Dissertations / Theses on the topic 'Ovarian Follicular Development'

The studies described in this thesis have been concerned with several aspects of follicular development in the mammalian ovary. Chapters 2, 3 6 4 deal with mathematical modelling of ovarian follicle dynamics in normal animals and comparisons with experimentally manipulated animals. Chapter 5 describes a novel aethod for estimating the clonal origin of the mouse ovarian follicle. In the final two chapters, the comparative physiology and anatomy of follicular numbers and sizes and the incidence of polyovular follicles are described for a number of species. The unifying theme of these studies is that they reveal patterns existing in follicular development and utllisation by detailed examination of one species, (CBA/ca mouse), and broadly by interspeci,fic comparisons (with relation to scaling). A detailed mathematical description of the follicular dynamics of virgin CBA/ca mice up to 98 days of age has been obtained by the application of compartmental modelling to differential follicle counts. The rates of follicle growth (migration) and death have been estimated for five ?stages of development (primordial to Graafian). The model predicts age changes in follicle growth and death rate, there being transitions in the parameters at 20 days second at 60 days. The parameters for normal animals have been compared with those of anilllals under two experimental conditions: 1) by unilateral ovariectomy at 4-2 days of age, which abruptly halves the numbers of ovarian follicles and alters the ratio of large : small follicles. 2) by blocking ovulation using progesterone implants. The dynamics of follicle growth were altered by both treatments in comparison with the controls. Follicles at all stages of development were affected by unilateral ovariectomy and differences may exist with time. The compensatory response by the remaining ovary was due to a combination of an increased preantral growth rate and a decrease in atresia at antral stages. Earlier stages of follicle development were affected this may have been incidental to the compensatory response. In progesterone treated animals follicles developed through to antral stages when they un~erwent atresia. The effects of treatment were observed at three levels of development: 1) The initiation of growth from the primordial pool, 2) Growth rate of small follicles and 3) deaths at larger stages of follicular development. Longer term observations indicated that these effects may not be constant. The modelling studies have looked at numerical changes in the follicle population with time but a greater understanding of the develomental biology of the follicle is required in order to explain the changes in growth and death rates observed. This problem has been tackled initially by studying the clonal origin of the follicular epithelium. The technique used is based on the principle that cells in females. are generally mosaic as a result of X-chromosome inactivation the use of X linked cell markers phospho-glycerate kinase-1 (PGK-1). Granulosa cells were found to be polyclonal in origin with the number of progenitor cells numbering 5 on average. Analysis of cumulus and mural granulosa cells showed that substantial cell mixing had occurred and cuaulus cells were generally founded by more than one clone. Finally, comparative studies have been conducted to look at scaling of follicle sizes and numbers and of polyovular follicles. Ovarian follicle and oocyte sizes were scaled according to body weight (ranging from .005-500Kg) using data from 22 species. Primordial and Graafian follicle sizes varied with body weight but closer correlations for the latter were obtained when the sum of the surface areas or volUiles for a preovulatory set were considered as opposed to the values for individual follicles. The numbers of nongrowtng follicles 1n reserve at young adult ages were correlated with maximum longevity of the species and related to body weight. The frequency of polyov~lar follicles varied 1n species studied and were most abundant 1n the domestic bitch. The overall incidence of polyovular folUcles 1n young bitches was 14 S, being reduced to 5~ 1n bitches at 7-11 years. The frequency of the various types of polyovular preantral folUcle varied inversely with the numbers of oocytes per follicle.

