犬黄体功能调控机制研究进展

犬黄体具有异于其他家畜的生理结构和功能表现,其中最特别的是犬缺乏急性黄体溶解机制。犬黄体发生过程受催乳素、促黄体素和前列腺素E₂的复杂调控,溶解退化过程则对其他家畜主要的溶黄体激素--前列腺素F_2α不敏感。而黄体的存留对母犬的生殖活动却具有重要的意义,其自分泌或旁分泌的类固醇激素也在妊娠调控和发情周期的更迭中发挥重要作用。犬黄体的退化机制尚未明确,但国外已有包括被动退化在内的诸多假说。此外,黄体是为数不多具有完整生命周期的功能性组织（或器官）,其发生过程、功能实现和衰老溶解都在周而复始地进行,这对炎症反应、癌症发生和细胞凋亡等研究都具有重要的参考价值。作者综述了犬黄体的发生和退化过程中调控机制的研究进展,旨在为进一步深入研究犬黄体发生和退化的分子机制提供研究背景和理论依据。     

前列腺素D2与F2α对绵羊黄体退化的影响及其机理研究

旨在研究前列腺素（prostaglandins,PGs）D₂与F_2α对绵羊黄体（corpus luteum,CL）组织形态、生殖激素及其关键基因与受体表达的影响,并解析其在黄体退化中的相互关系及机理,为保证母畜连续性繁育提供新的理论依据。将16只哈萨克绵羊随机分成4组,在发情周期的黄体期分别子宫肌内注射PGD₂、PGF_2α、PGD₂+PGF_2α及等量生理盐水（对照组）,采用HE染色结合物理拍照对比处理前后黄体组织形态变化,ELISA法检测外周血清中P₄、E₂、PGD₂和PGF_2α浓度变化;并利用qRT-PCR和Western blot检测关键合成酶基因HPGDS、PGFS及其受体DP1、CRTH2、FP的mRNA和蛋白表达水平。结果显示,与对照组相比,发现PGD₂+PGF_2α组中黄体退化效果最明显,随后依次是PGF_2α组明显大于PGD₂组。ELISA结果显示,随着处理后时间的推移,不同试验处理组中,P₄浓度均呈显著下降趋势（P<0.05）,其中在PGD₂+PGF_2α组中该变化趋势最显著（P<0.05）;但E₂、PGD₂和PGF_2α浓度均呈现不同差异性变化,其中,PGD₂+PGF_2α组中PGD₂和PGF_2α浓度呈显著下降（P<0.05）,PGF_2α组中E₂浓度呈显著升高（P<0.05）、PGD₂浓度呈显著下降（P<0.05）,PGD₂组中E₂浓度呈显著下降趋势（P<0.05）、PGD₂浓度呈显著升高趋势（P<0.05）。qRT-PCR和Western blot结果显示,与对照组相比,PGD₂+PGF_2α组中HPGDS mRNA和蛋白表达量显著下调（P<0.05）,PGFS、CRTH2及FP mRNA和蛋白表达量显著上调（P<0.05）;PGF_2α组中HPGDS mRNA和蛋白表达量呈显著下调（P<0.05）,其它基因mRNA和蛋白表达量呈显著上调（P<0.05）;PGD₂组中HPGDS、DP1、PGFS及FP mRNA和蛋白表达量呈显著上调（P<0.05）。同时,在不同受体基因表达量检测时,发现PGD₂组中DP1受体表达量显著高于CRTH2受体（P<0.05）,而PGF_2α组中CRTH2表达量则显著高于DP1（P<0.05）。综上,PGD₂无论单独使用还是结合PGF_2α使用,均能够促进CL的退化,尤其是二者结合时有明显的协同促溶效应,其作用机制可能与其体内激素水平、关键合成酶及受体类型的表达有关,这为全面认识哺乳动物CL退化的调控机制奠定了基础,也为进一步优化高效繁殖技术（尤其是PGs方案）提供了新的思路。     

VEGF在家畜黄体早期发育过程中对血管生成的调控作用

对黄体时期VEGF依赖性血管生成的分子机制研究，将有助于我们开发新的策略用于治疗黄体相关的不孕症，以及改善动物的繁殖性能。论文对VEGF在家畜黄体血管生成过程中的调控作用进行综述，旨在为临床研究及畜牧生产提供理论依据及参考资料。     

