Hormonal stimulation of maturation and ovulation,gamete morphology,and raising of larvae in <Emphasis Type="Italic">Dascyllus trimaculatus</Emphasis> (Pomacentridae)

The experiments on hormonal stimulation of the maturation and ovulation of oocytes of Dascyllus trimaculatus (Pomacentridae) are carried out. Double injections of surfagon (LH-RH-a) (5 + 15 μg/kg of fish body weight) with (or without) the addition of eglonil (5 + 15 mg/kg) are used, and the interval between the injections ranges from 12 to 17 h. Ovulation is registered 33.5–42.0 h after injection I. Morphological changes in oocytes are followed during the process of their maturation. The ultrastructure of spermatozoa and oocyte envelopes is studied. The quality of ovulated oocytes is assessed after their in vitro storage at 25 and 5°C. Preliminary results on the transition of the larvae to exogenous feeding are obtained.     

Hormonal stimulation of maturation and Ovulation,egg quality,and development of larvae of <Emphasis Type="Italic">Abudefduf sexfasciatus</Emphasis> (Pomacentridae)

Experiments on the hormonal stimulation of oocyte maturation and ovulation of Abudefduf sexfasciatus (Pomacentridae) have been conducted. Both single and double injections of surfagon (LH-RH-a) are applied. Ovulation is registered more often 40–48 h after injection I. The morphological changes of oocytes during final maturation are followed. The egg quality is assessed after the storage of ovulated oocytes inside of the female’s body (in vivo) and in the external medium (in vitro) at 25 and 5°C. The development of free embryos and larvae is described.     

Mitochondrial distribution, ATP-GSH contents, calcium [Ca2+] oscillation during in vitro maturation of dromedary camel oocytes

Dromedary camel oocytes have the ability to spontaneous parthenogenetic activation and development in vivo and in vitro. The present study was conducted to investigate changes in mitochondrial distribution, adenosine triphosphate (ATP), and glutathione (GSH) contents and [Ca²⁺] oscillation during in vitro maturation and spontaneous parthenogentic activation of dromedary camel oocytes. Dromedary camel cumulus-oocyte complexes (COCs) were matured in TCM199 medium supplemented with 10% FCS + 10 μg/mL FSH + 10 IU hCG + 10 IU eCG + 10 ng/mL EGF and 50 μg/mL gentamycine. Maturation was performed at 38.5 °C under 5% CO₂ in humidified air for 40 h. After maturation and removal of cumulus cells, oocytes were classified into: immature cultured (Group 1); metaphase II (M II, Group 2); and spontaneously parthenogenetically activated (with 2 polar bodies, Group 3); cleaved embryos (Group 4); and immature oocytes served as a control (Group 5). Cytoplasmic mitochondrial distribution, ATP-GSH contents, calcium [Ca²⁺] oscillation were determined. Results indicated that M II and spontaneously parthenogenetically activated oocytes represent 37.53% and 32.67% of the cultured oocytes, respectively, and 3.3% cleaved and developed to 2-16-cell stage embryos. Mitochondrial distribution, ATP-GSH contents and [Ca²⁺] oscillation were significantly (P < 0.01) differ between immature and matured dromedary camel oocytes. Mitochondrial distribution showed clustering form in matured oocytes without polar body. High polarized mitochondrial distribution (HPM) was detected in M II and spontaneously parthenogenetically activated oocytes, and the intensity of MitoTracker Red was higher in spontaneously parthenogenetically activated than M II. ATP-GSH contents and the duration of [Ca²⁺] oscillation were significantly (P < 0.01) higher in spontaneously parthenogenetically activated than M II oocytes or that matured without polar body. In conclusion, the higher incidence of spontaneously parthenogenetically activated in vitro matured dromedary camel oocytes could be attributed to the high polarized mitochondrial distribution associated with significantly higher ATP-GSH contents and duration of [Ca²⁺] oscillation.     