Proper understanding of ovarian follicular and luteal development is essential to improve and optimally control reproductive function in domestic animals and to unravel causes of infertility in animals and humans. MicroRNAs (miRNAs) are key post-transcriptional gene regulators in multiple processes involving tissue growth and differentiation. The studies in this thesis were carried out to identify and characterise expression profiles of miRNAs and their potential roles during ovarian follicular development in the cow. The first study aimed to identify expression profiles of miRNAs during antral follicle growth. Follicles were collected from abattoir ovaries and their status (healthy/atretic) was assessed by measuring steroid levels and aromatase expression. An heterologous microarray approach followed by RT-qPCR validation was used to identify and compare miRNA profiles between large (13–16 mm) healthy and small (4–8 mm) follicles. A unique subset of 7 miRNAs (miR-144, miR-202, miR-210, miR-451, miR-652, miR-873 and miR-876) was identified that were enriched in the Large Healthy follicles relative to small follicles and Large Atretic follicles. Further characterisation revealed that many of these miRNAs were highly expressed in the granulosa compartment of the follicle, predominantly in the mural granulosa cells, indicating their potential involvement in regulating granulosa cell function. Expression patterns of miRNAs in the ovarian tissues were generally reflected in the follicular fluid, thus follicular fluid may be used to monitor follicular health. Finally, tissue-wide screening of miRNAs identified miR-202 as a gonad-specific miRNA in the cow. The aim of the second study was to identify potential roles of the miRNAs enriched in Large Healthy follicles. Using in silico approaches, about 1700 bovine genes were identified as the predicted targets of those miRNAs. Putatively targeted signalling pathways are primarily involved in cell survival, proliferation and differentiation. Further, as expected, top predicted gene targets (SPRED1, ATG7, TGFBR2) showed an expression pattern in follicular tissues that was opposite to the patterns of the targeting miRNAs. However, expression patterns of miR-202 or miR-210 during follicular growth could not be recapitulated in gonadotrophin-stimulated cells in vitro and moreover, over-expression or inhibition of miR-202 in these cells did not result in changes in target mRNA levels. The third study investigated the expression profiles of miRNAs during follicle-luteal transition. Microarray analyses identified 9 miRNAs that were upregulated and 14 miRNAs that were downregulated between Large Healthy follicles and early corpus luteum in the cow. Predicted targets of these miRNAs were associated with signalling pathways involved in cell survival, proliferation and differentiation, indicating that these miRNAs might play significant regulatory roles during ovulation and early luteal development. Further, expression of some of these miRNAs and their putative targets during luteinisation in vivo could be recapitulated in cultured granulosa cells providing a system to study the functional roles of these miRNAs during follicle-luteal development. In summary, this is the first study identifying unique subsets of miRNAs associated with follicular development and the follicle-luteal transition in the cow which may be important for female fertility.

Successful ovarian follicular growth and ovulation require controlled and directional proteolytic activity for cell migration and follicular wall rapture. Plasminogen activator (PA) system is known to be involved in tissue remodelling during various physiologic and pathologic processes. PA system comprises two activators (tissue-type, tPA and urokinase-type, uPA), three inhibitors (PAI-1, PAI-2 and protein nexin-I) and one receptor (uPAR). In vivo expression of the PA system during follicular development was studied in the immature female rat. Whereas tPA, PAI and uPAR mRNA expression and protein distribution increased in both granulosa and theca-interstitial layers during follicular maturation and reached maximum levels during the preovulatory period, the opposite was true for uPA. Urokinase PA was highly expressed at early stage of development and markedly decreased prior to the expected time of ovulation. Net secreted PA (PAs) and cell associated PA (PAc) activities were higher in differentiated cells and PAs accounted for 70-80% of the total PA activity in both cell stages of follicular development. Our results suggest that (1) a coordinated expression of ovarian PA system exists during follicular development when granulosa cells undergo transformation from undifferentiated and proliferatively active cells to highly differentiated but mitogenically relatively quiescent ones, and (2) in addition to gonadotropins, several intra-ovarian factors play an important role in the regulation of the ovarian PA system during follicular development. (Abstract shortened by UMI.)