哺乳动物发情周期黄体的组织学研究进展

黄体是排卵后形成的暂时性的内分泌组织 ,在一个较短的存在期限中经历了一系列的结构变化和分泌阶段 ,对于哺乳动物维持正常的生殖功能具有重要作用。人们对黄体细胞的来源、组成、形态及大小持有不同的观点。近年来 ,人们就黄体细胞的功能和退化、溶解进行了较为深入的研究 ,发现不同动物黄体细胞的功能各有不同 ,黄体退化、溶解与细胞凋亡有密切关系。了解哺乳动物发情周期黄体的组织结构对于控制动物的繁殖具有重要意义。文章对哺乳动物发情周期黄体中不同类型黄体细胞的来源、组成、形态、功能及退化、溶解 ,黄体细胞的分泌方式和超声波在黄体研究中的运用作了概述     

前列腺素F2α及其类似物对哺乳动物生殖影响的研究进展

前列腺素F_2α(prostaglandin F_2α,PGF_2α)是一种黄体溶解因子,广泛用于母猪的同期发情和分娩启动。近年来还陆续发现PGF_2α的新功能,内源性PGF_2α可作为哺乳动物的妊娠识别信号,与胎体和子宫内膜PGF_2α受体(PGF_2α receptor,FP)结合,启动下游信号通路,对早期胚胎发育、着床、妊娠维持发挥重要作用,缺乏或合成不足会导致妊娠中断或流产。鉴于PGF_2α对母畜繁殖活动的促进作用,兽药中心研发了多种PGF_2α药物,如氯前列醇钠、地诺前列腺素F_2α等,被广泛用来启动分娩和促进发情。根据氯前列醇钠的旋光性,可分为D-型和L-型两种对映体,其中D-型结构是氯前列醇钠的主要活性成分。外源性PGF_2α药物不仅可以诱导母猪同期发情与同步分娩、促进产后健康,提高断奶母猪发情率,还可以改善母猪泌乳力与初乳质量,提高仔猪活力等生产性状。作者综述了近几年国内外PGF_2α的研究进展,包括PGF_2α对周期黄体的双重作用,PGF_2α对早期胚胎的发育、着床、分娩的影响,PGF_2α对母猪泌乳力及仔猪活力等方面的重要作用等,同时也综述了几种PGF_2α及其类似物对母畜繁殖性能等方面的影响,旨在为PGF_2α及其类似物的合理运用提供依据。     

Microvascularization and angiogenic activity of equine corpora lutea throughout the estrous cycle

Corpus luteum growth and endocrine function are closely dependent on the formation of new capillaries. The objectives of this study were to evaluate (i) tissue growth and microvascular development in the equine cyclic luteal structures; (ii) in vitro angiogenic activity of luteal tissues in response to luteotrophic (LH, PGE₂) and luteolytic (PGF₂) hormones and (iii) to relate data to luteal endocrinological function. Our results show that microvascular density was increased in the early and mid luteal phase, followed by a fall in the late luteal phase and a further decrease in the corpus albicans. Hyperplasia of luteal tissue increased until the mid luteal phase and it was followed by tissue regression. Luteal explants were cultured with no hormone added, or with PGF₂, LH, PGE₂, LH + PGE₂ or LH + PGF₂. Media conditioned by equine luteal tissue from different stages of the luteal phase were able to stimulate mitogenesis of bovine aortic endothelial cells (BAEC), suggesting the presence of angiogenic activity. No difference was observed among luteal structures on their mitogenic capacity, for any treatment used. Nevertheless, Late-CL conditioned-media with PGF₂ showed a significant decrease in BAEC proliferation (p < 0.05) and LH + PGF₂ a tendency to reduce mitogenesis. Thus, prostaglandin F₂ may play a role on vascular regression of the CL during the late luteal phase in the mare. These data suggest that luteal angiogenesis and vascular regression in the mare are coordinated with the development of non-vascular tissue and might be regulated by many different factors.     

Pituitary Hormone Responses to Luteal Regression after Administration of Prostaglandin F2α-Analogue

As an experiment to elucidate the canine luteal function maintenance system, a PGF2alpha-analogue, fenprostalene (PGF-F), was administered 24 and 54 days after ovulation to induce luteal regression, and the responses of luteinizing hormone (LH) and prolactin (PRL), which are considered to support the canine corpus luteum, were investigated. The plasma progesterone (P(4)) level was rapidly decreased, by which the plasma PRL level was increased, but no change was observed in the plasma LH level. The decrease in the plasma P(4) level after PGF-F administration may have induced positive feedback to the superior system, and stimulated secretion of the pituitary hormones. These findings suggested that the canine corpus luteum is maintained by PRL, not by LH.     