Maturity and fertility of rhesus monkey oocytes collected at different intervals after an ovulatory stimulus (human chorionic gonadotropin) in in vitro fertilization cycles

In rhesus monkeys undergoing ovarian stimulation for in vitro fertilization (IVF), a midcycle injection of human chorionic gonadotropin (hCG) substitutes for the LH surge and induces preovulatory oocyte maturation. The time interval between injection and oocyte collection, ideally, allows for the completion of oocyte maturation without ovulation, which would reduce the number of oocytes available for harvest. To evaluate the influence of this time interval on oocyte parameters following hCG administration, we conducted a series of gonadotropin treatment protocols in 51 animals in which the interval from hCG administration to follicular aspiration was systematically varied from 27 to 36 hr. Follicle number and size, evaluated prior to hCG administration by sonography, did not vary significantly or consistently with preovulatory maturation time. Oocytes were harvested by laparotomy or laparoscopy, and scored for maturity before insemination. The percentage of mature, metaphase II (MII) oocytes at recovery increased significantly with increasing preovulatory time and was inversely proportional to that of metaphase I (MI) oocytes. However, oocyte yield tended toward a progressive decrease with increasing preovulatory maturation times from a high of 27 oocytes at 27 hr to a low of 17 oocytes/animal at the 36 hr time interval. Fertilization levels declined significantly from a high of 50% at 27 hr to a low of 30% at 36 hr. Thus, although higher percentages of mature oocytes were recovered at the longer time intervals, optimal oocyte/embryo harvests were realized after the shorter time intervals (27 and 32 hr) and are most compatible with the goal of achieving high yields of fertile oocytes and embryos following gonadotropin stimulation in rhesus monkeys. © 1996 Wiley-Liss, Inc.     

Pattern of sea bass oocyte development after ovarian stimulation by LHRHa

The cyclic pattern of oocyte development in the sea bass, Dicentrarchus labrax L., was studied after induction of spawning by two injections, 24 h apart, of a luteinizing hormone releasing-hormone analog (LHRHa) administered at the end of vitellogenesis. The first difference in the developmental stage of the ovary and in the size-frequency distribution of oocytes between the LHRHa treated group and the control group, was detected 32 h after the first injection, the LHRHa group showing a higher proportion of the 900 μm diameter oocyte class (maturing oocytes) ( P <0.01). At 48 h LHRHa-treated females showed an increase in the 1000 and 1100 μm classes (maturing oocyte and ovulated eggs) ( P <0.01) and at 72 h these females exhibited a bimodal pattern, reaching the highest proportions in the 1100 (27.4%) and the 600 (14.7%) μm classes (ovulated eggs and advanced vitellogenic oocytes, respectively). Bimodal distributions were present in 80% of the LHRHa-treated females. Once oocyte final maturation was triggered by LHRHa the time needed for ovulation was about 48 h and the interval between consecutive ovulations and spawnings seemed to be 48–72 h.     

Triggering ovulation with 250 μg or 500 μg of r-hCG in oocyte donors treated with antagonist protocol has no effect on the number of mature oocytes retrieved: a randomized clinical trial

The aim of this study is to compare two r-hCG doses to trigger ovulation (250 μg vs. 500 μg of r-hCG) in an oocyte donation program. A prospective, randomized study was conducted in 118 oocyte donors. Group DI received 250 μg and Group DII received 500 μg of r-hCG. Both the groups were homogeneous. No significant differences were found in the total dose of gonadotropins, duration of the treatment, total number of oocytes, or Metaphase II (MII)oocytes. The pregnancy rate per embryo transfer in the corresponding recipients was similar for both the groups (58.2% for DII recipients and 56.1% for DI recipients). Mild hyperstimulation was observed in 17 donors in Group DI (29%) and in 23 donors in Group DII (39%). No cases of severe ovarian hyperstimulation syndrome (SOHSS) were observed. In conclusion, a double dose of r-hCG in oocyte donors to trigger ovulation after stimulation with r-FSH and antagonist does not translate into a higher number of MII oocytes retrieved or into higher pregnancy rates among recipients. Our results confirm that the optimal dose to induce the final oocyte maturation with r-hCG is 250 μg, and that a higher dose does not add any benefit.     

