Equine Oocyte Competence for Nuclear and Cytoplasmic In Vitro Maturation: Effect of Follicle Size and Hormonal Environment'

Size: px
Start display at page:

Download "Equine Oocyte Competence for Nuclear and Cytoplasmic In Vitro Maturation: Effect of Follicle Size and Hormonal Environment'"

Transcription

1 BIOLOGY OF REPRODUCTION 57, (1997) Equine Oocyte Competence for Nuclear and Cytoplasmic In Vitro Maturation: Effect of Follicle Size and Hormonal Environment' Ghylene Goudet, 2 Jacqueline Bezard, Guy Duchamp, Nadine Gerard, and Eric Palmer I.N.R.A.-Haras Nationaux, Unit6 Reproduction Equine, F Nouzilly, France ABSTRACT Equine oocyte competence after in vitro maturation (IVM) was investigated in terms of the diameter of the follicle of origin and the stage of the estrous cycle, with three criteria of maturation: nuclear stage after DNA Hoechst staining, meiotic spindle morphology after tubulin immunocytochemical staining, and cortical granule localization after lectin labeling. Seven successive in vivo ultrasound-guided follicular punctures were performed on 10 cyclic saddle mares, alternatively at the end of the follicular phase (after induction of ovulation with a gonadotropin injection) and in midluteal phase (with or without a gonadotropin injection). Expanded cumulus-oocyte complexes (COCs) were stained at collection, and compact COCs were stained after in vitro culture. They were observed under a confocal microscope. Successive punctures on one mare provided 0.9 preovulatory COCs and 8 immature COCs per 22 days. Among the preovulatory oocytes, 55% had completed nuclear and cytoplasmic maturation, 86% of which displayed a normal meiotic spindle. Of the 262 oocytes cultured in vitro, 37% completed nuclear maturation. The nuclear and cytoplasmic maturation rate significantly increased with follicle diameter. The IVM rate tended to be higher in follicular phase and tended to increase in luteal phase with the gonadotropin injection. The meiotic spindle morphology was not significantly different between the classes of follicular diameters. This study provided the opportunity to increase the number of characterized oocytes collected per cycle and per mare. This is the first report showing the progressive acquisition of meiotic competence in the equine oocyte during antral follicle growth and is the only description of the equine meiotic spindle. INTRODUCTION In vitro maturation (IVM) and fertilization (IVF) are required methods for the study of the mechanisms involved in oocyte maturation, fertilization, and early embryo development. However, in the mare, such research has been slow to progress. In the horse as compared with other domestic species, IVF is far from being routinely used [1, 2], and the IVM rate is low. Several culture times and media have been evaluated without any great improvement [3-6]. Moreover, in vivo maturation conditions in the mare are different from those in other domestic mammals. The ovulatory LH surge is a progressive increase and decrease lasting many days, with a maximum concentration occurring 1 day after ovulation [7, 8]. The fine structure of the equine oocyte after IVM has not been well described; most studies only evaluate nuclear Accepted March 17, Received November 18, 'This work was supported by grants from the Institut National de la Recherche Agronomique, France, and the Haras Nationaux, France. Ghylene Goudet was supported by a fellowship from the Institut National de la Recherche Agronomique, France, and the Region Centre, France. 2Correspondence: Ghylene Goudet, I.N.R.A.-Haras Nationaux, Unite Reproduction Equine, station P.R.M.D., F Nouzilly, France. FAX: ; goudet@tours.inra.fr 232 maturation using chromatin labeling [3-6, 9]. Baka et al. [10] and Pickering et al. [11] reported meiotic spindle disorganization with normal chromosome morphology in human oocytes. Aged human oocytes [11] and mouse oocytes atypical in meiotic progression [12] often display changes in spindle length or organization. To our knowledge, the equine oocyte meiotic spindle organization has never been described. To obtain fertilizability, a final cytoplasmic maturation must run parallel to nuclear maturation. In mammalian oocytes, the migration of cortical granules (CG) is an important step in cytoplasmic maturation [13, 14], and has been used as a significant criterion in the assessment of maturity and fertilizability [15, 16]. In the horse, Neumann et al. [17] evaluated organelle distribution before and after IVM; however, they worked with a total of 14 oocytes. No other work on equine oocyte cytoplasmic maturation after IVM has been published. The lack of knowledge and studies in the mare can be largely explained by the limited availability of equine oocytes. Collection of oocytes from preovulatory follicles by transvaginal ultrasound-guided puncture yields only oocytes per cycle [18]. Superovulation treatments increase the number of preovulatory follicles; but they have not been very efficient in the mare [19-21], and they damage the follicular environment [18]. Within the female ovary, the numerous immature follicles contain a reserve of immature oocytes. One way of acquiring mature oocytes is through collection of immature oocytes with subsequent IVM. Gametes may be collected from slaughterhouse animals by dissecting the follicles and scraping the follicle wall. Slaughterhouse material has the obvious disadvantages of lack of repeatability, delay in the time between oocyte collection and placement in culture medium, and lack of information about the stage of the cycle and follicular growth. To gain an understanding of the reasons for the low maturation rate in the mare, and to select oocyte populations able to mature in vitro, the in vivo ultrasound-guided transvaginal puncture is an interesting tool for oocyte collection. It allows collection of a well-characterized population of immature and mature oocytes: the follicles are individually punctured, follicular growth can be regularly checked by echography, and one can choose the day of the estrous cycle on which to perform puncture [22, 23]. Moreover, since one can perform repeated punctures on the same mare, the method allows comparison of several populations of oocytes within the same animal [22]. Our study using this tool was designed with two aims: 1) development of an optimal rhythm of successive punctures, adjusted to the follicular growth pattern, to maximize the oocyte yield, and 2) investigation of oocyte competence after IVM in terms of follicle size and stage of the estrous cycle. Fluorescent microscopy was used to examine chromatin, microtubules, and CG as criteria of oocyte maturation.

2 EQUINE OOCYTE COMPETENCE FOR IN VITRO MATURATION 233 MATERIALS AND METHODS Experimental Animals Ten adult cyclic mares (Selle Franqais) in good body condition, from 8 to 17 yr old, were kept indoors and fed with concentrates for use from April through July. Ovarian activity was assessed by routine rectal ultrasound scanning [24] using an Aloka 210 (Socit6 Bernard, Nantes, France) with a 5-MHz linear probe. Blood samples were withdrawn every 2 days. Plasma progesterone concentrations were measured using the RIA method described by Palmer and Jousset [25]. Animal Treatment During the collection procedure, mares were sedated with detomidine (0.8 mg/100 kg BW i.v. Domosedan; Smithkline & French, Courbevoie, France) and butorphanol (1.4 mg/100 kg BW i.v. Torbugesic; Willows Francis Veterinary, Crawley, England), and the rectum was relaxed with prifinium bromide (45 mg/100 kg BW i.v. Prifinial; Vtoquinol, Lure, France). After puncture, the mares received an antibiotic injection (Mixtencilline: IU penicillin/100 kg BW, and 1.3 g dihydrostreptomycin/100 kg BW i.m.; Rh6ne M6rieux, Lyon, France). To induce ovulation, an injection (i.v.) of 25 mg of crude equine gonadotropin (CEG) [26] was given when the largest follicle reached 35 mm. Luteolysis was induced by a prostaglandin F 2. analogue injection (cloprostenol; 250 pig/ mare i.m.; Estrumate, Pitman-Moore, Meaux, France). Follicular Puncture and Oocyte Recovery At the beginning of the experiment, all follicles larger than 5 mm were punctured 34 h after induction of ovulation (i.e. just before ovulation) to render the ovaries free of atretic follicles and allow healthy follicles to develop. For each mare, seven successive ultrasound-guided punctures were performed alternately, three at the end of the follicular phase (group "F") and four during the luteal phase (group "L"). At the end of the follicular phase, all follicles larger than 5 mm were punctured 34 h after induction of ovulation. During the luteal phase, all follicles larger than 5 mm were punctured 34 h after the largest follicle reached 18 mm, i.e., before it reached the dominant stage. Just after the puncture in luteal phase, the corpus luteum was suppressed by a prostaglandin F 2. analogue injection. In order to test the effect of the CEG injection, two punctures in luteal phase were performed after an injection (i.v.) of 25 mg of CEG on the day the largest follicle reached 18 mm (group "L+"), and two punctures in luteal phase were performed without any injection (group "L-"). Five mares were punctured according to the pattern of L+, F, L-, F, L+, F, L- and five according to the pattern of L-, F, L+, F, L-, F, L+. The follicles were punctured using a transvaginal ultrasound-guided follicular aspiration technique [22] with a 7.5-MHz sectorial probe (Kretz; Soframed, Truchtersheim, France). To improve the recovery rate of oocytes, two types of aspiration needles were used [22]. A single-lumen needle (length, 600 mm; outer diameter, 1.8 mm; Thiebaud Freres, Jouvernex Margencel, France) was used for puncturing follicles > 25 mm, and a double-lumen needle (length, 700 mm; outer diameter, 2.3 mm; internal diameter, 1.35 mm; CASMED, Cheam Surrey, England) was used for follicles < 25 mm. After follicular fluid aspiration, the follicle was flushed with PBS (Dulbecco A; Unipath, Dardilly, France) and heparin (50 IU/ml; LEO S.A., St-Quentin Yvelines, France) at 37 C. The single-lumen system allowed five successive flushes with complete filling and emptying. The doublelumen system allowed continuous flushing of the follicle, but aspiration was interrupted five times so that the follicle could fill again. All aspirated fluids were individually examined for oocyte recovery. The follicular fluid from follicles larger than 30 mm was centrifuged (15 min at 2000 x g), and the supernatants were stored at C until assayed. Oocyte Culture and Fixation At recovery, oocytes were individually classified and processed according to cumulus aspect (compact or expanded). Compact cumulus-oocyte complexes (COCs) were cultured individually in a humidified atmosphere (95% air:5% CO 2 ) at C for 30 h in 500 jil of maturation medium: Medium 199 with Earle's salts, 2.2 g/l NaHCO 3, and L-glutamine (Gibco, Eragny, France) supplemented with 20% inactivated fetal calf serum (FCS; Gibco), antibiotics (100 IU/ml penicillin, 100 IU/ml streptomycin, and 0.25 RIg/ml fungizone; Gibco), CEG (9.5 jig/ml equine FSH and 15 jig/ml equine LH) [26], and estradiol-17 (1 jig/ml; Sigma, La Verpillere, France). After culture, the COCs were stripped with small glass pipettes in 500 jil of PBS solution supplemented with 525 IU/ml hyaluronidase (type III, 875 IU/mg; Sigma) at 37C. Totally denuded oocytes were rinsed in PBS with 1% FCS at 37 C, fixed for 20 min in 2.5% paraformaldehyde in PBS at 37 C, and rinsed again. The oocyte diameter was then measured. Oocytes were kept at 4 C in PBS containing 0.05% NaN 3 (sodium azide; Prolabo, Paris, France) and 1 mm PMSF (Serva, Heidelberg, Germany). Expanded COCs at recovery were stripped of their cumulus cells, fixed, and kept as just described for the compact COCs. Immunocytochemical Staining and Lectin Labeling The oocytes were permeabilized in Triton X-100, 0.1% in PBS, for 5 min at room temperature and washed in PBS containing 0.05% NaN 3 and 1 mm PMSE Microtubule localization was performed according to the following immunofluorescence protocol. 1) Nonspecific reactions were blocked by preincubation of oocytes for 2 h at room temperature in an incubation solution: PBS containing 2% BSA (A-7030; Sigma), 0.05% NaN 3, 0.05% saponin (Sigma), and 10% goat serum. 2) Incubation was conducted for one night at 4C with a mouse monoclonal anti-oa tubulin TU-01 antibody diluted 1:300 in incubation solution; this antibody was produced by Dr. V. Viklicky (Institute of Molecular Genetics, Praha, Czech Republic) [27]. The oocytes were then rinsed in a washing solution: PBS with 0.2% BSA, 0.05% NaN 3, and 0.05% saponin. 3) Incubation was performed for 1 h at room temperature with tetramethylrhodamine isothiocyanate-conjugated goat antimouse IgG (Biosys, Compiegne, France) diluted 1:150 in the incubation solution, followed by rinsing. Oocytes were then incubated for 30 min at room temperature in 100 jig/ml of fluorescein isothiocyanate-conjugated peanut agglutinin in washing solution to detect distribution of CG. After rinsing, oocytes were stained with 1 ig/ml bis-benzimide (Hoechst 33342; Sigma) in PBS for 6 min for DNA detection. They were then mounted between

