 6, 8,10,12 and 14 months of age in order to monitor four follicular parameters. In this experiment, secondary to primary (S/P) data show that the Bolivian llama population analysed possessed a complete and mature skin follicle apparatus at birth that remained essentially constant throughout the investigation period. Due to the variation of these traits inside the same genetic population, the present results showed that T and Q types could only be subjective on the basis of S/P ratio.     

Emergence and selection of the dominant follicle and gonadotropin dynamics in postpartum lactating versus non-postpartum cycling mares

Among female livestock, the mare has the shortest interval from parturition to first ovulation. Due to the scarcity of research on postpartum mares, little progress has been made on the characterization of the resumption of ovarian cyclicity after parturition. This study compared follicular and gonadotropin dynamics during follicle emergence and deviation in postpartum lactating (PP Lactating) versus non-postpartum cycling (N-PP Cycling) mares. On the day of parturition, every PP Lactating mare was paired with a N-PP Cycling mare. Comparisons were made by considering the partum-ovulation interval and the postpartum interovulatory interval for the PP Lactating mares, and two interovulatory intervals for the N-PP Cycling mares. The results presented herein demonstrate that during the postpartum period, lactating mares have some similarities in follicular and hormonal profiles around emergence and deviation when compared with non-postpartum cycling mares. However, some peculiar and important characteristics were noticed during the postpartum period in lactating mares: (1) The emergence of the DF occurs around the day of parturition; (2) follicle deviation in the ovulatory wave occurs earlier during the foal heat than in other intervals; (3) lower FSH and LH systemic concentrations were not detrimental enough to prevent the rapid resumption of ovarian activity just after parturition; and (4) the association between parturition and season can have an additional and confounding effect during postpartum ovarian activity in mares. The novel findings of this study provide better knowledge of the resumption of ovarian activity after parturition and may help provide insight into the reproductive management of this species.     

Relationships of follicle versus oocyte maturity to ultrasound morphology, blood flow, and hormone concentrations of the preovulatory follicle in mares

The effects of ultrasound morphology, vascularity, and follicular-fluid hormones of the preovulatory follicle on oocyte recovery rate and on follicle and oocyte maturity rates were studied for 60 spontaneous and solitary preovulatory follicles in mares. An ovulation-inducing dose of hCG was given when the follicle was >or=32 mm (Hour 0), and a procedure for oocyte recovery was done 30 h later (Hour 30). Between Hours 0 and 30, diameter of the follicle increased less and circulating estradiol (E2) concentrations decreased more in groups with successful versus nonsuccessful oocyte recovery and in groups with mature versus immature recovered oocytes, as indicated by significant interactions of group and hour. Significant differences in blood-flow end points between groups were not detected. At Hour 30, the frequency of granulosa serration, an indicator of impending ovulation, was higher (P < 0.001), and the number and expansion of granulosa cells in the lavaging fluid, indicators of follicle maturity, were greater in the oocyte-recovery group and in the oocyte-mature group. Follicular-fluid concentrations of E2, progesterone, and free insulin-like growth factor (IGF) 1 were not different between the oocyte-recovery and -nonrecovery groups. Concentration of progesterone was significantly greater, and E2 and free IGF1 were less in the oocyte-mature than in the immature groups. Results indicated that the post-hCG oocyte-recovery and oocyte-maturity rates were positively affected by follicle maturity. Greater follicular-fluid progesterone and lower E2 and free IGF concentrations were associated temporally with maturation of the oocyte but not with maturation of the follicle.     

Ovarian follicular wave characteristics in alpacas

The objectives were to describe in detail ovarian follicular growth characteristics and to establish the interval between successive large follicles in unmated alpacas. The ovarian follicular status of 16 non-pregnant, non-lactating mature alpacas was recorded using ultrasound every second day for between 46 and 100 days. An inverse relationship was observed between the diameter of the largest follicle and the total number of follicles indicating that follicular growth in alpacas occurs in waves. There were 15/38 (39%) inter-wave intervals of 12 days and 12/38 (32%) intervals of 16 days. The maximum follicular diameter in each follicular wave was 8.8±0.3 mm (n=38). Inter-wave intervals of longer duration were associated with a larger maximum follicle diameter (P<0.001). However, the growth rate of dominant follicles was consistent over the first 10 days after emergence. They reached a diameter capable of ovulation by this time, regardless of subsequent inter-wave interval. The latter observation suggested that the optimal time of mating might be predicted in alpacas, provided that the emergence of ovarian follicular waves was controlled.     