A new classification of a preovulatory oocyte maturation stage suitable for the synchronization of ovulation in controlled reproduction of Eurasian perch Perca fluviatilis L

To improve controlled reproduction of Eurasian perch Perca fluviatilis, the criteria for the evaluation of final oocyte maturation stages were revised. The new classification covers six preovulatory maturational stages (I -VI) from the end of vitellogenesis to germinal vesicle breakdown (GVBD) and was based on macroscopic changes of preovulatory oocytes (position of the germinal vesicle, GVBD, oil droplets coalescence). The observation was performed during out-of-season artificial reproduction with the use of a single hCG injection (500 IU/kg). The classification was subsequently verified with the controlled reproduction of wild female perch with the use of hormonal stimulation (500 IU hCG/kg of body weight at 12°C). The females were at different maturational stages and constituted respective experimental groups (I-VI). During the experiment, ovulation appeared to be considerably synchronized within particular groups. Statistical differences in latency time (time between hormonal treatment and ovulation) were found between experimental groups (mean latency time: 110, 92, 68, 49, 29 and 18 h in groups representing VI, V, IV, III, II and I stage of the proposed classification, respectively). The proposed classification and the results presented in the study allowed for effective synchronisation of ovulation. The use of our new oocyte maturation classification may positively influence the effectiveness of Eurasian perch production.     

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

The process of oocyte maturation in the canine species is unique among mammals: oocytes are immature at ovulation and the resumption and progression of meiotic maturation occur in the oviduct. This study was performed to investigate (i) the effect of co-culture with infundibulum and ampullar oviductal epithelial cells on the in vitro maturation of canine oocytes and (ii) the culture time necessary to reach full meiotic maturation. For this purpose the oocytes, collected from the ovaries of bitches undergoing ovariectomies, were divided into three groups and cultured for 48 and 72 h with the following systems: (A) TCM 199 + 10% oestrus bitch serum + FSH (0.1 IU.mL(-1)), LH (0.1 IU.mL(-1)) + progesterone (1 microg.mL(-1)) + oestradiol (1 microg.mL(-1)) + cysteamine (100 microM); (B) medium A plus infundibulum cells; (C) medium A plus ampullar cells. Infundibulum and ampullar cells were recovered from the oviducts of bitches at the oestrus stage of their cycle. The results showed that after 48 h of incubation, a significantly higher meiotic resumption (P < 0.01) was observed in the oocytes cultured with infundibulum (59%) and ampullar cells (60.0%), than in the control group (40.0%). There was also a significantly (P < 0.01) higher meiotic progression to the MII in systems B and C (15.6% and 16.7%) than in system A (4.0%). After 72 h of culture, the percentages of meiotic resumption and progression were unchanged. These results showed that both the infundibulum and the ampullar oviductal epithelial cells positively influence the meiotic resumption and progression of canine oocytes and that 48 h are sufficient for the completion of nuclear maturation.     

Stimulation of parthenogenesis in mouse ovarian follicles by inhibitors of inosine monophosphate dehydrogenase