3 234 GOUDET ET AL. TABLE 1. Oocyte recovery rate from nonpreovulatory follicles (< 35 mm) according to group.* Groups Luteal Follicular phase phase Item Luteal phase + CEG + CEG Follicular aspiration attempts (n) Punctured follicles (n) Recovered COCs (n) COCs per follicle COCs per attempt * The recovery rate per follicle was not significantly different between the 3 groups. slide and cover slide in a mixture of Mowiol V4-88 (133 mg/ml; Hoechst, Frankfurt, Germany) and n-propyl gallate (5 mg/ml; Sigma). The slides were kept at 4 0 C in darkness until observation. Controls for immunofluorescence were performed using no primary antibody, and those for lectin labeling were performed by means of a previous incubation with 100 mm D-galactose in PBS. Confocal Laser Scanning Microscopy The oocytes were observed under a confocal laser scanning microscope (CLSM 310; Carl Zeiss, Thornwood, NY). Immunolabeling and lectin labeling were visualized in the confocal mode, whereas Hoechst staining was detected by conventional epifluorescence. For fluorescein, an argon ion laser adjusted at 488-nm wave length was used; for rhodamine, a helium-neon ion laser adjusted at 543 nm was used. The size of the meiotic spindles was measured along the longitudinal and the lateral axis, respectively, with Visilog software (version V4.1.4; Neosis, Orsay, France). Each measure was performed twice, with an error between the measures of 0.6 pxm. Follicular Fluid Endocrinology Steroid concentrations in the follicular fluid were determined by RIA without extraction according to the methods of Saumande et al. [28] for progesterone, of Terqui et al. [29] for estradiol-17[, and of Hochereau de Reviers et al. [30] for testosterone. Intraassay variability and limits of sensitivity were, respectively, 8.1% and 0.10 ng/ml for progesterone, 6.3% and ng/ml for estradiol-1713, and 8.1% and 0.10 ng/ml for testosterone. For each steroid, all samples were analyzed in the same assay to avoid interassay variability. Statistical Analysis The chi-square test was used to compare oocyte recovery and maturation rate in the three groups (F, L+, and L-). Analysis of oocyte maturation after IVM according to follicle diameter was performed by logistical regression analysis. Nonparametric test (G-test = 21-test) was used for comparison of oocyte recovery, nuclear maturation, meiotic spindle morphology, and CG localization between the various classes of follicle diameters. Variance analysis was used to compare oocyte spindle sizes and oocyte diameters. Comparison of steroid concentrations in different follicle groups was performed by a nonparametric test (Kolmo 2). TABLE 2. Oocyte recovery rate from nonpreovulatory follicles (< 35 mm) according to follicular diameter.* Follicular diameter (mm) Item 5to9 10to14 15to19 20to24 25to29 30to34 Punctured follicles (n) Recovered COCs (n) COCs per follicle * The recovery rate per follicle was not significantly different between the follicular diameters. RESULTS Oocyte Recovery In the 10 mares, 708 follicles larger than 5 mm were punctured, alternatively at the end of the follicular phase (3 attempts per mare) and in midluteal phase (4 attempts per mare: 2 attempts with CEG injection and 2 attempts without). At the end of the follicular phase, preovulatory (larger than 35 mm in diameter) and nonpreovulatory follicles were punctured. In the midluteal phase, all punctured follicles were nonpreovulatory; two follicles were larger than 35 mm, however, but these were luteinized follicles with echogenic walls and irregular shape, and filled with fluid just after puncture. Preovulatory follicles (larger than 35 mm). From the 30 attempts in follicular phase, 36 preovulatory follicles were flushed and 28 oocytes were collected. Seventeen preovulatory follicles could not be flushed because they ovulated before puncture. Averages of 0.9 oocytes per attempt and 0.78 oocytes per preovulatory follicle were obtained. Nonpreovulatory follicles. From the 70 puncture attempts (30 in follicular phase, 20 in luteal phase with CEG injection, and 20 in luteal phase without CEG injection), 672 nonpreovulatory follicles were flushed and 276 COCs were recovered. Averages of 4 COCs per attempt and 0.41 COCs per follicle were obtained. The recovery rate per follicle was not significantly different between the three groups (Table 1). It tended to decrease with increasing diameter of aspirated follicles (Table 2). The COC recovery rate per mare ranged from 32.6% (29 of 89) to 60.0% (33 of 55). There was a significant effect of mare on the COC recovery rate (p < 0.05). Oocyte yield per cycle. The interval between attempts was days after puncturing in follicular phase, days after puncturing in luteal phase without CEG injection, and days after puncturing in luteal phase with CEG injection. These intervals were not significantly different. The mean interval between 2 attempts in the same cycle stage was 22.1 days, the duration of a cycle. Puncturing once in follicular phase and once in luteal phase yielded a mean of 8.9 COCs per 22 days. Moreover, the mean number of punctured follicles and the mean number of collected oocytes in each group showed no increase or decrease during the successive punctures. Plasma progesterone levels. Puncture of the preovulatory follicle was always followed by a rapid increase in plasma progesterone, secreted by the resulting corpus luteum (data not shown). The maximum level was ng/ml. The corpus luteum was suppressed by the prostaglandin injection on the luteal-phase puncture day. In one mare, the increase of progesterone did not occur until 11 days after the puncture of the preovulatory follicle, and the maximum concentration was 2.6 ng/ml.

4 EQUINE OOCYTE COMPETENCE FOR IN VITRO MATURATION 235 FIG. 1. Equine COCs. A) Oocyte with expanded cumulus at collection; x1 7. B) Oocyte with compact corona radiata at collection; x 170. C) Oocyte with compact cumulus and cells from follicular wall at collection; x170. D) Denuded and degenerated oocyte without a cytoplasmic membrane; x255. During the luteal phase, the follicles were punctured 34 h after the largest follicle reached 18 mm. However, as the follicles do not grow at a constant rate, some punctured follicles were larger than expected. Nine nonpreovulatory follicles punctured in luteal phase led to plasma progesterone concentrations greater than 2 ng/ml. This was the case for 2 of I follicles mm (18%), 5 of 11 follicles mm (45%), and 2 of 2 follicles larger than 29 mm (100%). On average, the largest follicle diameter was mm in puncture attempts during the luteal phase without CEG injection and mm in puncture attempts during the luteal phase with CEG injection. Progesterone concentrations were greater than 2 ng/ml after 3 of 15 punctures in luteal phase without CEG injection and after 6 of 15 punctures in luteal phase with CEG injection; these rates were not different. Cumulus Aspect at Recovery At collection, the 304 recovered COCs were classified into three groups: expanded cumulus (Fig. A), compact corona radiata (CR) (Fig. B), and compact cumulus and cells from the follicular wall (W) (Fig. 1C). Expanded cumulus. In follicular phase, 138 COCs were collected. From preovulatory follicles (larger than 34 mm), 24 of 28 recovered COCs (86%) had an expanded cumulus. In follicles with diameters from 30 to 34 mm, diameters from 25 to 29 mm, and diameters 24 mm and smaller, 4 of 4, 4 of 9, and 6 of 97 COCs, respectively, had an expanded cumulus. In luteal phase, 166 COCs were obtained and only 4 were expanded. Compact cumulus. Of the 70 puncture attempts made in luteal and follicular phases, 55 CR (21%) and 207 W (79%) were recovered. No significant difference in the portion of

5 236 GOUDET ET AL. FIG. 2. Chromatin configurations in equine oocytes. A) Germinal vesicle with diplotene chromatin; B) dense chromatin; C) metaphase I; D) metaphase 11 (m) and polar body (gp). x400. COCs in classes CR and W was observed according to group Evaluation of Oocytes from Expanded COCs (respectively, 19% and 81% in follicular phase, 20% and 80% in luteal phase with CEG, 24% and 76% in luteal phase Chromatin configuration. Of the 42 expanded COCs colwithout CEG). No clear relationship between follicle size lected, 3 were lost during staining and 39 were analyzed for and compact cumulus aspect (CR or W) could be estab- nuclear maturation. Of these, 14 were degenerated (Fig. 1D), lished. 2 were immature (Fig. 2A), 9 had resumed meiosis (Fig. 2,

6 EQUINE OOCYTE COMPETENCE FOR IN VITRO MATURATION 237 FIG. 4. CG localization on serial optical sections in equine oocytes. A) CG migration achieved: the majority of the CG are lining the oolemma. B) No CG migration: the CG are located in the medullary zone, and no CG are lining the oolemma; x250. B and C), and 14 were mature (Fig. 2D). The chromatin configuration in relation to follicular size is presented in Table 3. In follicles 30 mm and larger, 52% of expanded COCs (2/4 + 12/23) enclosed a mature oocyte (metaphase II or telophase I). No oocyte from follicles 29 mm and smaller were mature. In total, from the 30 attempts in follicular phase, 14 mature oocytes with expanded cumulus were recovered, giving an average of 0.5 mature oocytes per attempt. Meiotic spindle morphology. The 14 mature oocytes (telophase I or metaphase II) were examined for meiotic

7 238 GOUDET ET AL. FIG. 5. Influence of diameter of the follicle of origin upon the chromatin configurations of equine oocytes after IVM. Mature: telophase I or metaphase II; resumption of meiosis: oocytes with dense chromatin or metaphase I; immature: germinal vesicle with diplotene chromatin; degenerated: oocytes without a cytoplasmic membrane or fragmented. The maturation rate significantly increased with the follicle diameter (p < 0.1%). The maturation rate in diameter class 5-9 mm was significantly less than the maturation rate in the other classes (p < 1%). spindle morphology. Eight oocytes (57%) had a barrelshaped spindle with two poles and distinct microtubules between poles; the chromosomes were aligned along the metaphase plate. This was defined as a normal spindle (Fig. 3A). One oocyte was in telophase I (Fig. 3B), with the two sets of chromosomes arranged at the two spindle poles. Three oocytes had a barrel-shaped spindle with two poles and disorganized microtubules between poles; the chromosomes were arranged into the meiotic spindle. This was defined as a disorganized spindle (Fig. 3C). Two oocytes showed a dense network but no poles and no distinct microtubules; the chromosomes remained on this network. This was defined as scattered tubulin (Fig. 3D). The 5 metaphase I oocytes from expanded COCs had a normal spindle. CG localization. Of the 14 mature oocytes (metaphase II or telophase I), 11 were analyzed for CG localization; 3 were not analyzed due to technical problems. CG migration was achieved or in progress in 11 of 11 oocytes: in 10 of the oocytes, most of the CG were lining the oolemma (Fig. 4A); in 1 of them, the CG had an uniform distribution between the oolemma and the medullary zone. In none of the oocytes was all the CG located in the medullary zone with no CG lining the oolemma (see description of CG localization after IVM, below; Fig. 4B). Ability of Compact COCs to Mature In Vitro Cumulus aspect after IVM. Oocytes with compact cumulus at recovery were cultured for 30 h. After IVM, 95% of the COCs had an expanded cumulus (249 of 262), and 5% remained compact. Meiotic maturation. From the 70 puncture attempts in luteal and follicular phase, 262 compact COCs were col- TABLE 3. Chromatin configuration of expanded COCs according to follicular diameter.* Follicular diameter (mm) Chromatin configuration' 5 to to to to 50 Mature (n) Resumption of meiosis (n) Immature (n) Degenerated (n) Total (n) * No statistical analysis was performed because of the small number of oocytes. ' Mature: telophase I or metaphase II; resumption of meiosis: oocytes with dense chromatin or metaphase I; immature: germinal vesicle with diplotene chromatin; degenerated: oocytes without a cytoplasmic membrane or fragmented. FIG. 6. Influence of diameter of the follicle of origin upon the chromatin configurations of equine oocytes after IVM, according to the hormonal environment. Mature: telophase I or metaphase II; resumption of meiosis: oocytes with dense chromatin or metaphase I; immature: germinal vesicle with diplotene chromatin; degenerated: oocytes without a cytoplasmic membrane or fragmented. Regardless of group, the nuclear maturation rate increased with the follicle diameter (p < 5%).