Follicular growth and maturation are tightly regulated processes, which involve the participation of endocrine, autocrineparacrine factors and intracellular molecules. Due to the numerous research efforts, a large number of regulators and their mechanisms of regulation of follicular growth and differentiation have been established. Although the abnormal expression and activities of some of these regulators are believed to be associated with ovarian dysfunction diseases, such as polycystic ovarian syndrome (PCOS), the etiology and pathogenesis of this syndrome are not completely understood. In this thesis, we have identified two novel regulators of follicular growth and differentiation and examined the cellular and molecular mechanisms that contribute to the folliculogenesis. We present here that chemerin reduces FSH-induced steroidogenic enzyme expression and steroid hormone production in follicles and granulosa cells. Prohibitin expression is upregulated by chemerin and knockdown of prohibitin attenuates the suppressive role of chemerin on steroidogenesis, an action regulated by Akt. Using an androgenized rodent model, we also present the dysregulation of chemerin and prohibitin and their association with dysregulated follicular steroidogenesis. Our data and preliminary clinical studies demonstrate the potential involvement of chemerin and prohibitin in the etiology of PCOS. These studies significantly improve the knowledge of ovarian functions and the pathophysiology of PCOS, and provide important clues for the development of novel diagnosis biomarkers and new treatment strategies for this complex syndrome.

Infertility is a major issue in both human and animal medicine with a great economic impact on reproduction. In order to better understand the common causes of infertility it is necessary to understand the basic physiology underlying the complex process of folliculogenesis and luteogenesis (Campbell et al., 2003). The lack of extensive understanding of the factors involved in regulating follicular and oocyte maturation has resulted in the slow process of developing in vitro systems for the study of folliculogenesis (Campbell et al., 2004). However, domestic ruminants represent a valuable in vivo model system for the study of the endocrine and local mechanisms regulating follicular development not only in ruminants but also in humans (Campbell et al., 2003) and have thus been extensively used in reproduction research. Improved understanding of the basic cellular mechanisms of ovarian physiology may improve biotechnology strategies in livestock reproduction and enhance fertility protocols for endangered species. Similarly, a better understanding of these reproductive processes could lead to innovative strategies to improve reproductive health in women.

Luteinising hormone (LH) is a crucial regulator of ovarian follicle maturation, ovulation and luteinisation. Development of healthy follicles and fertile ovulation can only occur within a specific range of circulating LH concentrations, with differing upper and lower limits depending on the stage of the oestrous cycle. The objective of the three studies in this thesis was to investigate the effects of both physiological and non-physiological circulating LH levels on equine follicular maturity by examining ovulatory and steroidogenic capacity, gene expression profiles and miRNA expression in ovulatory-size follicles at various stages of the oestrous cycle and/or in response to supplementation with LH. The aim of the first study was to investigate the hypothesis that deficient circulating LH is a primary cause for the inability of equine follicles to ovulate during the physiological anovulatory season. A LH-rich equine pituitary fraction (eLH) given twice daily to early transitional mares did not restore steroidogenic capacity of the ovulatory-size follicle or advance the onset of the natural breeding season; however, it significantly stimulated follicular growth to a level similar to that occurring during the normal oestrous cycle. The results demonstrated that a deficiency in LH is critically involved in reduced follicle growth during the anovulatory season. The second study examined the effects of elevated circulating LH levels early during follicle development on follicle maturation and ovulatory ability in cycling mares, with the hypothesis that excessive LH would disrupt ovulation and produce haemorrhagic anovulatory follicles (HAFs). Treatment with eLH or a luteolytic dose of prostaglandin F2α (to stimulate an increase in endogenous levels of LH) did not have any effects on follicle growth or ovulation, but did impair follicular production of androstenedione and insulin-like growth factor 1 (IGF1), suggesting a deleterious effect of high LH on follicle and oocyte maturation. The third study examined the expression of different follicular factors associated with follicle maturation as well as microRNAs (miRNAs) in ovulatory-size follicles naturally developing under different LH milieus (oestrus, dioestrus and spring transitional period). Progesterone and IGF1 were significantly reduced in follicles developing in a low LH environment (dioestrus and transition). All four miRNAs measured, miR-378, miR-542, miR-202 and miR-21 were found at higher levels in subordinate follicles than in preovulatory follicles during oestrus. In addition miR- 202 and miR-21 were significantly increased in transitional follicles relative to oestrous follicles. The results of this study indicate that follicles developing during both the spring transitional and dioestrous periods are developmentally immature and suggested potential important roles of miRNAs in follicle maturation in the horse. In summary, although LH is a key factor promoting follicular growth, it is by itself not sufficient to restore steroidogenic activity in transitional follicles. Elevated LH levels during follicle development do not disrupt ovulation, but induce changes in follicular fluid factors related to follicle maturation and oocyte quality. Follicles developing under different LH milieus show altered miRNA expression, suggesting an important role of miRNAs in follicle maturation.