The effects of hormonal priming and inosine monophosphate (IMP) dehydrogenase inhibitors on the meiotic maturation and parthenogenetic activation of mouse oocytes were examined in this study. In the first series of experiments, unprimed mice or mice primed 24 h with equine chorionic gonadotropin (eCG) received injections of the IMP dehydrogenase inhibitors, bredinin (Br) or mycophenolic acid (MA), followed by histological examination at 24 h, 48 h, and 72 h after drug administration. In both treatment groups, oocytes from nonatretic antral follicles were stimulated to undergo germinal vesicle breakdown by 24 h and became parthenogenetically activated as manifested by pronuclear formation and early cleavage divisions. The parthenotes underwent degeneration by 72 h. In the second part of this study, the effects of priming and drug treatment on parthenogenetic activation and subsequent developmental potential in vitro were examined. Mice were primed with eCG, and 24 or 48 h later received injections of Br or MA. Cumulus cell-enclosed oocytes were isolated 21-22 h later and assessed for maturation; those having undergone germinal vesicle breakdown were cultured and subsequently examined for embryonic development. In mice primed for 24 h, but not 48 h, Br and MA stimulated a significant number of oocytes to resume maturation in vivo; these subsequently underwent activation and developed to blastocysts in vitro. In another series of experiments, germinal vesicle-stage oocytes were isolated from primed or unprimed mice and cultured in vitro to permit spontaneous meiotic maturation. Nine percent of mature ova from 24-h-primed mice developed to 2-cell parthenotes; activation in ova from unprimed and 48-h-primed mice was considerably lower. A time-course experiment demonstrated that the extent of parthenogenetic activation in vivo following Br treatment was related to the period of time between drug injection and isolation of ova, the optimal period being 12 h. Neither Br nor MA had a direct activating effect on the oocytes as evidenced by an inability to induce parthenogenesis in vitro. Simultaneous injection of hCG with either Br or MA stimulated ovulation and prevented the parthenogenetic response. These data are consistent with the idea that conditions within the follicle promote parthenogenetic activation when the oocyte matures in the absence of gonadotropin stimulation.     

Nuclear maturation of domestic cat ovarian oocytes in vitro.

Using the domestic cat as a model for salvaging genetic material from rare Felidae, we collected oocytes from ovarian tissue and placed them in 1 of 3 treatments to observe time-related, meiotic changes of in vitro oocyte maturation. Oocytes obtained from ovaries collected at ovario-hysterectomy were assigned to 1 of 3 treatment groups: 1) modified Krebs-Ringer bicarbonate buffer (mKRB) + 4% BSA and 5 micrograms/ml FSH (+FSH, n = 499); 2) mKRB + 4% BSA (-FSH, n = 502); or 3) mKRB + 5% natural estrus cat serum (NE, n = 873). They were placed in the respective media in a 5% CO2 humidified environment at 38 degrees C. Beginning at 16 h, oocytes were removed at 4-h intervals through 48 h, and the meiotic status was evaluated by means of cytogenetic analysis. On the basis of chromosomal analysis, each cell was placed into one of the following categories: metaphase II (MII); metaphase I (MI); pre-MI (germinal vesicle [GV], GV breakdown, or diakinesis); degenerate or unidentifiable. The percentage of oocytes with degenerate chromatin increased over time in all culture treatments, but was always greatest (p less than 0.05) in the NE group. In the +FSH and -FSH treatments, the proportion of oocytes with nuclear material reaching MII increased with time in culture to 32 h and was equal to or greater than the proportion of oocytes with pre-MI + MI chromatin at this time interval (-FSH, 55%; +FSH, 38%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of LH releasing hormone on the state of gametes and the maturation of follicular oocytes in vitro

Ovulation, gametogenesis and maturation of rat follicular oocytes were examined in vitro under the effect of single and repeated injections of LH-RF. It was shown that LH-RF injection led to incomplete ovulation and to alteration of the heterogeneity of the gametes as regards the degree of maturation and the number of the degenerating forms. At the same time it did not produce any substantial effect on the rate of chromosomal abnormalities. The pattern of maturation of follicular oocytes from rats given LH-RH attests to the enhancement of atresia in the ovaries.     

The effect of growth hormone (GH) and insulin-like growth factor-I (IGF-I) on in vitro maturation of equine oocytes