8 EQUINE OOCYTE COMPETENCE FOR IN VITRO MATURATION 239 FIG. 7. Influence of diameter of the follicle of origin on metaphase II and telophase I spindle morphology after IVM. The spindle morphology was not significantly different between the classes of follicle diameters. lected and cultured in vitro; 258 of these were analyzed for nuclear maturation after IVM and 4 were lost. Their meiotic stages according to diameter of the follicle of origin are shown in Figure 5. The maturation rate significantly increased with an increase in follicular diameter (p < 0.001). In follicles 5-9 mm, 20% (17 of 83) of the oocytes reached metaphase II or telophase I, whereas in follicles mm, 45% (78 of 175) of the oocytes were mature; these two rates were significantly different (p < 0.01). The maturation rate did not vary significantly with the cumulus aspect before IVM: in class CR, 17 of 55 (31%) oocytes reached metaphase II or telophase I vs. 78 of 203 (38%) in class W. Of the 12 oocytes whose cumulus remained compact after IVM, 3 (25%) were in metaphase II. This maturation rate was not significantly different from that of oocytes whose cumulus was expanded after IVM (37%, 92 of 246). The maturation rate did not vary significantly between the different mares. Figure 6 shows the meiotic stage after IVM according to diameter of the follicle of origin and group (in follicular phase, in luteal phase with CEG injection, and in luteal phase without CEG injection). The results for follicle diameter classes from 20 to 50 mm were combined because of the small number of oocytes collected. Regardless of the group, the nuclear maturation rate increased with the follicular diameter (p < 0.05), and the maturation rate of oocytes from 5-9-mm follicles was less than that for the other follicles (not significant for follicular phase and for luteal phase without CEG; p < 0.05 for luteal phase with CEG). The maturation rate tended to be different among the three groups. Only 28% (25 of 90) of the oocytes recovered in luteal phase without CEG injection were able to mature in vitro. The CEG injection in luteal phase increased the maturation rate (38%, 27 of 71), especially for oocytes from follicles 10 mm and larger. The maturation rate of oocytes recovered in follicular phase with CEG injection was the highest (44%, 43 of 97). Nevertheless the difference between these three maturation rates was not significant. Oocytes with dense chromatin and oocytes in metaphase I, telophase I, or metaphase II resumed meiosis. Within all three groups, the rate of oocytes able to resume meiosis significantly increased with the follicle diameter (p < 0.001), and this rate was significantly lower in follicle diameter class 5-9 mm than in classes mm (p < 0.01). Meiotic spindle morphology. After IVM, 95 oocytes were in metaphase II and telophase I, of which 93 were examined for meiotic spindle morphology. As outlined above, we defined spindles as normal spindles, telophase I spindles, disorganized spindles, and scattered tubulin (Fig. 3, A-D, respectively). After IVM, some oocytes had a multipolar spindle (Fig. 3E). The meiotic spindle morphology was analyzed in terms of the diameter of the follicle of origin (Fig. 7). In follicles smaller than 10 mm, 14 of 16 oocytes had a normal metaphase II or telophase I spindle (88%). In follicles 10 mm or larger, only 55 of 77 oocytes had a normal spindle (71%). Oocytes from follicles 5-9 mm tended to be different from oocytes from follicles mm, but the difference was not significant. The oocyte number was too low to allow any analysis according to group. After IVM, 65 oocytes were in metaphase I. Among them, 41 had a normal metaphase spindle (63%). The meiotic spindle morphology was not significantly different between the classes of follicle diameters (Fig. 8). CG localization. Of the 258 oocytes analyzed for nuclear maturation after IVM, 196 were examined for CG localization in the ooplasm in relation to follicular size (Fig. 9). In 68% (40 of 59) of the analyzed oocytes recovered from follicles smaller than 10 mm, CG migration was achieved or in progress after IVM. In follicles 10 mm or larger, 89% (122 of 137) of the analyzed oocytes were in this state. These two follicular populations were significantly different (p < 0.01). CG localization after IVM according to nuclear stage is presented in Figure 10. CG migration was achieved or in progress after IVM in 95% (77 of 81) of mature oocytes, FIG. 8. Influence of diameter of the follicle of origin upon metaphase I spindle morphology after IVM. The spindle morphology was not significantly different between the classes of follicle diameters.

9 240 GOUDET ET AL. FIG. 9. Influence of diameter of the follicle of origin on CG localization after IVM. The percentage of oocytes with CG migration achieved or in progress in diameter class 5-9 mm was significantly different from the percentage in the other classes (p < 1%). in 89% (58 of 65) of oocytes showing resumption of meiosis, and in 54% (26 of 48) of immature oocytes. The oocyte number was too low to allow any analysis according to group. Meiotic Spindle Size Of the COCs analyzed at recovery, i.e., after in vivo maturation, 8 of 14 (57%) metaphase II and telophase I oocytes and 4 of 5 (80%) metaphase I oocytes had a normal spindle. Of the COCs cultured in vitro, 54 of 93 (58%) metaphase II and telophase I oocytes and 41 of 65 (63%) metaphase I oocytes had a normal spindle after IVM. Among these 107 oocytes, 2 were lost and only 105 were analyzed for spindle width and length (8, 3, 53, and 41 oocytes, respectively). Collectively, COCs analyzed after in vivo maturation and COCs cultured in vitro demonstrated metaphase I spindles that were significantly wider than metaphase II spindles ( tlm vs pxm, respectively, p < 0.001). Also, when all metaphase I and metaphase II oocytes were grouped, spindles from oocytes cultured in vitro were significantly wider and longer than spindles from oocytes analyzed after in vivo maturation (width: 17.9 ± 5.1 Lm vs pxm, respectively, p < 0.001; length: pxm vs pxm, respectively, p < 0.001). FIG. 10. Influence of nuclear stage on the CG localization after IVM. M II: metaphase II; T I: telophase I; M : metaphase I; D Ch: dense chromatin; GV: germinal vesicle with diplotene chromatin. Oocyte Diameter The oocyte diameter was measured in COCs analyzed after in vivo maturation and in COCs analyzed after IVM (Table 4). The oocytes from COCs analyzed after in vivo maturation were significantly smaller than those from COCs analyzed after IVM (p < 0.01). Among the COCs cultured in vitro, no clear relationship between diameter of the follicle of origin and oocyte diameter could be established. Overall oocyte diameter decreased as nuclear maturation progressed: the oocytes that remained in germinal vesicle stage were larger than the oocytes that resumed meiosis (p < 0.05), and the metaphase I oocytes were larger than the metaphase II oocytes (p < 0.001). Follicular Fluid Endocrinology The concentrations of steroids were measured in follicular fluid from follicles larger than 30 mm. The results ranged as follows: progesterone, ng/ml with a mean of 900 ng/ml; estradiol, ng/ml with a mean of 1575 ng/ml; testosterone, ng/ml with a mean of 14 ng/ml. No relationship was found between steroid concentrations and oocyte recovery rate or between steroid concentrations and oocyte nuclear maturation. DISCUSSION The first aim of this study was to find an optimal rhythm of successive punctures, adjusted to the follicular growth pattern, in order to maximize the oocyte yield. Early attempts at in vivo collection of equine oocytes were made via standing flank transcutaneous puncture [31-33]. In these studies, ovaries were aspirated only during estrus, and only the preovulatory follicle was punctured. The procedure did not allow visualization of tissues, and therefore aspirations were conducted blindly. Recovery rates were approximately 65%. Recently, efforts have been made to develop transvaginal ultrasound-guided follicular puncture in the mare [34]. Using this technique, recovery of oocytes from preovulatory follicles after induction of ovulation has been 50-85% [18, 35]. In this study, we performed transvaginal ultrasound-guided punctures at the end of the follicular phase after induction of ovulation. The recovery rate for oocytes from preovulatory follicles was 78%, a rate similar to previous results [18, 35], but the yield was only 0.9 preovulatory oocytes per cycle. The puncture of small follicles was first proposed in the cow [36] and

10 EQUINE OOCYTE COMPETENCE FOR IN VITRO MATURATION 241 TABLE 4. Oocyte diameter (m) according to nuclear stage and maturation conditions (mean + SD).* Nuclear stage t Maturation M II T I M I D Ch GV Deg Total In vitro (80) (15) (65) (6) (63) (9) d In vivo 110 ± ± ± ± e (13) (1) (5) (4) (2) (2) Total 114 7a ab 118 8b ab 123 ± 7c ab * Statistical analysis was performed on total values; sample size in parentheses. M II: metaphase II; T I: telophase I; M I: metaphase I; D Ch: dense chromatin; GV: germinal vesicle with diplotene chromatin; Deg: degenerated. abc Values with different superscripts differ significantly (p < 0.05). de Values with different superscripts differ significantly (p < 0.01). was adapted to the mare by Cook et al. [35]. Several studies were then conducted to further develop this method and investigate the influence of various parameters (different vacuums, single- or double-lumen needles with various diameters) [22, 23, 35, 37]. Recovery rates displayed high variability, ranging from as low as 12% [23, 37] to as high as 47% [38]. The technique used in this study was established in our laboratory by Duchamp et al. [22], who obtained an average of 0.29 COCs per nonpreovulatory follicle. In the present study, 0.41 COCs per nonpreovulatory follicles were collected. This higher yield is probably due to the increased experience of the technicians. The recovery rate in nonpreovulatory follicles tended to decrease with increasing follicle diameter, a finding that agrees with several previous studies [22, 23, 35]. This may be the case because it is easier to scrape the whole follicular wall when the surface is smaller. The recovery rate from preovulatory follicles was higher than from nonpreovulatory follicles because of the looser connections between the oocyte and the follicular wall during cumulus expansion caused by administration of exogenous gonadotropins. According to our results, exogenous hormones and the hormonal environment had no effect on nonpreovulatory follicles, as the recovery rate was not significantly influenced by the CEG injection or by the cycle stage. Additionally, the recovery rates for immature oocytes from mares are generally lower than those obtained in cattle, which range from 55% [36] to 70% [39]. Hawley et al. [40] reported that the cumulus of equine oocytes has a broader base and closer attachment to the follicle wall than that of bovine oocytes, and that unique cumulus cell projections into the thecal pad anchor the equine oocyte to the follicle wall. To improve the oocyte recovery potential for cyclic mares, several patterns of successives punctures have been tested. In 1993, Cook et al. [35] performed aspirations during estrus and diestrus on light-horse mares. An average of 1.5 oocytes were recovered per cycle. Two years later, Duchamp et al. [22] collected 3 oocytes from saddle mares and 1.8 oocytes from pony mares per 21 days with weekly punctures of all follicles larger than 8 mm. But they obtained irregular responses with alternating follicular growth in terms of follicle number and follicle size. Punctures on any occasion when a follicle had reached 15 mm allowed an average collection of 10 oocytes per 21 days [22]. However, some follicles had an irregular shape and echogenic dots, 27% of oocytes had an expanded cumulus, half of which had resumed meiosis. In our study, the puncture protocol was determined by follicular size rather than by day interval, and aspirations were performed during estrus and diestrus on all follicles larger than 5 mm in diameter; smaller follicles were too difficult to puncture. This puncture protocol improved the recovery rate to an average of 8.9 COCs per 22 days. This rhythm is adjusted to the follicular growth pattern so that we obtain well-characterized populations of oocytes. The puncture of preovulatory follicles results in an active corpus luteum [41]. The delay in the increase of progesterone in one mare may have been due to damage in the granulosa cells. As previously described [22], luteinization occurred after puncture in some follicles between 22 and 27 mm. The occurrence of luteinization was not increased by exogenous gonadotropins. Little information on steroid concentrations in equine preovulatory follicles has been published. Our results are similar to previous data on preovulatory follicles from pony mares [18]. The great variability between follicles and the low number of follicles did not allow us to find any relationship between steroid concentrations and oocyte recovery rate or oocyte nuclear maturation. At collection, the oocytes were surrounded either by an expanded cumulus, by a compact corona radiata, or by a compact cumulus attached to cells from the follicular wall. As in many mammals, induction of ovulation in the mare leads to cumulus expansion in the dominant follicle [1, 18]. In preovulatory follicles as well as in follicles with diameters from 30 to 34 mm, most recovered COCs had an expanded cumulus, in agreement with findings of Duchamp et al. [26]. Therefore, it appears that CEG injection caused follicular maturation and cumulus expansion in follicles whose diameter is around 30 mm or larger. In our study, only 4 follicles from 35 to 38 mm contained COCs with a compact cumulus. They were all contemporaneous with a preovulatory follicle that had ovulated or that contained a mature oocyte. Thus, they may not have been dominant follicles and, in spite of their large size, they may not be responsive to the exogenous gonadotropins. From 27 expanded COCs (follicles 30 mm and larger), 14 mature oocytes were collected (Table 3). Moreover, 17 preovulatory follicles ovulated before puncture. One can speculate that these follicles enclosed mature oocytes with expanded cumulus complexes. With these taken into account, 70% of expanded COCs enclosed a mature oocyte ( / ). This result is low compared to findings in pony mares [18, 42] and could be explained by the fact that more spontaneous multiple large follicles occur in large mares than in pony mares [43]. The unique large follicle in pony mares is undoubtedly the follicle that will ovulate and that contains a mature oocyte, whereas in large mares, not all the large follicles are intended for ovulation. They may grow, and cumulus expansion may occur, but the oocytes may be unable to complete maturation and ultimately will degenerate. In this study, 13 nonmature or degenerated oocytes surrounded by an expanded cumulus were collected from follicles 30 mm and larger. Of these,