Wnts are secreted extracellular signaling molecules that act locally to control diverse developmental processes such as cell fate specification, cell proliferation, and cell differentiation. Three WNT signaling pathways have been identified. The best characterized is the canonical or WNT/β-catenin signaling pathway. Recently, the WNT signaling pathway has been implicated in ovarian development and differentiation. However, little is known about the expression or role of β-catenin/Tcf-signaling activity within ovarian compartments. In order to aid in the ongoing pursuit of elucidating the mechanisms that govern ovarian differentiation and development, I explored the possible roles of the canonical WNT signaling pathway in the development of the ovarian surface epithelium (OSE) and follicular ovulatory capability. To examine canonical Wnt-signaling within the ovary, I utilized the Tcf-lacZ-reporter mice. In Manuscript I, I present a detailed spatio-temporal pattern of β-catenin/Tcf mediated expression in the OSE throughout development. Cells covering the indifferent gonad at E11.5 were β-catenin/Tcf signaling (lacZ-positive). With further development and sexual differentiation, lacZ staining was lost over the testis but maintained on embryonic ovaries. This staining became dispersed and the proportion of lacZ-positive OSE cells decreased to relative constancy when female mice reached maturity. FACS analyses revealed the lacZ-positive cell population exhibits cytoprotective mechanisms as indicated by enrichment within a side population. The results indicate that the mouse OSE is heterogeneous and may contain a population of progenitor cells. In Manuscript II, I investigated the molecular connection between β-catenin and Tcf-mediated lacZ activity and assessed whether β-catenin stabilization regulates β-catenin/Tcf-mediated gene expression and OSE proliferation. β-catenin was detected on the lateral membranes of ovarian epithelium. I demonstrated that treatment of OSE cells with Wnt agonist stabilized β-catenin but failed to induce β-catenin/Tcf-lacZ expression. Furthermore, E-cadherin expression was down-regulated and the proliferative potency of OSE cells increased. Of four ovarian cancers cell lines screened, only the HEY cell line demonstrated induction of luciferase reporter expression upon canonical WNT stimulation. These studies indicate that nuclear localization of β-catenin is insufficient for β-catenin/Tcf mediated gene expression in OSE cells, suggesting GSK-3β inhibition as a result of altered WNT signaling is of major importance in the control of epithelial-mesenchymal transition and cell proliferation leading to tumorigenesis. In Manuscript III, I investigated the role of the canonical WNT signaling pathway in the development of follicular ovulatory capability. Oocytes in primordial and primary follicles did not show active WNT signaling. β-catenin/Tcf-signaling was activated at the secondary follicle stage and the proportion of β-catenin/Tcf-signaling (lacZ-positive) follicles increased with follicular maturation. In contrast, the majority (>90%) of oocytes recovered from the oviducts at estrus and following hormone stimulation were lacZ-negative. The results indicate that the canonical WNT signaling pathway is active in growing oocytes and suggest that canonical WNT signaling may be involved in the development of follicular ovulatory capability and identifies non-ovulatory follicles.