Summary The objective of this study was to test the hypothesis that equine growth hormone (eGH), in combination with insulin growth factor-I (IGF-I), influences positively in vitro nuclear and cytoplasmic maturation of equine oocytes. Cumulus-oocyte complexes were recovered from follicles that were < 25 mm in diameter, characterized by morphology and were allocated randomly as follow: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 400 ng/ml anti-IGF-I antibody. Oocytes were matured for 30 h at 38.5°C in air with 5% CO2 and then stained with 10 μg/ml propidium iodide (PI) to evaluate nuclear status and 10 μg/ml Lens culinaris agglutinin-fluorescein complex (FITC-LCA) to assess cortical granule migration by confocal microscopy. The proportion of immature oocytes that developed to the metaphase II (MII) stage in the eGH + IGF-I group (15 of 45) was greater than in the groups that were treated only with IGF-I (7 of 36, p = 0.03). Oocytes that reached MII in the control group (20 of 56; 35.7%) showed a tendency to be different when compared with eGH + IGF-I group (15 of 45; 33.3%, p = 0.08). The treated group that contained anti-IGF-I (15 of 33; 45.4%) decreased the number of oocytes reaching any stage of development when compared with eGH (47 of 72; 65.3%) and eGH + IGF-I (33 of 45; 73.3%) groups (p = 0.05) when data from MI and MII were combined. We concluded that the addition of eGH to in vitro maturation (IVM) medium influenced the in vitro nuclear and cytoplasmic maturation of equine oocytes. The use of GH and IGF-I in vitro may represent a potential alternative for IVM of equine oocytes.     

Effects of a GnRH agonist on oocyte number and maturation in mice superovulated with eCG and hCG

The objective was to investigate the effects of a gonadotropin-releasing hormone agonist (GnRH) on ovulation rate and the number and maturation of oocytes in mice superovulated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). Thirty 3-month-old BALB/C female mice (weight: 25-30 g) were assigned to three experimental groups: control, superovulated, and superovulated with GnRH pretreatment (n=10 per group). Control mice received an i.p. injection of 0.1 ml physiological saline solution. Superovulation was induced with 5 IU eCG (i.p.) and 5 IU hCG 48 h later. Mice in the superovulated with GnRH pretreatment group were given GnRH (20 mg/kg Fertirelin, i.m.), 24 h before superovulation. Thirteen hours after hCG administration, mice were sacrificed by cervical dislocation and blood samples were collected to determine serum progesterone concentration (by radioimmunoassay). Ovaries and oviducts were also harvested to enumerate corpora lutea and cumulus-enclosed oocytes. Progesterone concentrations were not significantly different among groups. The oocyte number and the maturation, ovulation rate, and the number of corpora lutea were higher in GnRH-treated mice than both controls and superovulated mice. In conclusion, GnRH given 24 h before superovulation with eCG-hCG increased the number and maturation of oocytes and the rate of ovulation in mice.     

Bovine oocyte development following different oocyte maturation and sperm capacitation procedures

Various procedures have been reported for successful in vitro maturation and in vitro fertilization (IVM/IVF) of bovine follicular oocytes. Direct comparisons of these different recommended procedures have been rare. In this research, involving a total of 5,128 oocytes, a series of experiments were conducted to compare oocyte maturation, fertilization, and development in vitro with 2 maturation systems (with or without added hormones) and 3 types of sperm treatment procedures. Oocytes were collected from ovarian antral follicles (2–7 mm in diameter) within 3 hr after slaughter of cows or heifers. Those with intact or at least 4 layers of cumulus cells were selected for IVM/IVF. Oocytes were incubated for 22 hr in either Medium 199 with 7.5% fetal calf serum (M199 + FCS) alone or M199 + FCS with added hormones (M199 + FCS + H; oFSH 0.5 μg/ml, oLH 5.0 μg/ml, and E₂ 1.0 μg/ml) at 39°C in 5% CO₂ and 95% air. For IVF, frozen-thawed sperm were treated with either 0.1 μM calcium ionophore A23187 (A23187) for 1 min, or 10 or 100 μg/ml heparin (H10 or H100) for 15 min. Our results demonstrated the following: (1) both M199 + FCS and M199 + FCS + H supported maturation development to the metaphase II stage (90–95%, P > 0.05); (2) when oocytes were matured in M199 + FCS without added hormones, A23187 sperm treatment was superior to H10 or H100 treatment for fertilization and blastocyst development of the inseminated oocytes (P < 0.05); (3) when oocytes were matured in M199 + FCS + H, A23187 treated sperm again produced a higher fertilization rate than the H10 group (P < 0.05), but the development to the blastocyst stage was similar among all 3 sperm treatment groups (P > 0.05); (4) direct comparison of the 2 maturation systems with A23187 treated sperm resulted in no difference in all criteria measured; however, (5) when compared retrospectively, beneficial effects of added hormones are evident for blastocyst development (but not for fertilization) when sperm were treated with heparin procedures. © 1993 Wiley-Liss, Inc.     