11 242 GOUDET ET AL. 9 were contemporaneous with a large follicle that either ovulated or contained a mature oocyte, or with a follicle that was larger than 35 mm but whose oocyte could not be recovered. One of them came from a luteinized follicle. Only 3 oocytes were collected from a unique preovulatory follicle and were degenerated with no apparent reason. In humans, there have been reports of oocytes with an abnormal spindle but normal, compact chromosomes [44, 45]. Therefore, the analysis of nuclear stage by DNA staining does not allow detection of alterations in the spindle that may contribute to induction of aberrant embryonic development [46, 47]. Research on spindle structure may then be of considerable interest. The cytoskeletal organization of the oocyte has been described in many species and seems to be common in most mammals. However, to our knowledge, no description has been reported in the equine species. As a first step, we analyzed meiotic spindle morphology in the 14 metaphase II and telophase I oocytes collected with an expanded cumulus from follicles 30 mm and larger. Eight oocytes had a barrel-shaped spindle with two poles and distinct microtubules between poles, defined as a normal spindle. This equine spindle is similar to the normal spindle described in Xenopus [48], mouse [49, 50], human [45, 51], bovine [52], and porcine [53] oocytes. One oocyte was in telophase I with a spindle structure typical of that of Xenopus [48], mouse [49, 50], and porcine [53] oocytes. Three oocytes had a barrel-shaped spindle with two poles and disorganized microtubules between poles. This structure may be a normal step in microtubule dynamics during equine meiotic progression, as it looks like the prometaphase spindle described in Xenopus [48], mouse [50], and porcine [53] oocytes. However, one must be careful with this interpretation, as Pickering et al. [11, 51] obtained barrel-shaped oocytes with internal disorganization of spindle microtubules in aged or cooled oocytes. This disorganization was nevertheless often associated with chromosome dispersion, which was not observed in the present study. Among the 14 oocytes we analyzed, 2 had scattered tubulin. This is undoubtedly a sign of spindle degeneration, because no interphase network of microtubules is ever formed during the normal transition from metaphase I to metaphase II [48, 49] and such a network was observed in aged human oocytes [54] and in cooled human [45, 51] and bovine [52] oocytes. The presence of an abnormal spindle in in vivomatured oocytes has been previously reported in the human [11, 45]. Among the 14 metaphase II and telophase I oocytes collected with an expanded cumulus from follicles 30 mm and larger, 11 could be analyzed for CG localization as a criterion of cytoplasmic maturation. All apparently progressed through a normal maturation, as Bezard et al. [18] showed that in equine preovulatory oocytes collected just before ovulation, CG line the oolemma or lie in a homogeneous distribution in the ooplasm. In follicles smaller than 30 mm, 14 of 106 COCs had an expanded cumulus. Comparison of this rate with findings in other reports is not reliable, as the definition of an expanded cumulus is highly variable-from a corona radiata with partially dissociated cumulus [23, 55] to a gelatinous mass providing structural support to detached cumulus cells ([56], this report). Hinrichs [56] recovered most of the expanded COCs in atretic follicles. In the present study, 6 of 9 of the expanded COCs that were collected from follicles smaller than 25 mm contained a degenerated oocyte; therefore the follicle of origin may be atretic. Of the expanded COCs collected in follicles of 25- to 29-mm diameter in follicular phase, 3 of 3 were in metaphase I. The expansion of cumulus cells and resumption of meiosis may be due to the appearance of LH receptors sensitive to the exogenous LH. Of the 70 puncture attempts performed in luteal and follicular phases, 55 CR (21%) and 207 W (79%) were recovered. No significant difference in the proportion of COCs in classes CR and W was observed relative to the three groups or the follicular diameter. Comparison with cumulus morphology as reported by others is difficult-first because the classification systems used to characterize the cumulus are highly variable, and second because the recovery methods are different and some may damage the COCs. However, in most reports, the morphology of compact cumulus did not depend on follicle size up to 30 mm [55, 56]. COCs with compact cumulus at recovery were cultured for 30 h in the present study. After IVM, most COCs had an expanded cumulus. However, as previously reported [3, 6, 57], some COCs remained compact. The degree of cumulus expansion did not necessarily correlate with nuclear maturation, as the maturation rate of oocytes whose cumulus remained compact after IVM was not significantly different from that of oocytes whose cumulus became expanded during IVM. However, these two conditions are necessary, as Testart et al. [58] found that a higher proportion of human oocytes were able to cleave following IVF when cumulus expansion was complete. In this study, the IVM rate was about 37%. In several previous investigations, the maturation rate of equine oocytes reached 70% [6, 57, 59]. Our low maturation rate can be explained by the conditions of IVM and by the origin of the oocytes. The culture medium we used was developed in this laboratory [5] and seems to be quite well adapted to equine oocytes, as the positive effect of Tissue Culture Medium 199 containing serum, estradiol, and equine gonadotropins has been demonstrated (see [60] for review). However, equine gonadotropins are added as crude pituitary extract containing 91% unknown products [26] that could have a negative effect on oocyte maturation. Moreover, oocytes were cultured individually, whereas group culture is more often used [6, 57, 59]. In cattle, culture of oocytes individually, as compared with group culture, results in significantly reduced rates of morula formation [61]. Bruck et al. [62] cultured equine oocytes individually in medium supplemented with crude equine pituitary extract, and the IVM rate was about 38%. On the other hand, most experiments are carried out with oocytes obtained at slaughter. The ovaries are thus transported to the laboratory with a delay in the time between oocyte collection and placement in culture medium, leading to a heterogeneous population of oocytes from healthy and atretic follicles. A higher percentage of competent oocytes in follicles in early atresia has been suggested in cattle [61, 63]. Finally, oocytes are often selected before culture according to their cumulus morphology and the appearance of their cytoplasm [4, 59], and the maturation rate is calculated on selected oocytes only. In this study, all oocytes were used and the maturation rate was calculated over all the collected oocytes. We observed that the IVM rate significantly increased with antral follicle size. This is consistent with findings for ovine [64, 65], porcine [66, 67], bovine [68], human [69], and rhesus monkey [70] oocytes. On the other hand, in several rodent species, acquisition of meiotic competence is associated with antrum formation and maximum oocyte size [71-73], and developmental competence is acquired during antral follicular growth [74]. This difference be-

12 EQUINE OOCYTE COMPETENCE FOR IN VITRO MATURATION 243 tween species can be explained by oocyte size: rodent oocytes reach their full size before antrum formation, whereas porcine and bovine oocytes are still growing when follicles reach the early antral stage [68, 75]. In pigs and cattle, as in rodent species, the ability to resume meiosis is acquired during oocyte growth. In the current study, we were unable to establish any relationship between equine oocyte diameter and follicle size. Moreover, the oocytes that remained in germinal vesicle stage after IVM were larger than the oocytes that resumed meiosis. It seems that in the horse, as in the rhesus monkey [70], meiotic competence is not associated with maximum oocyte diameter. Oocytes that appear fully grown may still need to undergo cytoplasmic changes before they are competent for optimal maturation. The differences in oocyte diameter according to the nuclear stage after IVM may be linked to changes in the osmolarity of the cytoplasm, but whether these differences are detectable before IVM or whether they are due to differing behavior of the oocytes during IVM remains unknown. The expulsion of the polar body may account for a decrease in oocyte diameter. On the other hand, the smaller oocyte diameter after in vivo maturation versus IVM cannot be explained by a difference in osmolarity between the maturation medium and the follicular fluid, since both were evaluated (320 mos for the maturation medium and 290 mos for the follicular fluid). The nuclear maturation rate of oocytes collected in follicles from 5 to 9 mm was significantly lower than the maturation rate of oocytes from larger follicles. Similar results with equine oocytes obtained from slaughterhouse ovaries have been recently published [62]. Additionally, it was observed that CEG injection in the luteal phase tended to increase the IVM rate, but the increase was minimal in follicles smaller than 9 mm. On the other hand, data for prepubertal fillies 4-12 mo of age showed that, on average, the follicles did not exceed 10 mm [76]. Likewise, during deep anestrus, ovarian follicles usually do not develop above 10 mm [77]. Therefore, follicular populations less than 10 mm in diameter are relatively independent of the hormonal environment, and they represent the basal gonado-independent follicular growth. In these follicles, the cytoplasmic maturation rate of oocytes, using CG localization as a criterion, is significantly lower than that of oocytes from larger follicles. These results are not surprising, as CG localization seems to be linked with the stage of nuclear maturation. Finally, the rate of normal metaphase II or telophase I spindles in oocytes from follicles smaller than 9 mm tend to be higher from follicles larger than 10 mm, whereas they are less competent for IVM. It seems that, when they are able to mature, maturation takes place normally. On the other hand, the percentage of normal metaphase I spindles in oocytes cultured in vitro is low (63%) compared with the rate for normal metaphase II and telophase I spindles (74%). This can be explained by the aging of oocytes arrested in metaphase I. In this study, the mare's reproductive status did not significantly affect the percentage of metaphase II oocytes after in vitro culture. However, the hormonal environment in the follicular phase tended to increase the percentage of competent oocytes. Conflicting results are obtained with oocytes from slaughterhouse ovaries [62, 78]. The reason may be that the mares are slaughtered at different phases of the cycle, with cyclic stage determined relative to plasma progesterone concentrations and presence of corpora lutea on the ovaries. However, many other factors, not taken into account, bring about variations in the hormonal environment, such as the presence of a dominant follicle, of the LH rise, and of a persistent corpus luteum. The populations of oocytes are therefore heterogeneous. Repeated in vivo follicular punctures allow collection of well-characterized oocyte populations and lead to strict and reliable comparisons within the same animal. When we pooled oocytes with a metaphase II or metaphase I spindle at recovery and oocytes with a metaphase II or metaphase I spindle after IVM, metaphase I spindles were significantly wider than metaphase II spindles. As previously reported in the mouse oocyte [49], the size of the spindles is correlated with the number of chromatids present within them. Moreover, spindles obtained after IVM are significantly wider and longer than spindles obtained at recovery. It seems that a general change in the critical concentration of tubulin in the cytoplasm may induce an alteration in the size of the spindle [79]. In vitro culture may allow the same nuclear maturation as in vivo maturation but may induce a change in the concentration of cytoplasmic proteins, such as tubulin, and also other factors involved in regulation of the cell cycle. In conclusion, a systematic protocol for successive punctures that lead to well-characterized populations of oocytes was developed in this study. The protocol provides for an increase in the number of oocytes collected to an average of 8.9 COCs per 22 days. Moreover, our study demonstrates that the acquisition of meiotic competence in equine oocytes occurred progressively during antral follicle growth. The availability of equine oocytes originating from follicles of different sizes that grew in different hormonal environments represents an interesting tool for further studies on acquisition of meiotic competence as well as on the regulation of the cell cycle during oocyte maturation. ACKNOWLEDGMENTS We wish to thank Dr. Jacques Fl1chon (Institut National de la Recherche Agronomique, Jouy-en-Josas, France) for the kind donation of the anti-tubulin antibody, and Monique Ottogali and Dr. Christine Gaillot for generous supplies of crude equine gonadotropin. We are grateful to Dr. Pierre Adenot, who taught us manipulation of the confocal microscope, and to Olivier Bastien for the initiation into the Visilog software. We would like to thank Isabelle Couty and the staff of the experimental stud farm for technical assistance, Alain Beguey and Odile Moulin for photographic work, and Pat Lonergan for correction of the English in the manuscript. REFERENCES I. Palmer E, Bdzard J, Magistrini M, Duchamp G. In vitro fertilization in the horse. A retrospective study. J Reprod Fertil Suppl 1991; 44: Grondahl C, Host T, Bruck I, Viuff D, Bzard J, Fair T, Greve T, Hyttel P In vitro production of equine embryos. Biol Reprod 1995; Monogr 1: Zhang JJ, Boyle MS, Allen WR, Galli C. Recent studies on in vivo fertilisation of in vitro matured horse oocytes. Equine Vet J 1989; 8(suppl): Willis P, Caudle AB, Fayrer-Hosken RA. Equine oocyte in vitro maturation: influences of sera, time, and hormones. Mol Reprod Dev 1991; 30: Bzard J, Palmer E. In vitro maturation of horse oocytes from slaughtered ovaries. In: Proc 12th Int Cong Anim Reprod; 1992; The Hague, The Netherlands. 1: Shabpareh V, Squires EL, Seidel GE Jr, Jasko DJ. Methods for collecting and maturing equine oocytes in vitro. Theriogenology 1993; 40: Whitmore HL, Wentworth BC, Ginther OJ. Circulating concentrations of luteinizing hormone during estrous cycle of mares as determined by radioimmunoassay. Am J Vet Res 1973; 34: Irvine CHG, Alexander SL. The dynamics of gonadotrophin-releasing hormone, LH and FSH secretion during the spontaneous ovulatory