黄体退化的分子机制及早期黄体钝化的某些原因探讨

黄体退化是生殖过程中的一个重要过程 ,它控制着生殖周期的长短 ,与妊娠的建立和维持密切相关 ,已证实前列腺素F2α、催产素、内皮素 1及一氧化氮合酶等多种因素参与黄体退化过程。在黄体发育早期 ,外源PGF2α不能诱导黄体溶解 ,可能原因有 :(1)早期黄体内缺乏内皮素 1,(2 )黄体早期生物学反应减弱导致受体脱敏 ,(3)早期黄体中存在PGF2α代谢酶 ,(4)PGF2α不能激活黄体细胞内蛋白激酶C效应体系     

Specific binding of dl-cloprostenol and d-cloprostenol to PG F2α receptors in bovine corpus luteum and myometrial cell membranes

Prostaglandin F_2α receptors (PGF_2α Rs) were measured in bovine corpus luteum and myometrial cell membranes using a radiometric method. The inhibition of labelled PGF_2α binding exerted by d-cloprostenol, dl-cloprostenol, PGF_2α and PGE₁ (l0–¹¹ M to 10^–4 M) was evaluated in vitro. Results strongly suggest that cloprostenol binding to PGF_2αRs is stereospecific. d-Cloprostenol and PGF_2α were equipotent, about 150 times more potent than d-cloprostenol (P < 0.05) and approximately 280 times more potent than PGE, (P < 0.05) in inhibiting [3H]PGF_2α binding to corpus luteum cell membranes. Such differences were less evident in myometrial cell membranes, where d-cloprostenol and PGF_2α were about 10 times more potent than d-cloprostenol (P < 0.05) and approximately 95 times more potent than PGE_l (P < 0.05).     

Effect of homogenized embryos on IUD-induced luteal regression in the ewe

The effect of homogenized embryos on IUD-induced luteal regression in the ewe was studied. Plastic spiral IUDs were inserted in an uterine horn adjacent to an ovary containing a corpus luteum during various times of the estrous cycle. The ewes were sacrificed on Day 6 for examination of the corpus luteum. Estrous Cycle Day 3 was the last day in which the IUD insertion induced luteal regression. Corpus luteum was maintained in ewes when the IUD was inserted on Day 4, 5, or 6. Corpus luteum regression was not prevented when homogenized 14- or 15-day-old embryos were injected into the uterus at the time of IUD insertion. Ewes which received an IUD, a cannula, and an infusion of saline or homogenized embryos on Days 3, 4, and 5 maintained corpora lutea in 4 of 7 embryo-infused ewes and in none of 7 saline-infused ewes (p less than .05). It was determined that the IUD must remain in the uterine horn for longer than 1 day to induce corpus luteum regression and that embryos can sometimes prevent the IUD-induced regression.     

Extension of short cycles in postpartum beef cows by intrauterine treatment with catecholestradiol

Eight multiparous beef cows were used to examine the effects of intrauterine infusion of catecholestradiol (4-hydroxylated estradiol) on development and function of the first corpus luteum after parturition. Calves were weaned on day 1 (day 0 = parturition) to initiate formation of a corpus luteum (CL) by approximately day 10 or 11. Before CL formation, on days 5 to 9, cows received twice daily infusions of catecholestradiol (4 μg; n = 4) or vehicle (n = 4) into the uterine horn opposite the previous pregnancy. Plasma progesterone during the first estrous cycle was elevated longer (P<.001) and reached a higher (P<.001) concentration in cows treated with catecholestradiol. The decline in progesterone was associated with an increase in plasma 13,14-dihydro, 15-keto-prostaglandin F₂ (PGFM) in all cows infused with catecholestradiol. In contrast, a rise in PGFM at the end of the first short cycle was detected in only one of four cows treated with vehicle. Furthermore, PGFM concentrations were linearly related (R² = .870; P<.001) to concentrations of progesterone. Estradiol-17β concentrations were not different during the infusion period, but after formation of the first CL, estradiol remained elevated (P<.01) in cows that received vehicle. Results of this experiment suggest that exposure of postpartum beef cows to catecholestradiol extended luteal function in association with enhanced PGFM release.     