Les Wnts sont des molécules de signalisation sécrétées dans l'espace extracellulaire pour réagir localement et contrôler divers processus du développement comme la spécification, la prolifération et la différenciation des cellules. Trois voies de signalisation WNTS ont été identifiées. La plus connue est la voie canonique appelée aussi la voie WNT/β-catenin. Récemment des études ont montré que la voie de signalisation WNT serait impliquée dans le développement et la différenciation ovarienne. Malgré ces études, peu est connu sur l'expression et la fonction de l'activité β-catenin/Tcf dans les différents compartiments d'ovaire. Pour clarifier les mécanismes moléculaires responsables dans le développement d'ovaire, j'ai étudié le rôle de la signalisation WNT/β-catenin durant la formation d'épithélium superficiel ovarien et dans la capacité ovulatoire des follicules. Pour visualiser l'implication de la voie canonique dans l'activation ovarienne, nous avons utilisé des souris transgéniques qui expriment la protéine β-galactosidase (lacZ) en réponse des protéines β-catenin/Tcf.Le premier article que je présente (Manuscrit I) décrit l'activation spatiotemporal de β-catenin/Tcf dans l'épithélium superficiel ovarien au cours du développement de la souris. Au jour E11.5, la gonade non-différenciée est marquée positivement pour LacZ indiquant que β-catenin/Tcf est activée. Au cours de la différenciation sexuelle de la souris, l'expression de LacZ disparait dans les testicules. Par contre, dans l'ovaire embryonnaire l'expression de LacZ est maintenue. À la maturation sexuelle de la souris femelle, l'expression de LacZ est devenue plus diffuse et la quantité de cellules qui expriment LacZ a diminué. Une analyse de cytométrie en flux a indiqué que la population de cellules positive pour LacZ démontre des mécanismes cytoprotecteurs. En conclusion, les résultats suggèrent que l'épithélium superficiel ovarien est constitué d'une population de cellules hétérogène et mais aussi de cellules progénitrices. Le deuxième article que je présente (Manuscrit II) étudie la stabilisation de β-catenin, les gènes induit par le complexe de β-catenin/Tcf et la prolifération d'épithélium superficiel ovarien. β-catenin est localisée dans les membranes latérales d'épithélium ovarien. J'ai démontré que la stimulation des cellules d'épithélium superficiel ovarien avec une agoniste Wnt stabilise β-catenin mais n'induit pas l'expression de lacZ dans les souris transgéniques. En plus, le niveau d'expression du gène E-cadherin a baissé et la prolifération des cellules d'épithélium superficiel ovarien a augmenté. Des quatre lignées cellulaires de cellules ovariennes cancéreuses qu'on a examinées, seulement la lignée HEY avait une réponse transcriptionelle à la stimulation de protéines WNT canoniques. Nos études révèlent que la localisation nucléaire de β-catenin n'est pas suffisante pour induire l'expression de ces gènes cibles dans l'épithélium superficiel ovarien. Dans les cellules cancereuses la voie de signalisation WNT est altèrée de sorte que l'inhibition de GSK-3β est augmentée et β-catenin est activée ce que contrôle la prolifération et la transition d'éptihelium à mésenchyme durant la tumorigenèse.Le troisième article que je présente (Manuscrit III) examine le rôle de la voie canonique durant le développement folliculaire. Les ovocytes des follicules primordiaux et primaires ne montraient aucune activation de la signalisation WNT. L'activation de β-catenin/Tcf était présente dans les follicules secondaires et la proportion de follicules positifs pour l'expression de lacZ a augmenté durant la maturation des follicules. Au contraire, la majorité (>90%) d'ovocytes retrouvés dans les oviductes durant l'oestrus et aprés un traitement hormonal n'exprimait pas le gène lacZ. Nos résultats indiquent que la voie de signalisation canonique de WNT est activée durant la croissance des ovocytes et peut identifier les follicules non-ovulatoires.