13 244 GOUDET ET AL. surge of the mare as revealed by intensive sampling of pituitary venous blood. J Endocrinol 1994; 140: Hinrichs K, Schmidt AL, Friedman PP, Selgrath JP, Martin MG. In vitro maturation of horse oocytes: characterization of chromatin configuration using fluorescence microscopy. Biol Reprod 1993; 48: Baka SG, Toth TL, Veeck LL, Jones HW Jr, Muasher SJ, Lanzendorf SE. Evaluation of the spindle apparatus of in vitro matured human oocytes following cryopreservation. Hum Reprod 1995; 10: Pickering SJ, Johnson MH, Braude PR, Houliston E. Cytoskeletal organization in fresh, aged and spontaneously activated human oocytes. Hum Reprod 1988; 3: Albertini DF, Eppig JJ. Unusual cytoskeletal and chromatin configurations in mouse oocytes that are atypical in meiotic progression. Dev Genet 1995; 16: Thibault C, Szoll6si D, G6rard M. Mammalian oocyte maturation. Reprod Nutr Dev 1987; 27: Cran DG. Cortical granules during oocyte maturation and fertilization. J Reprod Fertil Suppl 1989; 38: Sathananthan AH, Trounson AO. Ultrastructural observations on cortical granules in human follicular oocytes cultured in vitro. Gamete Res 1982; 5: Long CR, Damiani P, Pinto-Correia C, MacLean RA, Duby RT, Robl JM. Morphology and subsequent development in culture of bovine oocytes matured in vitro under various conditions of fertilization. J Reprod Fertil 1994; 102: Neumann H, Alm H, Schnurrbusch U, Torner H, Greising T, Kanitz W. The ultrastructure of equine oocytes before and after in vitro maturation. Reprod Domest Anim 1995; 30:428 (abstract). 18. B6zard J Goudet G, Duchamp G, Palmer E. Preovulatory maturation of ovarian follicles and oocytes in unstimulated and superovulated mares. In: Sharp DC, Bazer FW (eds.), Equine Reproduction VI. Madison, WI: Society for the Study of Reproduction; 1995: Woods GL, Scraba ST, Ginther OJ. Prospects for induction of multiple ovulations and collection of multiple embryos in the mare. Theriogenology 1982; 17: Palmer E, Hajmeli G, Duchamp G. Gonadotrophin treatments increase ovulation rate but not embryo production from mares. Equine Vet J 1993; 15(suppl): Douglas RH. Review of induction of superovulation and embryo transfer in the equine. Theriogenology 1979; 11: Duchamp G, Bzard J, Palmer E. Oocyte yield and the consequences of puncture of all follicles larger than 8 millimeters in mares. In: Sharp DC, Bazer FW (eds.), Equine Reproduction VI. Madison, WI: Society for the Study of Reproduction; 1995: Kanitz W, Becker F, Am H, Torner H. Ultrasound-guided follicular aspiration in mares. In: Sharp DC, Bazer FW (eds.), Equine Reproduction VI. Madison, WI: Society for the Study of Reproduction; 1995: Palmer E, Driancourt MA. Use of ultrasonic echography in equine gynecology. Theriogenology 1980; 13: Palmer E, Jousset B. Urinary oestrogen and plasma progesterone levels in non-pregnant mares. J Reprod Fertil Suppl 1975; 23: Duchamp G, Bour B, Combarnous Y, Palmer E. Alternative solutions to hcg induction of ovulation in the mare. J Reprod Fertil Suppl 1987; 35: Viklicky V, Draber P, Hasek J, Bartek J. Production and characterization of a monoclonal antitubulin antibody. Cell Biol Int Rep 1982; 6: Saumande J, Tamboura D, Chupin D. Changes in milk and plasma concentrations of progesterone in cows after treatment to induce superovulation and their relationships with number of ovulations and of embryos collected. Theriogenology 1985; 23: Terqui M, Dray F, Cotta J. Variations de la concentration en oestradiol- 173 dans le sang pripherique de la Brebis au cours du cycle oestral. C R Acad Sci 1973; 277: Hochereau de Reviers MT, Copin M, Seek M, Monet-Kuntz C, Cornu C, Fontaine I, Perreau C, Elsen JM, Boomarov A. Stimulation of testosterone production by PMSG injection in the ovine male: effect of breed and age and application to males carrying or not carrying the "F" Booroola gene. Anim Reprod Sci 1990; 23: Palmer E, Duchamp G, Bezard J, Magistrini M, King WA, Bousquet D, Betteridge KJ. Non-surgical recovery of follicular fluid and oocytes of mares. J Reprod Fertil Suppl 1987; 35: Vogelsang MM, Kreider JL, Bowen MJ, Potter GD, Forrest DW, Kraemer DC. Methods for collecting follicular oocytes from mares. Theriogenology 1988; 29: Hinrichs K, Kenney DE Kenney RM. Aspiration of oocytes from mature and immature preovulatory follicles in the mare. Theriogenology 1990; 34: Briick I, Raun K, Synnestvedt B, Greve T Follicle aspiration in the mare using a transvaginal ultrasound-guided technique. Equine Vet J 1992: 24: Cook NL, Squires EL, Ray BS, Jasko DJ. Transvaginal ultrasoundguided follicular aspiration of equine oocytes. Equine Vet J 1993; 15(suppl): Pieterse MC, Vos PLAM, Kruip TAM, Wurth YA, van Beneden TH, Willemse AH, Taverne MAM. Transvaginal ultrasound-guided follicular aspiration of bovine oocytes. Theriogenology 1991; 35: Bracher V, Parlevliet J, Fazeli AR, Pieterse MC, Vos PLAM, Dieleman SJ, Taverne MAM, Colenbrander B. Repeated transvaginal ultrasound-guided follicle aspiration in the mare. Equine Vet J 1993; 15(suppl): Meintjes M, Bellow MS, Paul JB, Broussard JR, Li LY, Paccamonti D, Eilts BE, Godke RA. Transvaginal ultrasound-guided oocyte retrieval from cyclic and pregnant horse and pony mares for in vitro fertilization. In: Sharp DC, Bazer FW (eds.), Equine Reproduction VI. Madison, WI: Society for the Study of Reproduction; 1995: Looney CR, Lindsey BR, Gonseth CL, Johnson DL. Commercial aspects of oocyte retrieval and in vitro fertilization (IVF) for embryo production in problem cows. Theriogenology 1994; 41: Hawley LR, Enders AC, Hinrichs K. Comparison of equine and bovine oocyte-cumulus morphology within the ovarian follicle. In: Sharp DC, Bazer FW (eds.), Equine Reproduction VI. Madison, WI: Society for the Study of Reproduction; 1995: Hinrichs K, Rand WM, Palmer E. Effect of aspiration of the preovulatory follicle on luteinization, corpus luteum function, and peripheral plasma gonadotropin concentrations in the mare. Biol Reprod 1991; 44: King WA, Bezard J, Bousquet D, Palmer E, Betteridge KJ. The meiotic stage of preovulatory oocytes in mares. Genome 1987; 29: Ginther OJ. Reproductive Biology of the Mare. Basic and Applied Aspects. Cross Plains, WI: Equiservices; 1979: Almeida PA, Bolton VN. The effect of temperature fluctuations on the cytoskeletal organization and chromosomal constitution of the human oocyte. Zygote 1995; 3: Baka SG, Toth TL, Veeck LL, Jones HW Jr, Muasher SJ, Lanzendorf SE. Evaluation of the spindle apparatus of in-vitro matured human oocytes following cryopreservation. Hum Reprod 1995; 10: Winston NJ, McGuinness O, Johnson MH, Maro B. The exit of mouse oocytes from meiotic M-phase requires an intact spindle during intracellular calcium release. J Cell Sci 1995; 108: Webb M, Howlett SK, Maro B. Parthenogenesis and cytoskeletal organization in ageing mouse eggs. J Embryol Exp Morphol 1986; 95: Gard DL. Microtubule organization during maturation of Xenopus oocytes: assembly and rotation of the meiotic spindles. Dev Biol 1992; 151: Kubiak JZ, Weber M, G6raud G, Maro B. Cell cycle modification during the transitions between meiotic M-phase in mouse oocytes. J Cell Sci 1992; 102: Messinger SM, Albertini DE Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte. J Cell Sci 1991; 100: Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990; 54: Aman RR, Parks JE. Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro-matured bovine oocytes. Biol Reprod 1994; 50: Kim N, Funahashi H, Prather RS, Schatten G, Day BN. Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation. Mol Reprod Dev 1996; 43: Eichenlaub-Ritter U, Stahl A, Luciani JM. The microtubular cytoskeleton and chromosomes of unfertilized human oocytes aged in vitro. Hum Genet 1988; 80: Torner H, Alim H. Meiotic configuration of horse oocytes in relation to the morphology of the cumulus-oocyte complex. In: Sharp DC, Bazer FW (eds.), Equine Reproduction VI. Madison, WI: Society for the Study of Reproduction; 1995:

14 EQUINE OOCYTE COMPETENCE FOR IN VITRO MATURATION Hinrichs K. The relationship of follicle atresia to follicle size, oocyte recovery rate on aspiration, and oocyte morphology in the mare. Theriogenology 1991; 36: Okolski A, Slonina D, Banasinska K. In vitro maturation of equine oocytes in co-culture with granulosa and theca interna cells. Equine Vet J 1993; 15(suppl): Testart J, Lassalle B, Frydman R, Belaisch JC. A study of factors affecting the success of human fertilization in vitro. II. Influence of semen quality and oocyte maturity on fertilization and cleavage. Biol Reprod 1983; 28: Dell'Aquila ME, Fusco S, Lacalandra GM, Maritato E In vitro maturation and fertilization of equine oocytes recovered during the breeding season. Theriogenology 1996; 45: Squires EL. Maturation and fertilization of equine oocytes. Vet Clin North Am Equine Pract 1996; 12: Blondin P, Sirard MA. Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes. Mol Reprod Dev 1995; 41: Bruck I, Grondahl C, Host T, Greve T. In vitro maturation of equine oocytes: effect of follicular size, cyclic stage and season. Theriogenology 1996; 46: Sirard MA, Blondin P. Oocyte maturation and IVF in cattle. Anim Reprod Sci 1996; 42: De Smedt V, Crozet N, Gall L. Morphological and functional changes accompanying the acquisition of meiotic competence in ovarian goat oocyte. J Exp Zool 1994; 269: Moor RM, Trounson AO. Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. J Reprod Fertil 1977; 49: Tsafriri A, Channing CP. Influence of follicular maturation and culture conditions on the meiosis of pig oocytes in vitro. J Reprod Fertil 1975; 43: Motlik J, Crozet N, Fulka J. Meiotic competence in vitro of pig oocytes isolated from early antral follicles. J Reprod Fertil 1984; 72: Arlotto T, Schwartz J-L, First NL, Leibfried-Rutledge ML. Aspects of follicle and oocyte stage that affect in vitro maturation and development of bovine oocytes. Theriogenology 1996; 45: Tsuji K, Sowa M, Nakano R. Relationship between human oocyte maturation and different follicular sizes. Biol Reprod 1985; 32: Schramm RD, Tennier MT, Boatman DE, Bavister BD. Chromatin configurations and meiotic competence of oocytes are related to follicular diameter in nonstimulated rhesus monkeys. Biol Reprod 1993; 48: Erickson GF, Sorensen RA. In vitro maturation of mouse oocytes isolated from late, middle, and pre-antral graafian follicles. J Exp Zool 1974; 190: Iwamatsu T, Yanagimachi R. Maturation in vitro of ovarian oocytes of prepubertal and adult hamsters. J Reprod Fertil 1975; 45: Bar-Ami S, Tsafriri A. Acquisition of meiotic competence in the rat: role of gonadotropin and estrogen. Gamete Res 1981; 4: Eppig JJ, Schroeder AC, O'Brien MJ. Developmental capacity of mouse oocytes matured in vitro: effects of gonadotrophic stimulation, follicular origin and oocyte size. J Reprod Fertil 1992; 95: Motlik J, Fulka J. Factors affecting meiotic competence in pig oocytes. Theriogenology 1986; 25: Wesson JA, Ginther OJ. Influence of season and age on reproductive activity in pony mares on the basis of a slaughterhouse survey. J Anim Sci 1981; 52: Driancourt M-A, Prunier A, Palmer E, Mariana J-C. Seasonal effects on ovarian follicular development in pony mares. Reprod Nutr Dev 1983; 23: Del Campo MR, Donoso X, Parrish JJ, Ginther OJ. Selection of follicles, preculture oocyte evaluation, and duration of culture for in vitro maturation of equine oocytes. Theriogenology 1995; 43: Eichenlaub-Ritter U, Chandley AC, Gosden RG. Alterations to the microtubular cytoskeleton and increased disorder of chromosome alignment in spontaneously ovulated mouse oocytes aged in vivo: an immunofluorescence study. Chromosoma (Berl) 1986; 94:

Animal Science 434! Tonic and Preovulatory Surge of GnRH! Tonic and Preovulatory Surge of GnRH! Lecture 11: The Follicular Phase of the Estrous Cycle!

Animal Science 434! Tonic and Preovulatory Surge of GnRH! Tonic and Preovulatory Surge of GnRH! Lecture 11: The Follicular Phase of the Estrous Cycle! Tonic and Preovulatory Surge of GnRH! Animal Science 434! Lecture 11: The Follicular Phase of the Estrous Cycle!! (-)! Hypothalamus! GnRH! Estradiol! (-)! Tonic and Preovulatory Surge of GnRH! Anterior!