In vitro maturation of pig oocytes with different media, hormone and meiosis inhibitors

This study evaluated in vitro maturation of pig oocytes in two maturation media (TCM199 and NCSU23) supplemented with 10% porcine follicular fluid (pFF) or 0.1% polyvinyl alcohol (PVA) and four hormonal treatments. The best media was then used to evaluate the effect of reversible meiosis inhibitors cycloheximide (5 microgram/ml) [DOSAGE ERROR CORRECTED]and butyrolactone I (12.5M) on the maturation of pig oocytes was evaluated. After maturation for 44 h, the oocytes were fixed, stained, and examined under epifluorescence microscopy. The comparison of the proportion of oocytes in metaphase II revealed that hormonal treatment 2(incubation for 22 h - 10 ng EGF/ml, 10 IU hCG/ml and 10 IU eCG/ml, followed by incubation for 22 h - 10 ng EGF/ml) presented higher repeatability percentages: TCM+ PVA (54.5% - 61/112); TCM+ pFF (65.0% - 63/97);NCSU23 + PVA (54.6% - 65/119), and NCSU23 + pFF (58.1% - 61/105). The comparison of maturation media showed that TCM199 presented more constant results than NCSU23. Regarding supplementation with pFF or PVA, TCM199 with pFF presented better results. The comparison between butyrolactone I and cycloheximide demonstrated that both drugs effectively inhibited meiosis; however, only cycloheximide presented metaphase II percentages similar to the control (70.29% and 75.49%, respectively). In conclusion, it is recommended the use of TCM199 medium supplemented with pFF and hormonal treatment with 10 ng EGF/ml, 10 UI hCG/mland 10 UI eCG/ml during the first 22 h and more 22 h with 10 ng EGF/ml for the pig oocytes maturation. Butyrolactone I and cycloheximide effectively arrested/resumpted maturation; however, the oocytes percentages in metaphase II was the same for both cycloheximide and the control groups.     

In-vivo and in-vitro maturation rate of oocytes from two strains of mice

Female mice of the KE and CBA strains were used to examine the rate of oocyte maturation in vivo and in vitro. In CBA females killed just before ovulation most preovulatory oocytes were already in the metaphase II stage, while the oocytes of KE mice were arrested at metaphase I until the time of ovulation, and further stages of maturation occurred in the oviduct, reaching the metaphase II stage 3-5 h later. A similar strain difference in oocyte maturation rate was observed from in-vitro culture of cumulus-free oocytes, isolated from the ovaries of PMSG-primed females and intact females killed at the metoestrous phase of the cycle. This indicates that the strain-specific course of maturation is determined in the oocyte by a few days before ovulation. Therefore, if the rate of oocyte maturation is influenced by somatic components of the follicle, this must occur at some earlier stages of follicle development.     

In vitro maturation of oocytes from a marsupial,the tammar wallaby (Macropus eugenii)