Recent experimental evidence has suggested that the control of ovarian folliculogenesis and ovulation rate in cattle cannot be accounted for solely through changes in pituitary gonadotrophins, gonadal steroids and/or proteins. The aim of this project was to investigate the role of growth hormone (GH), insulin-like growth factor-I (IGF-I) and insulin in the control of ovarian follicular growth and development in heifers. Daily treatment of heifers with 25mg recombinant bovine somatotropin (BST), for a period of 2 oestrous cycles, significantly increased the population of small ovarian follicles (2-5mm in diameter) although ovulation rate ws unaltered. While peripheral concentrations of GH, IGF-I and insulin were significantly increased, there was no effect of BST on either circulating concentrations of oestradiol, progesterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) or number of FSH and LH binding sites in granulosa and thecal cells. A further study using real-time ultrasound demonstrated that in our population of heifers the majority (9/12) have 3 waves of follicular growth and development per oestrous cycle, whilst the remainder displayed a 2-wave pattern. The growth of each dominant follicle was always associated with a marked reduction in the number and growth of the subordinate follicles. While there was no effect of BST on both the number of follicular waves and the number of large (> 10mm) and medium-sized (5-10mm) follicles, BST treatment significantly increased the number of small follicles throughout the treatment period. However, the inhibitory effect of the dominant follicle on subordinate follicles was not affected by BST treatment. Treatment of heifers with a single dose of BST (320mg in a sustained release formulation) demonstrated that the increase in the number of small follicles was temporally correlated with changes in peripheral IGF-I and insulin concentrations.

The present study was undertaken to examine the effect of ovary type on in vitro production of embryos. Experiment 1 examined the effect of the presence or absence of a corpus luteum and/or large follicle(s) on an ovary, on the development of oocytes to embryos at the blastocyst stage. Experiment 2 investigated whether the ovary type used as the source of granulosa cells in culture (as supplementary cells and for monolayers), affected in vitro production of embryos at the blastocyst stage. The concentrations of progesterone, oestradiol-17B and testosterone in follicular fluid (experiments 1 and 2) "spent" maturation and culture media (experiment 2) in relation to embryonic production were also examined. Ovaries obtained from heifers at presumed random stages of the oestrous cycle were grouped into treatments CL, LF and N as described below: CL:-Ovaries with a corpus luteum at stages II and III of the oestrous cycle (Ireland et al., 1980). Ovaries with follicles>12 mm diameter were excluded. LF:-Ovaries with a large follicle>12 mm diameter (excluding cystic follicles). N:-Ovaries with follicles <12 mm diameter and/or a newly formed or regressing corpus luteum. The number of oocytes matured, inseminated and cleavage rates on a per oocyte recovered basis were not significantly different among the three ovarian treatments, nor for the three monolayer types.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第9018号
農博第1200号
新制||農||823(附属図書館)
学位論文||H13||N3537(農学部図書室)
UT51-2001-F348
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 宮本 元, 教授 今井 裕, 教授 矢野 秀雄
学位規則第4条第1項該当

Alors que le protéome de l'ovocyte isolé, aux stades VG et MII a déjà été étudié, celui du follicule en croissance n'a jamais été décrit.Dans cette étude, nous avons cherché à identifier, comparer et caractériser les profils protéiques de follicules ovariens de souris à trois stades de leur développement in vitro distincts morphologiquement : follicules secondaires en début de culture - stade initial (IS), follicules avec une rupture complète de la membrane de Slavjanski (RMS) et follicules avec une cavité similaire à l'antrum (FA).Après un préfractionnement par IEF et une analyse LC-MS/MS en deux configurations (1D et 2D), 1403 protéines ont pu être identifiées dans le follicule ovarien de souris. 43,4 % (609) des protéines identifiées étaient communes aux trois stades et d'autres ont été identifiées uniquement à un stade : 71 au stade IS, 182 au stade RMS et 193 au stade FA. De plus, on a identifié 365 protéines qui n'avaient pas été décrites antérieurement dans le protéome de l'ovocyte ce qui pourrais indiquer qu'elles sont exprimées dans les cellules somatiques du follicule. Des analyses qualitatives et quantitatives complémentaires ont démontré une surreprésentation pour 44 fonctions biologiques par rapport aux fonctions biologiques des gènes constituant le génome de Mus musculus et mis en évidence des différences d'expression et d'abondance des protéines liées au cycle cellulaire, à la fixation des ions de calcium et à la glycolyse selon le stade de développement. Ces résultats représentent un point de départ pour beaucoup d'autres études de caractérisation moléculaire du développement folliculaire.