More information

A comparison of the effects of estrus cow. nuclear maturation of bovine oocytes

A comparison of the effects of estrus cow. nuclear maturation of bovine oocytes A comparison of the effects of estrus cow serum and fetal calf serum on in vitro nuclear maturation of bovine oocytes J Spiropoulos, SE Long University of Bristol, School of Veterinary Science, Department

More information

Effect of Bovine Follicular Fluid Added to the Maturation Medium on Sperm Penetration in Pig Oocytes Matured In Vitro

Effect of Bovine Follicular Fluid Added to the Maturation Medium on Sperm Penetration in Pig Oocytes Matured In Vitro Article Effect of Bovine Follicular Fluid Added to the Maturation Medium on Sperm Penetration in Pig Oocytes Matured In Vitro Abstract Naoki ISOBE Research Associate Graduate School for International Development

More information

Ovarian Characteristics, Serum Hormone Concentrations, and Fertility in Lactating Dairy Cows in Response to Equine Chorionic Gonadotropin

Ovarian Characteristics, Serum Hormone Concentrations, and Fertility in Lactating Dairy Cows in Response to Equine Chorionic Gonadotropin Ovarian Characteristics, Serum Hormone Concentrations, and Fertility in Lactating Dairy Cows in Response to quine Chorionic Gonadotropin S. L. Pulley, L. D. Wallace, H. I. Mellieon, and J. S. Stevenson

More information

Treatment 3 Days After Ovulation In Mares

Treatment 3 Days After Ovulation In Mares Luteal Regression And Follicle Development Following Prostaglandin-F 2α Treatment 3 Days After Ovulation In Mares D.R. Bergfelt a, R.A. Pierson b, and O.J. Ginther a a University of Wisconsin, Madison,

More information

Clinical ICSI in the horse:

Clinical ICSI in the horse: Clinical ICSI in the horse: differences and similarities to human in an in vitro maturation-based system Katrin Hinrichs College of Veterinary Medicine & Biomedical Sciences Texas A&M University Standard

More information

Proceedings of the Society for Theriogenology Annual Conference 2014

Proceedings of the Society for Theriogenology Annual Conference 2014 www.ivis.org Proceedings of the Society for Theriogenology Annual Conference 2014 Aug. 6-9, 2014 Portland, OR, USA Next SFT Meeting: Aug. 5-8, 2015 San Antonio, TX, USA Reprinted in the IVIS website with

More information

Chapter 27 The Reproductive System. MDufilho

Chapter 27 The Reproductive System. MDufilho Chapter 27 The Reproductive System 1 Figure 27.19 Events of oogenesis. Before birth Meiotic events 2n Oogonium (stem cell) Mitosis Follicle development in ovary Follicle cells Oocyte 2n Primary oocyte

More information

OVARY The surface of the ovary is covered with surface epithelium

OVARY The surface of the ovary is covered with surface epithelium OVARY Cow The ovary, or female gonad, is: 1. an exocrine gland, producing oocytes 2. an endocrine gland, secreting hormones, i.e., estrogen and progesterone OVARY OVARY The surface of the ovary is covered

More information

The reproductive lifespan

The reproductive lifespan The reproductive lifespan Reproductive potential Ovarian cycles Pregnancy Lactation Male Female Puberty Menopause Age Menstruation is an external indicator of ovarian events controlled by the hypothalamicpituitary

More information

Concentrations of Luteinizing Hormone and Ovulatory Responses in Dairy Cows Before Timed Artificial Insemination

Concentrations of Luteinizing Hormone and Ovulatory Responses in Dairy Cows Before Timed Artificial Insemination Concentrations of Luteinizing Hormone and Ovulatory Responses in Dairy Cows Before Timed Artificial Insemination S. L. Pulley, D. H. Keisler, S. L. Hill, and J. S. Stevenson Summary The objective of this

More information

CASE 41. What is the pathophysiologic cause of her amenorrhea? Which cells in the ovary secrete estrogen?

CASE 41. What is the pathophysiologic cause of her amenorrhea? Which cells in the ovary secrete estrogen? CASE 41 A 19-year-old woman presents to her gynecologist with complaints of not having had a period for 6 months. She reports having normal periods since menarche at age 12. She denies sexual activity,

More information

Oocyte maturation within stimulated immature bovine

Oocyte maturation within stimulated immature bovine Oocyte maturation within stimulated immature bovine follicles in vivo W. D. FOOTE C. D. MILLS, D. A. PHELPS F. D. TIBBITTS Max C. Fleischmann College of Agriculture, University of Nevada, Reno, Nevada

More information

Page 1. A wide variety of ovarian abnormalities are encountered in clinical practice

Page 1. A wide variety of ovarian abnormalities are encountered in clinical practice A wide variety of ovarian abnormalities are encountered in clinical practice Common Problems Anovulatory follicles Persistent anovulatory follicles Hemorrhagic/Luteinized follicles Persistent corpus luteum

More information

Meiotic competence of in vitro grown goat oocytes

Meiotic competence of in vitro grown goat oocytes Journal of Reproduction and Fertility (2000) 118, 367 373 Meiotic competence of in vitro grown goat oocytes N. Crozet, M. Dahirel and L. Gall Institut National de la Recherche Agronomique, Unité de Physiologie

More information

Female Reproductive Physiology. Dr Raelia Lew CREI, FRANZCOG, PhD, MMed, MBBS Fertility Specialist, Melbourne IVF

Female Reproductive Physiology. Dr Raelia Lew CREI, FRANZCOG, PhD, MMed, MBBS Fertility Specialist, Melbourne IVF Female Reproductive Physiology Dr Raelia Lew CREI, FRANZCOG, PhD, MMed, MBBS Fertility Specialist, Melbourne IVF REFERENCE Lew, R, Natural History of ovarian function including assessment of ovarian reserve

More information

Theriogenology Department, Faculty of Veterinary Medicine, Beni Suef University, Egypt 2

Theriogenology Department, Faculty of Veterinary Medicine, Beni Suef University, Egypt 2 Theriogenology Insight: 3(1):11-16. April, 2013 Ultrasonic monitoring and biometry of ovaries and ovarian structures during superovulation following transvagianl follicle ablation in Murrah buffaloes S.M.

More information

Maturation and Freezing of Bovine Oocytes

Maturation and Freezing of Bovine Oocytes Maturation and Freezing of Bovine Oocytes D. Mapes and M. E. Wells Story in Brief Immature bovine oocytes were aspirated from small to medium size follicles of bovine ovaries by needle and syringe. The

More information

Concentrations of luteinizing hormone and ovulatory responses in dairy cows before timed artificial insemination

Concentrations of luteinizing hormone and ovulatory responses in dairy cows before timed artificial insemination Kansas Agricultural Experiment Station Research Reports Volume 0 Issue Dairy Research (98-0) Article 8 0 Concentrations of luteinizing hormone and ovulatory responses in dairy cows before timed artificial

More information

Effects of Preservation of Porcine Oocytes by Dibutyryl Cyclic AMP on in vitro Maturation, Fertilization and Development

Effects of Preservation of Porcine Oocytes by Dibutyryl Cyclic AMP on in vitro Maturation, Fertilization and Development JARQ 45 (3), 295 300 (2011) http://www.jircas.affrc.go.jp of Porcine Oocytes Using dbcamp Effects of of Porcine Oocytes by Dibutyryl Cyclic AMP on in vitro Maturation, Fertilization and Development Dai-ichiro

More information

In Vitro Growth of Mouse Ovarian Preantral Follicles and the Capacity of Their Oocytes to Develop to the Blastocyst Stage

In Vitro Growth of Mouse Ovarian Preantral Follicles and the Capacity of Their Oocytes to Develop to the Blastocyst Stage FULL PAPER Theriogenology In Vitro Growth of Mouse Ovarian Preantral Follicles and the Capacity of Their Oocytes to Develop to the Blastocyst Stage Christopher BISHONGA 1), Yoshiyuki TAKAHASHI 1)*, Seiji

More information

Research Article Efficacy of Tuohy Needle in Oocytes Collection from Excised Mare Ovaries

Research Article Efficacy of Tuohy Needle in Oocytes Collection from Excised Mare Ovaries SAGE-Hindawi Access to Research International Volume 2010, Article ID 102591, 4 pages doi:10.4061/2010/102591 Research Article Efficacy of Tuohy Needle in Oocytes Collection from Excised Mare Ovaries F.

More information

Ovarian follicular development in cattle

Ovarian follicular development in cattle Ovarian follicular development in cattle John P Kastelic Professor of Theriogenology Head, Department of Production Animal Health University of Calgary Calgary, Alberta, Canada Overview Prenatal development

More information

Effects of superovulation with FSH. Abstract

Effects of superovulation with FSH. Abstract Chapter 2 Effects of superovulation with ofsh and norgestomet/gnrh-controlled release of the LH surge on hormone concentrations, and yield of oocytes and embryos at specific developmental stages Effects

More information

REPRODUCTION & GENETICS. Hormones

REPRODUCTION & GENETICS. Hormones REPRODUCTION & GENETICS Hormones http://www.youtube.com/watch?v=np0wfu_mgzo Objectives 2 Define what hormones are; Compare and contrast the male and female hormones; Explain what each hormone in the mail

More information

Available online at Theriogenology xxx (2009) xxx xxx

Available online at   Theriogenology xxx (2009) xxx xxx Available online at www.sciencedirect.com 1 2 3 4 5 6 7 8 9 10 11 The effect of hormone treatments (hcg and cloprostenol) and season on the incidence of hemorrhagic anovulatory follicles in the mare: A

More information

Superovulation of Beef Heifers with Follicle Stimulating Hormone or Human Menopausal Gonadotropin: Acute Effects on Hormone Secretion

Superovulation of Beef Heifers with Follicle Stimulating Hormone or Human Menopausal Gonadotropin: Acute Effects on Hormone Secretion Superovulation of Beef Heifers with Follicle Stimulating Hormone or Human Menopausal Gonadotropin: Acute Effects on Hormone Secretion A.S. Leaflet R1362 Acacia A. Alcivar, graduate research assistant,

More information

Ghylène Goudet, François Belin, Jacqueline Bézard, and Nadine Gérard 2

Ghylène Goudet, François Belin, Jacqueline Bézard, and Nadine Gérard 2 BIOLOGY OF REPRODUCTION 60, 1120 1127 (1999) Intrafollicular Content of Luteinizing Hormone Receptor, -Inhibin, and Aromatase in Relation to Follicular Growth, Estrous Cycle Stage, and Oocyte Competence

More information

In-vitro maturation of camel oocytes using different media and sera

In-vitro maturation of camel oocytes using different media and sera In-vitro maturation of camel oocytes using different media and sera A. E. B. Zeidan 1, M. E. Hammad 2, Sh. A. Gabr 2, M. H. Farouk 3*, Sh. M. Shamiah 1, E. A. A. Ahmadi 1 and W. M. A. Nagy 1. 1 Dept. Camel

More information

THE MENSTRUAL CYCLE INA S. IRABON, MD, FPOGS, FPSRM, FPSGE OBSTETRICS AND GYNECOLOGY REPRODUCTIVE ENDOCRINOLOGY AND INFERTILITY

THE MENSTRUAL CYCLE INA S. IRABON, MD, FPOGS, FPSRM, FPSGE OBSTETRICS AND GYNECOLOGY REPRODUCTIVE ENDOCRINOLOGY AND INFERTILITY THE MENSTRUAL CYCLE INA S. IRABON, MD, FPOGS, FPSRM, FPSGE OBSTETRICS AND GYNECOLOGY REPRODUCTIVE ENDOCRINOLOGY AND INFERTILITY REFERENCE Comprehensive Gynecology 7 th edition, 2017 (Lobo RA, Gershenson

More information

Web Activity: Simulation Structures of the Female Reproductive System

Web Activity: Simulation Structures of the Female Reproductive System differentiate. The epididymis is a coiled tube found along the outer edge of the testis where the sperm mature. 3. Testosterone is a male sex hormone produced in the interstitial cells of the testes. It

More information

Why Cycle Control?" Manipulating Ovulation and Estrous Synchronization" Manipulating Ovulation" Cattle" Principle of PGF 2α Use"

Why Cycle Control? Manipulating Ovulation and Estrous Synchronization Manipulating Ovulation Cattle Principle of PGF 2α Use Why Cycle Control?" Manipulating Ovulation and Estrous Synchronization" John Parrish 1. Group females for parturition: " a) Decrease labor, calving period Reduce calving season" b) More uniform weaning

More information

F ertilizability of Rabbit Ova after Removal of the Corona Radiata

F ertilizability of Rabbit Ova after Removal of the Corona Radiata F ertilizability of Rabbit Ova after Removal of the Corona Radiata M. C. CHANG, Ph.D., and J. M. BEDFORD, M.R.C.V.S." FRESHLY ovulated rabbit ova are surrounded by a mass of follicular cells in a mucous

More information

LH (Bovine) ELISA Kit

LH (Bovine) ELISA Kit LH (Bovine) ELISA Kit Catalog Number KA2280 96 assays Version: 05 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Background... 3 Principle of the Assay...