黄体对牛胚胎移植效果影响的探究

黄体释放的黄体酮对母畜发情周期、启动发情行为和维持妊娠起着至关重要的作用,但目前国内外对黄体是否影响胚胎移植效果有着不同报道。本文以探究黄体对牛胚胎移植效果的影响为主线,查阅大量文献,归纳黄体的检查和级别判断方法,分别分析黄体大小、黄体位置、黄体质量和黄体生产溶解过程激素水平与胚胎移植受胎率的关系,为提高胚胎移植效果提供科学的参考依据。生产上常通过B超扫描结合直肠检查法判断受体牛黄体发育和位置作为能否移植的依据;由各学者研究结果可见,不同直径黄体的胚胎移植受胎率差异不显著,为提高受体牛的利用,在对受体牛黄体大小选择时可适当放宽标准;胚胎移植到黄体同侧的受胎率比移植到黄体对侧的高,另外,可能因为操作者左右手操作习惯不同,发现左侧黄体移植受胎率比右侧的要高;较多学者认为质量好的胚胎选择黄体质量好的受体移植才能得到更高的受胎率;胚胎移植后能否成功受孕,还与黄体生成溶解过程中孕酮含量及孕酮与雌二醇的比例等有关。     

Population of Follicles and Luteal Structures during the Oestrous Cycle of Mares Detected by Three-Dimensional Internal Structure Microscopy

The structure of the equine ovary is different from that of other mammals in its extremely large size, the presence of ovarian fossa and the inverted location of its cortex and medulla. A three-dimensional internal structure microscopy (3D-ISM), which consists of a computer-controlled slicer, a CCD camera, a laser disc recorder and a PC, is very useful for the observation of the internal structures in equine ovaries. In addition, the three-dimensional images of follicles and corpus luteum (CL) reconstructed by the segmentation technique can clarify the spatial arrangement in the equine ovary. In this study, to understand the changes in the ovarian internal structures of the mare during the oestrous cycle, the size and numbers of follicles and luteal structures were analysed by 3D-ISM in addition to the concentrations of progesterone (P₄) and oestradiol-17β. As a result, many small follicles (<10 mm in diameter) were detected. It was recognized that the luteal structures were distinguished into three types, such as the corpus haemorragicum (CH), which is formed by blood elements at the cavity after ovulation, CL and corpus albican (CA). There were some CHs and CL in the group, which had the concentration of P₄ > 1 ng/ml. CHs were also observed in the group, which had low level of P₄ (P₄ < 1 ng/ml). CAs were found regardless of the P₄ level. In conclusion, 3D-ISM enabled the internal observation of the ovarian structures in detail, and estimation of the stage of the ovarian cycle with complementary physiological information. The findings by 3D-ISM provide basic information for clinical applications.     

Beta-carotene uptake and changes in ovarian steroids and uterine proteins during the estrous cycle in the canine

The uptake of beta-carotene by reproductive tissues and the effects of beta-carotene on reproductive function in the dog are unknown. We studied the uptake of beta-carotene by blood, corpus luteum, and uterine endometrium and the role of dietary beta-carotene in influencing ovarian steroid and uterine protein production during the estrous cycle in the dog. Mature female Beagle dogs (n = 56) were fed diets containing 0, 2, 20, or 50 mg of beta-carotene daily for approximately 6 wk before estrus detection. Blood was sampled at regular intervals from estrus through d 45 after ovulation (d 0 = ovulation), when laparotomy was performed. The ovaries were obtained for the isolation of corpus luteum. The uterus was flushed with phosphate-buffered saline and the endometrium obtained by scraping. Beta-carotene was not detectable in plasma, corpus luteum, or endometrium of unsupplemented dogs. However, beta-carotene and alpha-carotene in plasma, corpus luteum, and uterine endometrium increased in a dose-dependent manner. Alpha-carotene made up a high percentage of total carotenoids even though the alpha-carotene content in the dietary source was very low. Dogs fed 50 mg of beta-carotene had significantly higher concentrations of plasma progesterone between d 12 and 26 compared with unsupplemented dogs. Dietary beta-carotene did not influence plasma estradiol-17beta and total uterine proteins. Therefore, beta-carotene is absorbed into plasma, corpus luteum, and uterine endometrium of dogs. Furthermore, dietary beta-carotene increased plasma progesterone concentrations during the estrous cycle. It is possible that dietary beta-carotene may improve reproductive function in the canine.     