MSCAGR (Animal Science)
Department of Animal Science
The main purpose of this study was to assess the efficiency of ultrasonography in monitoring reproductive organs, pregnancy diagnosis, and foetal gender identification and to verify its reliability by laparoscopy and laparotomy, where applicable. Reproductive organs, pregnancy diagnosis and gender of the foetus were examined by A-mode ultrasound using 3.0 - 8.0 MHz trans-rectal transducer. A Sony Olympus Model laparoscope with a camera transducer was used to monitor the reproductive organs and pregnancy diagnosis. In monitoring the follicular dynamics, daily ultrasonography (ULTS) scanning was done for 17 days in sheep and for 21 days in both goats and cattle. Follicles of diameter ≥ 3 mm were selected for analysis of growth, ovulation and regression. For determining the efficiency of the techniques, laparoscopy (LAPSC) and laparotomy (LAPT) were used on days 3 and 10 of the goats and sheep oestrous cycle. The follicles were grouped into three categories according to their diameter as 3 - 4.9 mm, 5 - 7.9 mm and ≥ 8 mm, whereas the follicles of cattle were grouped as 3 - 4.9 mm, 5 - 9.9 mm and ≥ 10 mm. Early pregnancy diagnosis examinations were carried out from day 18 post insemination until pregnancy was confirmed. Foetal gender examinations were conducted from day 40 of pregnancy until the day the gender of the foetus was confirmed. Follicular development was accompanied by the occurrence of waves of follicular growth at different period of the oestrous cycle. The first follicular wave emerged on day 1.0 ± 0.4 in goats, 1.2 ± 0.4 in sheep and 2.2 ± 0.4 in cattle. The maximum diameter of the dominant follicles of observed follicular waves in goats was 7.3 ± 0.4 mm, 6.6 ± 0.2 mm, 7.3 ± 0.2 mm; in sheep was 6.4 ± 0.4 mm, 6.6 ± 0.4 mm and 6.7 ± 0.7 mm and in cattle was 13.1 ± 0.8 mm, 14.2 ± 0.6 mm and 15.7 ± 0.6 mm in wave 1, 2 and 3, respectively. However, the maximum size of the dominant follicle of the ovulatory wave in cattle was larger than the dominant follicles of both first and second waves, but in goats and sheep the dominant follicles were of similar size throughout the waves. In cattle, the ovulatory wave was shorter (p ˂ 0.05) than the duration of the first and second waves, while in sheep and goats were similar throughout the waves. In goats the total number of follicles counted in right and left ovaries under category 3 - 4.9 mm was lower with ULTS and LAPSC than with LAPT method (p ˂ 0.05). In sheep the mean number of follicles between 3 - 4.9 mm category in both right and left ovaries were different (p ˂ 0.05) between ULTS and LAPT. However, for categories 5 - 7.9 mm and ≥ 8 mm in both goats and sheep the mean numbers of follicles observed by all techniques were similar (p ˃ 0.05). In goats, pregnancy diagnosis accuracy improved from zero percent on day 18 to 100% on day 26 - 28, in sheep pregnancy diagnosis was 40% on day 18 and improved to 100% on day 20 - 22 vi of gestation. In cattle accuracy of pregnancy diagnosis was not possible at day 18 and gradually increased to 100% on day 30 - 32 of gestation. Out of 5 (100%) goat’s foetuses whose gender was determined, the diagnosis was correct in 100% (3/3) of the male foetuses and 100% (2/2) of the female foetuses. In sheep two foetuses were sexed as males while the other three were sexed as females and were both 100%. Out of 60% (3/5) of foetuses examined in cattle, 1 (100%) was identified as male and the remaining 2 (100%) were identified as females. The results obtained confirmed that the accuracy for foetal gender by ultrasonography was 100% in all foetuses observed. The current study demonstrated that trans-rectal ultrasonography examination is an efficient method for monitoring follicular dynamics, diagnosing pregnancy and foetal gender identification and that it is as reliable as laparoscopy and laparotomy where they were applied together.
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