More information

Superovulation of Beef Heifers with Follicle Stimulating Hormone or Human Menopausal Gonadotropin: Acute Effects on Hormone Secretion

Superovulation of Beef Heifers with Follicle Stimulating Hormone or Human Menopausal Gonadotropin: Acute Effects on Hormone Secretion Beef Research Report, 1996 Animal Science Research Reports 1997 Superovulation of Beef Heifers with Follicle Stimulating Hormone or Human Menopausal Gonadotropin: Acute Effects on Hormone Secretion Acacia

More information

Oocyte maturation. A.Trounson 1 ' 3, C.Anderiesz 1, G.MJones 1, A.Kausche 1, N.Lolatgis 2 and C.Wood 2

Oocyte maturation. A.Trounson 1 ' 3, C.Anderiesz 1, G.MJones 1, A.Kausche 1, N.Lolatgis 2 and C.Wood 2 A.Trounson 1 ' 3, C.Anderiesz 1, G.MJones 1, A.Kausche 1, N.Lolatgis 2 and C.Wood 2 Centre for Early Human Development, Institute of Reproduction and Development, Monash University, Monash Medical Centre,

More information

Effect of Warming on the Survivability and Fertilizability of Vitrified Matured Bovine Oocytes

Effect of Warming on the Survivability and Fertilizability of Vitrified Matured Bovine Oocytes International Journal of Agricultural Technology 2014 Vol. 10(1):49-58 Available online http://www.ijat-aatsea.com ISSN 2630-0192 (Online) Fungal Diversity Effect of Warming on the Survivability and Fertilizability

More information

REPRODUCTIVE CYCLE OF FEMALE MAMMAL

REPRODUCTIVE CYCLE OF FEMALE MAMMAL REPRODUCTIVE CYCLE OF FEMALE MAMMAL Fig. 8-12 Secondary follicles growing follicles increase in number of layers of granulosa cells Tertiary follicles maturing follicles antrum formation fluid filled space

More information

Follicle profile and plasma gonadotropin concentration in pubertal female ponies

Follicle profile and plasma gonadotropin concentration in pubertal female ponies Brazilian Journal of Medical and Biological Research (0) 37: 913-9 Puberty in female pony ISSN 00-79X 913 Follicle profile and plasma gonadotropin concentration in pubertal female ponies Departamento de

More information

Why Cycle Control? Manipulating Ovulation and Estrous Synchronization. Manipulating Ovulation. Cattle. Principle of PGF 2a Use

Why Cycle Control? Manipulating Ovulation and Estrous Synchronization. Manipulating Ovulation. Cattle. Principle of PGF 2a Use Why Cycle Control? Manipulating and Estrous Synchronization John Parrish 1. Group females for parturition: a) Decrease labor, calving period Reduce calving season b) More uniform weaning weights. 2. Reduce

More information

FAILED OOCYTE MATURATION. A Fekih, N Farah, D Chardonnens, F Urner, D De Ziegler, PG Bianchi, P Mock, A Campana, H Lucas

FAILED OOCYTE MATURATION. A Fekih, N Farah, D Chardonnens, F Urner, D De Ziegler, PG Bianchi, P Mock, A Campana, H Lucas FAILED OOCYTE MATURATION A Fekih, N Farah, D Chardonnens, F Urner, D De Ziegler, PG Bianchi, P Mock, A Campana, H Lucas INTRODUCTION In our laboratory,we perform (X) IVF and (Y) ICSI per year with a success

More information

Mohammad. Renad zakaria ---

Mohammad. Renad zakaria --- 13 Mohammad Renad zakaria --- Before we start: - I didn t follow the record order, for organizing purposes. - I added extra information from our text box which is Guyton 12 th edition, pages 987-997, actually

More information

Meiotic competence in vitro of pig oocytes isolated from early antral follicles

Meiotic competence in vitro of pig oocytes isolated from early antral follicles Meiotic competence in vitro of pig oocytes isolated from early antral follicles J. Motlik, Nicole Crozet and J. Fulka Czechoslovak Academy of Sciences, Institute of Animal Physiology and Genetics, Department

More information

In vitro Embryo Production in Calves

In vitro Embryo Production in Calves In vitro Embryo Production in Calves Reuben J. Mapletoft 1, Ana Rita Tavares Krause 1 and Pietro S. Baruselli 2 1 WCVM, University of Saskatchewan, Saskatoon, SK S7N 5B4 CANADA 2 Departamento de Reprodução

More information

Reproduction and Development. Female Reproductive System

Reproduction and Development. Female Reproductive System Reproduction and Development Female Reproductive System Outcomes 5. Identify the structures in the human female reproductive system and describe their functions. Ovaries, Fallopian tubes, Uterus, Endometrium,

More information

Effects of Label-Dose Permethrin Administration on Reproductive Function and Embryo Quality on Superovulated Beef Heifers

Effects of Label-Dose Permethrin Administration on Reproductive Function and Embryo Quality on Superovulated Beef Heifers Animal Industry Report AS 662 ASL R3050 2016 Effects of Label-Dose Permethrin Administration on Reproductive Function and Embryo Quality on Superovulated Beef Heifers Tyler M. Dohlman Iowa State University,

More information

Influence of large follicles on oestrus induction and ovulation after embryo collection in superovulated Japanese Black cows

Influence of large follicles on oestrus induction and ovulation after embryo collection in superovulated Japanese Black cows J. Reprod. Engineer. 2015; 17: 1 5. http://sreprod.jp/contents.htm = Original Article = Journal of REPRODUCTION ENGINEERING Influence of large follicles on oestrus induction and ovulation after embryo

More information

The Why s, What s, and How s of Timed Artificial Insemination Programs

The Why s, What s, and How s of Timed Artificial Insemination Programs Kansas Agricultural Experiment Station Research Reports Volume 1 Issue 8 Dairy Research Article 5 January 2015 The Why s, What s, and How s of Timed Artificial Insemination Programs J. Stevenson Kansas

More information

Influence of co-culture with oviductal epithelial cells on in vitro maturation of canine oocytes

Influence of co-culture with oviductal epithelial cells on in vitro maturation of canine oocytes Reprod. Nutr. Dev. 42 (2002) 265 273 265 INRA, EDP Sciences, 2002 DOI: 10.1051/rnd:2002024 Original article Influence of co-culture with oviductal epithelial cells on in vitro maturation of canine oocytes

More information

The Cell Life Cycle. S DNA replication, INTERPHASE. G 2 Protein. G 1 Normal THE CELL CYCLE. Indefinite period. synthesis. of histones.

The Cell Life Cycle. S DNA replication, INTERPHASE. G 2 Protein. G 1 Normal THE CELL CYCLE. Indefinite period. synthesis. of histones. Mitosis & Meiosis The Cell Life Cycle INTERPHASE G 1 Normal cell functions plus cell growth, duplication of organelles, protein synthesis S DNA replication, synthesis of histones THE CELL CYCLE M G 2 Protein

More information

Distributions of Mitochondria and the Cytoskeleton in Hamster Embryos Developed In Vivo and In Vitro

Distributions of Mitochondria and the Cytoskeleton in Hamster Embryos Developed In Vivo and In Vitro J. Mamm. Ova Res. Vol. 23, 128 134, 2006 128 Original Distributions of Mitochondria and the Cytoskeleton in Hamster Embryos Developed In Vivo and In Vitro Hiroyuki Suzuki 1 *, Manabu Satoh 1 ** and Katsuya

More information

Mouse sperm extraction:

Mouse sperm extraction: Mouse sperm extraction: This method of extraction is used for acrosome reaction assays, immunocytochemistry and biochemical assays. Collect two cauda epidydimus from one male, cut them 5 times and place

More information

Foundational questions Oocyte-derived functional mediators of early embryonic development (EST and candidate gene) JY-1 Nobox Importin 8 Oocyte and cu

Foundational questions Oocyte-derived functional mediators of early embryonic development (EST and candidate gene) JY-1 Nobox Importin 8 Oocyte and cu Models for study of oocyte competence: George W. Smith (Smithge7@msu.edu) Foundational questions Oocyte-derived functional mediators of early embryonic development (EST and candidate gene) JY-1 Nobox Importin

More information

Manipulation of Ovarian Function for the Reproductive Management of Dairy Cows

Manipulation of Ovarian Function for the Reproductive Management of Dairy Cows Veterinary Research Communications,28(2004) 111 119 2004 Kluwer Academic Publishers. Printed in the Netherlands Manipulation of Ovarian Function for the Reproductive Management of Dairy Cows W.W. Thatcher1*,

More information

Prostaglandin F 2α. J. S. Stevenson, S. L. Pulley, and H. I. Mellieon, Jr.

Prostaglandin F 2α. J. S. Stevenson, S. L. Pulley, and H. I. Mellieon, Jr. Prostaglandin F 2α and GnRH Administration Improved Progesterone tatus, Luteal Number, and Proportion of Ovular and Anovular Dairy Cows with Corpora Lutea efore a Timed Artificial Insemination Program

More information

INDUCTIONS OF SUPEROVULATION USING SEVERAL FSH REGIMENS IN HOLSTEIN-FRIESIAN HEIFERS

INDUCTIONS OF SUPEROVULATION USING SEVERAL FSH REGIMENS IN HOLSTEIN-FRIESIAN HEIFERS lpn. l. Vet. Res., 33, 45-50 (1985) INDUCTIONS OF SUPEROVULATION USING SEVERAL FSH REGIMENS IN HOLSTEIN-FRIESIAN HEIFERS Yoshiyuki TAKAHASHI and Hiroshi KANAGAWA (Received for publication, February 2,

More information

Growth of small follicles and concentrations of FSH during the equine oestrous cycle

Growth of small follicles and concentrations of FSH during the equine oestrous cycle Growth of small follicles and concentrations of FSH during the equine oestrous cycle O. J. Ginther and D. R. Bergfelt Department of Animal Health and Biomedicai Sciences, Veterinary Science Building, University

More information

REPRODUCCIÓN. La idea fija. Copyright 2004 Pearson Education, Inc., publishing as Benjamin Cummings

REPRODUCCIÓN. La idea fija. Copyright 2004 Pearson Education, Inc., publishing as Benjamin Cummings REPRODUCCIÓN La idea fija How male and female reproductive systems differentiate The reproductive organs and how they work How gametes are produced and fertilized Pregnancy, stages of development, birth

More information

GONADOTROPHIN (LUTEINISING)- RELEASING HORMONE AND ANALOGUES (GnRH OR LHRH)

GONADOTROPHIN (LUTEINISING)- RELEASING HORMONE AND ANALOGUES (GnRH OR LHRH) GONADOTROPHIN (LUTEINISING)- RELEASING HORMONE AND ANALOGUES (GnRH OR LHRH) Naturally occurring hormone, produced by the hypothalamus and transferred to the anterior pituitary gland in the hypophyseal

More information

S. M. Quirk, G. J. Hickey and J. E. Fortune. Summary. Ultrasonography was used to monitor the growth, ovulation and regression

S. M. Quirk, G. J. Hickey and J. E. Fortune. Summary. Ultrasonography was used to monitor the growth, ovulation and regression Growth and regression of ovarian follicles during the follicular phase of the oestrous cycle in heifers undergoing spontaneous and PGF-2\g=a\-induced luteolysis S. M. Quirk, G. J. Hickey and J. E. Fortune

More information

SISTEMA REPRODUCTOR (LA IDEA FIJA) Copyright 2004 Pearson Education, Inc., publishing as Benjamin Cummings

SISTEMA REPRODUCTOR (LA IDEA FIJA) Copyright 2004 Pearson Education, Inc., publishing as Benjamin Cummings SISTEMA REPRODUCTOR (LA IDEA FIJA) How male and female reproductive systems differentiate The reproductive organs and how they work How gametes are produced and fertilized Pregnancy, stages of development,

More information

Is it the seed or the soil? Arthur Leader, MD, FRCSC

Is it the seed or the soil? Arthur Leader, MD, FRCSC The Physiological Limits of Ovarian Stimulation Is it the seed or the soil? Arthur Leader, MD, FRCSC Objectives 1. To consider how ovarian stimulation protocols work in IVF 2. To review the key events

More information

Raoul Orvieto. The Chaim Sheba Medical Center Tel Hashomer, Israel. Declared no potential conflict of interest

Raoul Orvieto. The Chaim Sheba Medical Center Tel Hashomer, Israel. Declared no potential conflict of interest Raoul Orvieto The Chaim Sheba Medical Center Tel Hashomer, Israel Declared no potential conflict of interest LH in antagonist cycles; is the story really written? Raoul Orvieto M.D. Israel Overview Role

More information

Morphometric analysis of ovarian follicles of Black Bengal goats during winter and summer season

Morphometric analysis of ovarian follicles of Black Bengal goats during winter and summer season Morphometric analysis of ovarian follicles of Black Bengal goats during winter and summer season MA Bari 1, ME Kabir 1, MB Sarker 1, AHNA Khan 2 and M Moniruzzaman* 1 1 Department of Animal Science, Bangladesh

More information

Gametogenesis. Dr Corinne de Vantéry Arrighi Dr Hervé Lucas

Gametogenesis. Dr Corinne de Vantéry Arrighi Dr Hervé Lucas WHO Collaborating Center for Research in Human Reproduction Clinic for Infertility and Gynecological Endocrinology University Hospital, Geneva, Switzerland Gametogenesis Dr Corinne de Vantéry Arrighi Dr

More information

Course: Animal Production. Instructor: Ms. Hutchinson. Objectives: After completing this unit of instruction, students will be able to:

Course: Animal Production. Instructor: Ms. Hutchinson. Objectives: After completing this unit of instruction, students will be able to: Course: Animal Production Unit Title: Hormones TEKS: 130.3 (C)(6)(A) Instructor: Ms. Hutchinson Objectives: After completing this unit of instruction, students will be able to: A. Define what hormones

More information

Genome Integrity in Mammalian Oocytes

Genome Integrity in Mammalian Oocytes Genome Integrity in Mammalian Oocytes ESHRE Workshop on mammalian folliculogenesis and oogenesis April 19 21 Stresa Italy 2003 Workshop Lisbon Genome Integrity Structure is chromatin in open or closed

More information

Proceedings, The Applied Reproductive Strategies in Beef Cattle Workshop, September 5-6, 2002, Manhattan, Kansas

Proceedings, The Applied Reproductive Strategies in Beef Cattle Workshop, September 5-6, 2002, Manhattan, Kansas 20 10 0 Proceedings, The Applied Reproductive Strategies in Beef Cattle Workshop, September 5-6, 2002, Manhattan, Kansas REVIEW OF FOLLICULAR GROWTH AND THE BOVINE ESTROUS CYCLE Milo C. Wiltbank Department

More information

FREE-ROAMING HORSE AND BURRO FERTILITY CONTROL WORKSHOP Albuquerque, NM November 8, 2018

FREE-ROAMING HORSE AND BURRO FERTILITY CONTROL WORKSHOP Albuquerque, NM November 8, 2018 FREE-ROAMING HORSE AND BURRO FERTILITY CONTROL WORKSHOP Albuquerque, NM November 8, 2018 Current Contraceptive Use pzp GonaCon Porcine Zona Pellucida Antibodies to ZP3 Cons: Requires boosters Continuous

More information

Dr. Julio Giordano. Ovulation. Follicle. Corpus Luteum. GnRH

Dr. Julio Giordano. Ovulation. Follicle. Corpus Luteum. GnRH Dr. Julio Giordano Follicle Corpus Luteum LH FSH E2 Hypothalamic hormones Gonadotropin releasing hormone () Hormone Concentration CL LH (ng/ml) 12 10 8 6 4 2 LH Response Cows Treated with 28 h (22-36)

More information

In Vitro Production of Equine Embryos: State of the Art

In Vitro Production of Equine Embryos: State of the Art Reprod Dom Anim 45 (Suppl. 2), 3 8 (2010); doi: 10.1111/j.1439-0531.2010.01624.x ISSN 0936-6768 In Vitro Production of Equine Embryos: State of the Art K Hinrichs Departments of Veterinary Physiology and

More information

Investigation: The Human Menstrual Cycle Research Question: How do hormones control the menstrual cycle?