Effect of day of the estrous cycle at the initiation of a timed artificial insemination protocol on reproductive responses in dairy heifers

Our objectives were to identify stages of the estrous cycle at which initiation of a timed artificial insemination (Ovsynch/TAI) protocol may reduce pregnancy rates and to monitor ovarian follicle dynamics and corpus luteum development after initiation of the Ovsynch/TAI protocol at different stages of the cycle. Cycling Holstein heifers (n = 24) were injected twice with prostaglandin F2alpha to induce estrus and were scanned by ovarian ultrasonography to determine the day of ovulation (d 0). Heifers were assigned to initiate the Ovsynch/TAI protocol at d 2 (n = 5), 5 (n = 5), 10 (n = 4), 15 (n = 5), or 18 (n = 5) of the cycle. The Ovsynch/TAI was initiated with an injection of gonadotropin-releasing hormone agonist followed 7 d later with an injection of prostaglandin F2alpha. At 36 h after injection of prostaglandin F2alpha, heifers were injected with gonadotropin-releasing hormone agonist and inseminated 16 h later. Heifers were scanned daily during the Ovsynch/TAI protocol and every other day after insemination until 16 d later. Blood samples were collected daily starting at the 1st day heifers were scanned and continued until 16 d after insemination. Initiation of the Ovsynch/TAI protocol at d 15 of the estrous cycle caused heifers to ovulate prior to insemination. A shortened return to estrus (< 16 d) was caused by ovulation failure to the second gonadotropin-releasing hormone injection, by incomplete regression of the corpus luteum, and by short life-span of the induced corpus luteum. Day of the cycle in which the Ovsynch/TAI protocol is initiated affects dynamics of follicular development, plasma progesterone profiles, and occurrence of premature ovulation. Size of the pre-ovulatory follicle was associated positively with subsequent progesterone concentrations following insemination.     

The Role of Uterine and Ovarian Hormones in Luteolysis: A Comparison among Species

Luteolytic mechanisms have evolved in mammals to improve reproductive efficiency. The hormonal interactions that control the onset and progress of luteolysis are complex. They involve endocrine and paracrine signals that link the corpus luteum, uterus and posterior pituitary gland. Current concepts concerning these interactions will be examined in the five major domestic ungulate species commonly raised in Europe and North America (cattle, sheep, goats, pigs and horses). Some of these interactions are similar across species. All five depend on prostaglandin F_2α secreted from the uterus, to induce luteolysis. Three hormones, progesterone, estradiol and oxytocin interact to regulate uterine secretion of PGF_2α. Oxytocin is an acute stimulus for uterine PGF_2α secretion. Progesterone and estradiol interact to regulate uterine secretary responsiveness to oxytocin. Precisely how these hormones interact varies across species.     

Morphological study of the corpus luteum in the rat

The cyclic related growth and regression of the corpus luteum during four consecutive oestrous cycles of the regular four-day-cycling (ie, 16 days), virgin albino Wistar rat were followed up by light and electron microscopic investigation of the ovaries. After ovulation, follicular granulosa cells differentiated into luteal cells in the newly formed corpus luteum. Based on their specific histological characteristics, four various types of corpus luteum in each stage of the oestrous cycle could be identified. As soon as the luteal cells started to degenerate, the number of fibroblasts progressively increased and apoptotic degeneration of luteal cells was initiated and became most prominent during oestrus. Complete regression of the corpus luteum was seen after 15 days. This study shows a strictly organised pattern of luteal cell growth and degeneration in the corpus luteum of the regular four-day-cycling, virgin Wistar rat. The morphological alterations may be regulated by a hormonal fluctuation.     

Regulation of Luteal Function and Corpus Luteum Regression in Cows: Hormonal Control, Immune Mechanisms and Intercellular Communication

The main function of the corpus luteum (CL) is production of progesterone (P4). Adequate luteal function to secrete P4 is crucial for determining the physiological duration of the oestrous cycle and for achieving a successful pregnancy. The bovine CL grows very fast and regresses within a few days at luteolysis. Mechanisms controlling development and secretory function of the bovine CL may involve many factors that are produced both within and outside the CL. Some of these regulators seem to be prostaglandins (PGs), oxytocin, growth and adrenergic factors. Moreover, there is evidence that P4 acts within the CL as an autocrine or paracrine regulator. Each of these factors may act on the CL independently or may modify the actions of others. Although uterine PGF_2α is known to be a principal luteolytic factor, its direct action on the CL is mediated by local factors: cytokines, endothelin-1, nitric oxide. The changes in ovarian blood flow have also been suggested to have some role in regulation of CL development, maintenance and regression.     