Transition to the ovulatory season in mares: An investigation of antral follicle receptor gene expression in vivo

The inability to obtain in vivo samples of antral follicle wall layers without removing the ovaries or sacrificing the animals has limited more in‐depth studies on folliculogenesis. In this study, a novel ultrasound‐guided follicle wall biopsy (FWB) technique was used to obtain in vivo follicle wall layers and follicular fluid samples of growing antral follicles. The expression of proliferative, hormonal, angiogenic, and pro‐/antiapoptotic receptors and proteins in the follicular wall among three follicle classes were compared during the spring transitional anovulatory (SAN) and spring ovulatory (SOV) seasons in mares. The main findings observed in the granulosa, theca interna, and/or all follicle layers during the SOV season compared with the SAN season were (a) small‐sized follicles (10–14 mm) had greater epidermal growth factor receptor (EGFR) and Bcl‐2 expression; (b) medium‐sized follicles during the expected deviation/selection diameter (20–24 mm) had greater expression of EGFR, Ki‐67, luteinizing hormone receptor (LHR), and Bcl‐2; and (c) dominant follicles (30–34 mm) had greater EGFR, Ki‐67, vascular endothelial growth factor, LHR, and Bcl‐2 expression. Estradiol related receptor alpha expression and intrafollicular estradiol concentration increased, along with an increase in follicle diameter in both seasons. In this study, the application of the FWB technique allowed a direct comparison of different receptors’ expression among follicles in different stages of development and between two seasons using the same individuals, without jeopardizing their ovarian function. The successful utilization of the FWB technique and the mare as an experimental animal offer a great combination for future folliculogenesis studies on mechanisms of follicle selection, development, and ovulation in different species, including women.     

Systemic treatment with high dose of flunixin-meglumine is able to block ovulation in mares by inducing hemorrhage and luteinisation of follicles

Prostaglandins play an obligatory role during the process of ovulation in mammals. Ovulation can be blocked by intrafollicular administration of non-steroidal anti-inflammatory drugs (NSAIDs) in several domestic species including the mare as well as by systemic administration of these drugs in women. In the mare, the effect of systemic NSAIDs treatment on ovulation has not been critically studied. The objectives of this study were: a) to determine whether high dose of flunixin-meglumine (FM) administered systemically to mares during the periovulatory period was able to block ovulation; and b) to study the follicular ultrasound characteristics of FM treated mares. Six mares were used in the study during two consecutive estrous cycles. Each mare received 2 mg FM/kg i.v. twice a day starting at the time of treatment with hCG when the follicle reached a diameter of ≥ 32 mm and continuing until ovulation. During the consecutive control cycle (CON) the mares received the same dose of hCG but were not administered FM. During the FM cycles five of six mares failed to ovulate and collapse the preovulatory follicle; but echoic specks were observed within the follicles, which continued to grow until a mean diameter of 55 mm. Eventually, the follicular contents were organised and luteinised. All CON mares ovulated normally. In conclusion, when mares were treated with FM, they had a higher incidence of ovulatory failure and development of luteinised unruptured follicles (83%, P = 0.015) compared with untreated mares.     

Progesterone exposure of seasonally anoestrous ewes alters the expression of angiogenic growth factors in preovulatory follicles

Small-dose, multiple injections of GnRH given to seasonally anoestrous ewes induce final stages of the preovulatory follicle development, but result in an high incidence of defective CL unless animals are primed with progesterone, which completely eliminates luteal dysfunction. Progesterone priming upregulates luteal vascularization; however, its effect on follicular angiogenesis is poorly understood. This study tested the hypothesis that progesterone priming of seasonally anoestrous ewes treated with dose multiple injections of GnRH eliminates defective luteal function by altering the expression of vascular endothelial growth factor (VEGF), VEGF receptor-2, angiopoietin (ANG)-1, ANG-2, and TIE-2 during early and late preovulatory follicle development. Ten seasonally anoestrous ewes were given 20 mg of progesterone im 3 days before the start of GnRH treatment; 10 other animals served as controls. Intravenous injections of 500 ng GnRH were given to all animals every 2 hours for 28 hours, followed at 30 hours with a 300-μg GnRH bolus injection to synchronize the preovulatory LH surge. Ovaries were collected at 24 and 46 hours after the start of GnRH treatment. Small (2–2.5 mm) and large (>2.5 mm) follicles were analyzed for protein and mRNA expression of the angiogenic factors using immunohistochemistry and in situ hybridization assays. Progesterone priming did not have an influence on angiogenic factor levels in small follicles. However, progesterone-primed animals showed significantly (P ≤ 0.05) higher levels of VEGF, VEGFR-2, ANG-1, and ANG-2 in large follicles compared with nonprimed ones. These data suggest that progesterone priming alters the expression of angiogenic factors in large preovulatory follicles, ensuring adequate luteal development and function.     