Investigation: The Human Menstrual Cycle Research Question: How do hormones control the menstrual cycle? Investigation: The Human Menstrual Cycle Research Question: How do hormones control the menstrual cycle? Introduction: The menstrual cycle (changes within the uterus) is an approximately 28-day cycle that

More information

Female Reproductive System. Justin D. Vidal

Female Reproductive System. Justin D. Vidal Female Reproductive System Justin D. Vidal If you cannot identify the tissue, then it is probably part of the female reproductive system! Introduction The female reproductive system is constantly changing,

More information

Effect of the Dominant Follicle Aspiration before or after Luteinizing Hormone Surge on the Corpus Luteum Formation in the Cow

Effect of the Dominant Follicle Aspiration before or after Luteinizing Hormone Surge on the Corpus Luteum Formation in the Cow Journal of Reproduction and Development, Vol. 52, No. 1, 2006 Research Note Effect of the Dominant Follicle Aspiration before or after Luteinizing Hormone Surge on the Corpus Luteum Formation in the Cow

More information

Chapter 14 The Reproductive System

Chapter 14 The Reproductive System Biology 12 Name: Reproductive System Per: Date: Chapter 14 The Reproductive System Complete using BC Biology 12, page 436-467 14. 1 Male Reproductive System pages 440-443 1. Distinguish between gametes

More information

Abstracts for the KSAR and JSAR Joint Symposium. Fertility control in female domestic animals: From basic understanding to application

Abstracts for the KSAR and JSAR Joint Symposium. Fertility control in female domestic animals: From basic understanding to application Abstracts for the KSAR and JSAR Joint Symposium Fertility control in female domestic animals: From basic understanding to application Current Research Orientation in Livestock Reproduction in Korea Choong-Saeng

More information

In vitro maturation of human oocytes for assisted reproduction

In vitro maturation of human oocytes for assisted reproduction MODERN TRENDS Edward E. Wallach, M.D. Associate Editor In vitro maturation of human oocytes for assisted reproduction Marcus W. Jurema, M.D., and Daniela Nogueira, Ph.D. Department of Obstetrics and Gynecology,

More information

The Human Menstrual Cycle

The Human Menstrual Cycle The Human Menstrual Cycle Name: The female human s menstrual cycle is broken into two phases: the Follicular Phase and the Luteal Phase. These two phases are separated by an event called ovulation. (1)

More information

Male Reproduction Organs. 1. Testes 2. Epididymis 3. Vas deferens 4. Urethra 5. Penis 6. Prostate 7. Seminal vesicles 8. Bulbourethral glands

Male Reproduction Organs. 1. Testes 2. Epididymis 3. Vas deferens 4. Urethra 5. Penis 6. Prostate 7. Seminal vesicles 8. Bulbourethral glands Outline Terminology Human Reproduction Biol 105 Lecture Packet 21 Chapter 17 I. Male Reproduction A. Reproductive organs B. Sperm development II. Female Reproduction A. Reproductive organs B. Egg development

More information

STUDIES OF THE HUMAN UNFERTILIZED TUBAL OVUM*t

STUDIES OF THE HUMAN UNFERTILIZED TUBAL OVUM*t FERTILITY AND STERILITY Copyright @ 1973 by The Williams & Wilkins Co. Vol. 24, No.8, August 1973 Printed in U.S.A. STUDIES OF THE HUMAN UNFERTILIZED TUBAL OVUM*t C. NORIEGA, M.D., AND C. OBERTI, M.D.

More information

Reproductive cyclicity 19. Introduction. Page 1. repro and its story lines. Male repro: a simpler way of control

Reproductive cyclicity 19. Introduction. Page 1. repro and its story lines. Male repro: a simpler way of control Reproductive cyclicity 19 Male repro: a simpler way of control Menstrual cycles: ovary / uterine anatomy and cell types, follicular phase, ovulation, luteal phase, cyclicity Race events: removal of P4

More information

Influence of Epidermal Growth Factor in the In vitro Development of Bovine Preimplantation Embryos

Influence of Epidermal Growth Factor in the In vitro Development of Bovine Preimplantation Embryos Available online at www.ijpab.com DOI: http://dx.doi.org/10.18782/2320-7051.6638 ISSN: 2320 7051 Int. J. Pure App. Biosci. 6 (5): 584-589 (2018) Research Article Influence of Epidermal Growth Factor in

More information

Ovarian follicular dynamics and superovulation in cattle

Ovarian follicular dynamics and superovulation in cattle Ovarian follicular dynamics and superovulation in cattle John P Kastelic Professor of Theriogenology Head, Department of Production Animal Health University of Calgary Calgary, Alberta, Canada Factors

More information

Oocyte development in cattle: physiological and genetic aspects. Jack H. Britt

Oocyte development in cattle: physiological and genetic aspects. Jack H. Britt Revista Brasileira de Zootecnia ISSN impresso: 1516-3598 R. Bras. Zootec., v.37, suplemento especial p.110-115, 2008 ISSN on-line: 1806-9290 www.sbz.org.br Oocyte development in cattle: physiological and

More information

Effect of Resistin on Granulosa and Theca Cell Function in Cattle

Effect of Resistin on Granulosa and Theca Cell Function in Cattle 1 Effect of Resistin on Granulosa and Theca Cell Function in Cattle D.V. Lagaly, P.Y. Aad, L.B. Hulsey, J.A. Grado-Ahuir and L.J. Spicer Story in Brief Resistin is an adipokine that has not been extensively

More information

IVM in PCOS patients. Introduction (1) Introduction (2) Michael Grynberg René Frydman

IVM in PCOS patients. Introduction (1) Introduction (2) Michael Grynberg René Frydman IVM in PCOS patients Michael Grynberg René Frydman Department of Obstetrics and Gynecology A. Beclere Hospital, Clamart, France Maribor, Slovenia, 27-28 February 2009 Introduction (1) IVM could be a major

More information

Effects of Catecholamines and Dibenamine on Ovulation in the Perfused Fowl Ovary

Effects of Catecholamines and Dibenamine on Ovulation in the Perfused Fowl Ovary Effects of Catecholamines and Dibenamine on Ovulation in the Perfused Fowl Ovary Tomoki HIGUCHI, Tomoki SOH, Frank HERTELENDY* and Kousaku TANAKA Faculty of Agriculture, Kyushu University, Higashi-ku,

More information

Concentrations of Circulating Gonadotropins During. Various Reproductive States in Mares

Concentrations of Circulating Gonadotropins During. Various Reproductive States in Mares BIOLOGY OF REPRODUCTION, 744-75 (19) Concentrations of Circulating Gonadotropins During Various Reproductive States in Mares KURT F. MILLER, S. L. BERG, D. C. SHARP and. J. GINTHER Department of Veterinary

More information

Article Effect of gonadotrophin priming on in-vitro maturation of oocytes collected from women at risk of OHSS

Article Effect of gonadotrophin priming on in-vitro maturation of oocytes collected from women at risk of OHSS RBMOnline - Vol 13. No 3. 2006 340 348 Reproductive BioMedicine Online; www.rbmonline.com/article/2328 on web 12 June 2006 Article Effect of gonadotrophin priming on in-vitro maturation of oocytes collected

More information

Kulnasan Saikhun 1 and Kampon Kaeoket 2

Kulnasan Saikhun 1 and Kampon Kaeoket 2 Effect of Different Charged Groups of Cow Follicular Fluid Proteins on In Vitro Oocyte Maturation Saranya Satitmanwiwat 1*,Chinarat Changsangfah 1, Tassanee Faisaikarm 1, Kulnasan Saikhun 1 and Kampon

More information

Synchronization of Ovulation and Fixed-Time Insemination for Improvement of Conception Rate in Dairy Herds with Poor Estrus Detection Efficiency

Synchronization of Ovulation and Fixed-Time Insemination for Improvement of Conception Rate in Dairy Herds with Poor Estrus Detection Efficiency Journal of Reproduction and Development, Vol. 45, No. 1, 1999 Synchronization of Ovulation and Fixed-Time Insemination for Improvement of Conception Rate in Dairy Herds with Poor Estrus Detection Efficiency

More information

Relevance of LH activity supplementation

Relevance of LH activity supplementation Relevance of LH activity supplementation in ovulation induction Franco Lisi Servizio di Fisiopatologia della Riproduzione Clinica Villa Europa Roma, Italia Comprehension of the role of LH in follicular

More information

Summary. Mouse eggs were fertilized in vitro, in the presence and

Summary. Mouse eggs were fertilized in vitro, in the presence and THE R\l=O^\LEOF CUMULUS CELLS AND THE ZONA PELLUCIDA IN FERTILIZATION OF MOUSE EGGS IN VITRO A. PAVLOK and ANNE McLAREN Czechoslovak Academy of Sciences, Laboratory of Animal Genetics, Libechov, Czechoslovakia,

More information

OVARIAN RESPONSES AND CONCEPTION RATES IN RESPONSE TO GnRH, hcg, AND PROGESTERONE 1

OVARIAN RESPONSES AND CONCEPTION RATES IN RESPONSE TO GnRH, hcg, AND PROGESTERONE 1 Dairy Research 2006 OVARIAN RESPONSES AND CONCEPTION RATES IN RESPONSE TO GnRH, hcg, AND PROGESTERONE 1 J. S. Stevenson, M. A. Portaluppi, D. E. Tenhouse, A. Lloyd, D. R. Eborn, S. Kacuba 2 and J. M. DeJarnette

More information

Physiology of Male Reproductive System

Physiology of Male Reproductive System Physiology of Male Reproductive System the anterior pituitary gland serves as the primary control of reproductive function at puberty Ant Pituitary secretes FSH & large amounts of LH (ICSH) FSH & LH cause

More information

RECOVERY OF MIDCYCLE HUMAN FOLLICULAR OOCYTES: CORRELATION OF THEIR MORPHOLOGY WITH ENDOMETRIAL AND FOLLICULAR HISTOLOGY

RECOVERY OF MIDCYCLE HUMAN FOLLICULAR OOCYTES: CORRELATION OF THEIR MORPHOLOGY WITH ENDOMETRIAL AND FOLLICULAR HISTOLOGY r FERTILITY AND STERILITY Copyright ~ 1978 The American Fertility Society Vol. 29,.5, May 1978 Printed in U.S.A. RECOVERY OF MIDCYCLE HUMAN FOLLICULAR OOCYTES: CORRELATION OF THEIR MORPHOLOGY WITH ENDOMETRIAL

More information

M. Irfan-ur-Rehman Khan, M. A. Rana and N. Ahmad. Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore, Pakistan

M. Irfan-ur-Rehman Khan, M. A. Rana and N. Ahmad. Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore, Pakistan 82 ULTRASONIC MONITORING OF FOLLICLES AND CORPORA LUTEA DURING SYNCHRONIZATION IN SUMMER ANOESTROUS NILI RAVI BUFFALOES AND THEIR SUBSEQUENT SUPEROVULATORY RESPONSE M. Irfan-ur-Rehman Khan, M. A. Rana

More information

Microtubule and microfilament organization in maturing human oocytes

Microtubule and microfilament organization in maturing human oocytes Human Reproduction vol.13 no.8 pp.2217 2222, 1998 Microtubule and microfilament organization in maturing human oocytes Nam-Hyung Kim 1, Hyung Min Chung 2, Kwang-Yul Cha 2 and Kil Saeng Chung 1,3 1 Animal

More information

Reproductive Hormones

Reproductive Hormones Reproductive Hormones Male gonads: testes produce male sex cells! sperm Female gonads: ovaries produce female sex cells! ovum The union of male and female sex cells during fertilization produces a zygote

More information

Hormonal Control of Human Reproduction

Hormonal Control of Human Reproduction Hormonal Control of Human Reproduction Bởi: OpenStaxCollege The human male and female reproductive cycles are controlled by the interaction of hormones from the hypothalamus and anterior pituitary with

More information