This study examined the competence of oocytes from the tammar wallaby, Macropus eugeniio mature in vitro. Oocytes were collected from follicles >1 mm diameter 24 h after pregnant mare serum gonadotrophin (PMSG) treatment and incubated in Eagle's minimum essential medium supplemented with 10% fetal calf serum, at 35°C in 5% CO₂ in air for 24, 36, or 48 h. Oocytes were incubated either granulosa cell-intact or granulosa cell-free or in the presence of 10 IU ml^?1 PMSG or 10 μg ml^?1 porcine luteinizing hormone (LH) + 10 μg ml^?1 porcine follicle stimulating hormone (FSH). The ability of oocytes recovered from small (<1.5-mm-diameter) and large (≥1.5-mm-diameter) follicles to mature in vitro was also examined. The nuclear status of oocytes was assessed using the DNA-specific dye Hoechst 33342. Initially, all oocytes examined contained a germinal vesicle. After 24 h of culture, 60% of oocytes had progressed to metaphase I or anaphase I. After 36 h, approximately 20% of oocytes possessed metaphase II chromosomes, and 20% of oocytes were at metaphase I or anaphase I. At the completion of the 48 h culture period, 40% of oocytes had completed maturation to the metaphase II stage. In vitro oocyte maturation after 48 h was not affected by the presence of granulosa cells, PMSG, or LH and FSH. More oocytes from large follicles (55%) completed maturation by 48 h than from small follicles (20%). Approximately 50% of oocytes remained at the GV stage at all times under all conditions. Marsupial oocytes thus undergo spontaneous nuclear maturation once removed from the follicular environment, suggesting a basically similar control system to that in placental mammals. © 1993 Wiley-Liss, Inc.     

Effect of intermittent injections of gonadotrophin releasing hormone at various frequencies on the release of luteinizing hormone and ovulation in dairy cows

Gonadotrophin releasing hormone (GnRH, 5 μg every 4 h) was administered to six dairy cows between days 5 and 10 post-partum and the release of luteinizing hormone (LH) and the onset of ovulation were determined. LH was measured using a specific radioimmunoassay and the occurrence of ovulation was assessed from changes in the concentration of progesterone in milk. Treatment with GnRH resulted in a median time of first ovulation of 17.0 days after calving. This was less (P < 0.05) than that observed for control cows (21.5 days, n = 7). Determinations of plasma LH concentrations over an 8-h period on days 6 and 10 post-partum indicated that there was a tendency for GnRH-treated cows to have higher levels of LH on these days. The 5 μg dose of GnRH did not repeatably induce a release of LH between days 6 and 10. Endogenous pulsatile release of LH did, however, increase in frequency from 3.18 pulses per 8 h on day 6 to 5.18 pulses per 8 h on day 14 post-partum (P < 0.01).In a second experiment groups of 20 cows were treated with either 5 μg GnRH every 4 h or 15 μg GnRH every 12 h from days 5 to 10 post-partum. Seventeen untreated cows served as controls. The median times to first ovulation were 27.0 days for the control cows, 22.5 days for those cows treated with 5 μg GnRH every 4 h and 17.0 days for cows treated with 15 μg every 12 h. The latter treatment significantly advanced the time of first ovulation (P < 0.05) relative to controls. This difference had, however, disappeared by the time of the second and third ovulations. Primiparous cows ovulated later (P < 0.01) than the pluriparous cows in the group treated with 5 μg GnRH every 4 h. This was a major reason for the lack of effect of this treatment. Some treated cows were blood sampled at frequent intervals on day 8 to evaluate the LH responses to GnRH injections. The administration of 5 μg GnRH on day 8 did not elicit a pulse of LH which could be distinguished from endogenous pulsatile secretion at this time. The dose of 15 μg on this day did, however, elicit a more defined pulse on some, but not all, occasions.The injection of a small dose of GnRH twice a day from day 5 to day 10 after calving, therefore, advanced the time of first ovulation in dairy cows by 10 days.     

Influence of culture medium osmolality on maturation and ovulation of common frog oocytes stimulated in vitro by pituitary extract or progesterone

The influence of diluted Ringer solution on ovulation and maturation of common frog oocytes stimulated in vitro by homologous pituitary extract (0.005 pit/ml) or progesterone (1 μg/ml) was studied. During hibernation, the dilution of Ringer solution led to a decreased percentage of oocytes ovulated and matured under the influence of both inducers. As the season of reproduction approached, the dependence of oocyte maturation and ovulation on the Ringer solution dilution was reduced. Possible causes of different dependence of the ovulation of amphibian and sturgeon oocytes stimulated by gonadotropic hormones or progesterone on the culture medium osmolality is discussed.