Relative luteinizing hormone-stimulable adenylyl cyclase of the preovulatory follicle: a predictor of ovulation in the domestic hen

The regulation of the ovulatory cycle of the hen (Gallus domesticus) is an enigma. The hen's ovulatory cycle is approximately 26 h in length. She lays an egg each day at a progressively later time. The hen then skips a day, resets her "clock", and a new sequence is started. We investigated if the ovary regulates the ovulatory cycle. Our biologic endpoint was the measurement of basal and luteinizing hormone (LH)-stimulable adenylyl cyclase (AC) activity in granulosa layers of the largest (F1) and second largest (F2) follicles. F1 and F2 follicles were obtained at lights off on nights before the first (C1; n = 7), second (C2; n = 7), or terminal ovulation (CT; n = 5) or the night before the day when no ovulation was expected (Cskip; n = 6). F1 and F2 follicles removed on C1, C2, CT, and Cskip had been these specific follicles for 32 h, 12 h, 10 h, and 8 h, respectively. Mean basal activity (pmol/min/mg protein) for the follicles was: C1 = 27.2, C2 = 44.1, CT = 60.5, and Cskip = 68.7. No significant differences were found in LH-stimulable AC activities of these F1 follicles. Relative LH (expressed as fold increase over basal) stimulation was significantly correlated (P less than 0.001) with maturity of the F1 follicle (C1 greater than C2 greater than CT greater than Cskip). No differences in AC activity were found for the F2 follicles whether they were C1, C2, CT or Cskip. For the Cskip, relative LH AC activity for the F1 follicle (2.8) was similar to that for the F2 follicle (2.7).(ABSTRACT TRUNCATED AT 250 WORDS)     

Short-term feed restriction decreases the systemic and intrafollicular concentrations of leptin and increases the vascularity of the preovulatory follicle in mares

The objective of this study was to evaluate the effect of short-term feed restriction on characteristics of the preovulatory follicle and on concentrations of systemic hormones (leptin, follicle-stimulating hormone [FSH], luteinizing hormone [LH]) and follicular fluid hormones and growth factors (leptin, estradiol, inhibin-A, activin-A, free insulin-like growth factor-1 [IGF1], insulin-like growth factor binding protein 2 [IGFBP2], vascular endothelial growth factor [VEGF]). Mares were submitted to a short-term (48 h) feed restriction when the expected ovulatory follicle was ≥27 mm (Hour 0) or served as controls (n = 8/group). No effect of short-term feed restriction was detected for systemic concentrations of FSH and LH and for intrafollicular concentrations of estradiol, activin-A, free IGF1, and IGFBP2. Restricted mares had decreased systemic concentrations of leptin at Hour 24 (approached significance) and at Hours 36 and 48 (P < 0.04). Follicular fluid of restricted mares at Hour 48 had lower (P < 0.02) concentration of leptin and a tendency (P < 0.1) for greater concentrations of inhibin-A and VEGF. The percentage of wall of the preovulatory follicle with color-Doppler signals of blood flow at Hour 48 was greater (P < 0.04) in the restricted group. Intrafollicular concentration of leptin (combined groups) was positively correlated with score for body condition (r = +0.60; P < 0.002) and negatively correlated with the percentage of the follicle wall with blood-flow signals (r = −0.60; P < 0.02). Our favored interpretation is that the preovulatory follicle seems to compensate for a nutritional deficiency by increasing the blood flow in the follicle wall.     

Ultrasound characteristics of experimentally induced luteinized unruptured follicles (LUF) and naturally occurring hemorrhagic anovulatory follicles (HAF) in the mare

The development of hemorrhagic anovulatory follicles (HAF) involves luteinization and hemorrhage of the follicle. This is observed on ultrasound as an increase in the echogenicity of the granulosa layer and formation of echoic particles in the antrum. The inhibition of prostaglandin synthesis with flunixin meglumine (FM) during the periovulatory period induces ovulatory failure with development of luteinized unruptured follicles (LUF). These two types of anovulatory follicles appear to share similar ultrasound features but they have not been compared critically. The following endpoints: follicle diameter, follicular contents score, interval from hCG administration to beginning of follicular hemorrhage, interval from hemorrhage to organization of follicular contents, and cycle length were studied and compared in mares with HAF (n = 11) and LUF (n = 13). The objective of this study was to elucidate whether these two unruptured follicles have a consistent clinical pattern of development and therefore can be considered as part of the same anovulatory syndrome. None of the endpoints analyzed differed significantly between HAF and LUF. However, there was a greater individual variation in HAF as compared with LUF in regards to interval from hCG to hemorrhage, follicular diameter at the administration of hCG, and beginning of hemorrhage. In conclusion, HAF share a similar cascade of ultrasound characteristics with the experimentally induced LUF. This finding may provide new insights in elucidating the pathogenesis of HAF.