Angiogenesis and Interstitial Pressure in a Rat Tumour Model

Introduction and Aim:  The corpus luteum is one of the most intensely vascularized tissues. Luteal angiogenesis is strictly controlled and blood vessels regress completely within a short period of time. The aim of this study was to investigate vascular dynamics in relation to cellular and molecular mechanisms of luteal angiogenesis and anti-angiogenesis.
Material and Methods:  Endothelial cells of blood vessels in paraffin sections of bovine corpora lutea from different stages were examined by labelling with the lectin Bandeiraea simplicifolia agglutinin I. Angiogenesis was studied by morphometry of the capillaries, and immunolocalization of the angiogenic factor VEGF and VEGF-receptor 2. Presence of apoptotic luteal and endothelial cells was investigated using the TUNEL test and transmission electron microscopy.
Results:  During development of the corpus luteum (day 3–8 of the oestrous cycle) a dense capillary network (8–12% area ratio) is established and maintained until day 17. Early regression (day 18–24) is characterized by a remarkable decrease of capillaries (1% area ratio). In the regressing corpus luteum the number of apoptotic luteal cells is closely correlated ( r  = 0.9) to the number of apoptotic endothelial cells. VEGF is immunolocalized in luteal cells (day 3–17), smooth muscle cells and endothelial cells of arterioles of the regressing corpus luteum. During late luteal regression, a moderate increase of capillaries (2.5% area ratio) is obvious.
Conclusions:  The dynamic changes of the capillarity during development and regression of the cyclic corpus luteum correlate with VEGF and VEGF-R2 activities. In contrary to expectations the late stage of luteal regression is accompanied by angiogenesis. One reason for this phenomenon may be an increase in metabolic activity resulting in re-organization of blood vessels already regressed.     

Ultrasonic evaluation of the reproductive tract of the mare: Ovaries

Ultrasonography is useful for monitoring the dynamic follicular and luteal changes of equine ovaries, since it permits rapid, visual, non-invasive access to the reproductive tract. A 5 MHz transducer has greater resolving power and is far more suitable for evaluation of ovaries than a 3–3.5 MHz transducer. Follicles as small as 2–3 mm can be seen and the corpus luteum can usually be identified throughout its functional life. In a study involving daily ovarian examinations, there was a pronounced change in shape of the preovulatory follicle from a roughly spherical to a pear-shaped or oblong form in 66% of the ovulatory periods, This change usually occurred on the day preceding ovulation. The occurrence of ovulation was detectable by the disappearance of a large follicle. In addition, the ovulation site on day 0 was characterized by an intense echogenic area in 88% of 32 ovulations. The developing corpus luteum retained the echogenicity for a mean of 2.4 days. In a blind study, the location of the corpus luteum, as determined by ultrasound, agreed with a previous independent determination of the side of ovulation by palpation in 88% of the 40 bred mares on days 0–14. In all of the 12 mares that were in estrus, the location of the corpus luteum could not be ascertained. In another study, the corpus luteum was identified for a mean of 16 days in 14 estrous cycles. One or more days before the corpus luteum became ultrasonically unidentifiable, it developed increased echogenicity in 36% of the mares, indicating greater tissue density. It is concluded that ultrasonic evaluation of the corpus luteum is superior to digital evaluation by rectal palpation. Some of the potential applications of ultrasonic examination of the ovaries include: 1) obtaining important, sometimes definitive, information by a single examination for judging whether a mare has entered the ovulatory season, 2) aiding in estimating the stage of the estrous cycle, 3) detecting double preovulatory-sized follicles which are in close apposition and difficult to discern by palpation, 4) detecting failure of ovulation or anovulatory estrus by the absence of a corpus luteum, 5) differentiating a persistent corpus luteum from anovulatory or anestrous conditions, 6) diagnosing certain pathological conditions such as peri-ovarian cysts and ovarian tumors, and 7) diagnosing anovulatory hemorrhagic follicles.