Standardization and validation of an induced ovulation model system in buffalo cows: Characterization of gene expression changes in the periovulatory follicle

In bovines characterization of biochemical and molecular determinants of the dominant follicle before and during different time intervals after gonadotrophin surge requires precise identification of the dominant follicle from a follicular wave. The objectives of the present study were to standardize an experimental model in buffalo cows for accurately identifying the dominant follicle of the first wave of follicular growth and characterize changes in follicular fluid hormone concentrations as well as expression patterns of various genes associated with the process of ovulation. From the day of estrus (day 0), animals were subjected to blood sampling and ultrasonography for monitoring circulating progesterone levels and follicular growth. On day 7 of the cycle, animals were administered a PGF(2alpha) analogue (Tiaprost Trometamol, 750 microg i.m.) followed by an injection of hCG (2000 IU i.m.) 36 h later. Circulating progesterone levels progressively increased from day 1 of the cycle to 2.26+/-0.17 ng/ml on day 7 of the cycle, but declined significantly after PGF(2alpha) injection. A progressive increase in the size of the dominant follicle was observed by ultrasonography. The follicular fluid estradiol and progesterone concentrations in the dominant follicle were 600+/-16.7 and 38+/-7.6 ng/ml, respectively, before hCG injection and the concentration of estradiol decreased to 125.8+/-25.26 ng/ml, but concentration of progesterone increased to 195+/-24.6 ng/ml, 24h post-hCG injection. Inh-alpha and Cyp19A1 expressions in granulosa cells were maximal in the dominant follicle and declined in response to hCG treatment. Progesterone receptor, oxytocin and cycloxygenase-2 expressions in granulosa cells, regarded as markers of ovulation, were maximal at 24h post-hCG. The expressions of genes belonging to the super family of proteases were also examined; Cathepsin L expression decreased, while ADAMTS 3 and 5 expressions increased 24h post-hCG treatment. The results of the current study indicate that sequential treatments of PGF(2alpha) and hCG during early estrous cycle in the buffalo cow leads to follicular growth that culminates in ovulation. The model system reported in the present study would be valuable for examining temporo-spatial changes in the periovulatory follicle immediately before and after the onset of gonadotrophin surge.     

Initiation of follicular maturation during mid-pregnancy by the administration of LH in the rat

Pregnant rats were injected twice daily for 1-3 days (Days 13-16 of pregnancy) with various doses of ovine LH. Follicular maturation was determined by the ability of the follicles to ovulate in response to 10 i.u. hCG as well as by endogenous production of oestradiol-17 beta and inhibin. In control animals, no ovulation was induced by hCG given on Day 16 of pregnancy. An injection of hCG on Day 16 of pregnancy, however, induced ovulation in LH-treated animals (6.25-50.0 micrograms LH per injection, s.c. at 12-h intervals from Days 13 to 16). Concentrations of oestradiol-17 beta and inhibin activity in ovarian venous plasma increased after the administration of LH, indicating that development of ovulatory follicles had been induced. Abolishing the decline in plasma LH values therefore induced maturation of a new set of follicles or prevented the atresia of large antral follicles usually seen at this time of pregnancy. Plasma and pituitary concentrations of FSH decreased in LH-treated animals compared with those in control animals. Concentrations of progesterone, testosterone and oestradiol-17 beta in the peripheral plasma were not significantly different between the two groups. These results suggest that the increase in inhibin secretion from the ovary containing maturing follicles after LH treatment may suppress the secretion of FSH from the pituitary gland. These findings indicate that (1) the development of ovulatory follicles can be induced by the administration of exogenous LH during mid-pregnancy in the rat and (2) basal concentrations of FSH are enough to initiate follicular maturation even in the presence of active corpora lutea of pregnancy, when appropriate amounts of plasma LH are present.     

hCG treatment on day of mating improves embryo viability and fertility in ewe lambs

An investigation was conducted to determine the effects of supplementing hCG at mating time on luteal function, conceptus growth, placentation and reproductive performance in TexelxClun Forest ewe lambs. After oestrus synchronisation with progestagen sponges and PMSG ewe lambs were treated either with normal saline (n=102) or 150 IU of hCG (n=105) at mating time. At 25 days after mating 24 animals were slaughtered from each group to determine embryo viability and placental development. hCG significantly (P<0.05) increased crown-rump length (saline: 11.9+/-0.2 mm; hCG: 12.7+/-0.2 mm), amniotic sac width (saline: 11.4+/-0.4 mm; hCG: 12.0+/-0.3 mm) and the number of placentomes (saline: 90.8+/-7.3; hCG=122.4+/-6.3). Among the pregnant animals that were slaughtered on 25 days post-mating, ovulation rate tended to be higher in the hCG group compared to controls (saline: 1.16; hCG: 1.54). However, no difference was observed either in mean plasma progesterone concentrations (saline: 4.6 ng/ml; hCG: 4.9 ng/ml; sed 0.56) or in progesterone production from luteal slices when cultured in vitro (saline: 239.6+/-11.8 ng/mg; hCG: 263.2+/-13.6 ng/mg) between controls and hCG treated animals. Reproductive performance was also recorded in ewe lambs that were either treated with saline (n=78) or hCG (n=81). The total number of lambs born (saline: 38; hCG: 58) was significantly (P<0.05) greater in hCG group compared to saline-treated controls. Both lambing percentage (saline: 36%; hCG: 48%) and litter size (saline: 1.35; hCG: 1.48) tended to be greater (P<0.10) in hCG-treated animals compared to the controls. In conclusion, these data suggest that treatment of ewe lambs with hCG at the time of mating improves conceptus growth, placentation and number of lambs born.     

Inhibition of human chorionic gonadotropin-induced ovulation and steroidogenesis by short-term hyperprolactinemia in female rabbits

To clarify the possible direct effects of hyperprolactinemia on the ovulatory process, we experimentally established hyperprolactinemia in female rabbits with 4 daily injections of sulpiride (SLP) at different doses and induced ovulation with human chorionic gonadotropin (hCG). Plasma levels of prolactin (PRL) were increased significantly before hCG injection in each SLP-treated group compared with the corresponding values for the controls. The ovulation rates at 14 h after hCG were significantly reduced in the 16 and 24 mg/kg/day SLP-treated groups. An inverse correlation (r = -0.74, P less than 0.001) was found between the ovulation rate and the increasing in plasma PRL measured just prior to hCG injection. The increase in peripheral as well as ovarian venous progesterone and 20 alpha-hydroxypregn-4-en-3-one(20 alpha-OHP) at 4 and 14 h after hCG injection in inhibited ovulation groups was much less than in the control group. However, the estradiol, androstenedione and testosterone concentrations were comparable with the control values. These results indicate that hypersecretion of PRL induced by SLP has a direct effect on ovary by inhibiting follicular rupture induced by hCG and this inhibitory effect was partly due to the suppression of progesterone secretion during the course of ovulation. This may be one of the causes leading to hypogonadism during hyperprolactinemia.     

Dissociation of copulation from ovulation in pregnant rabbits

Experiments were designed to determine why copulation in the pregnant rabbit does not terminate pregnancy while treatment with ovulatory doses of luteinizing hormone (LH) human chorionic gonadotropin (hCG) or luteinizing hormone-releasing hormone (LHRH) is known to do so. Pregnant rabbits (Day 8) were mated or were injected with hCG (25 IU/doe) or LHRH (1, 10 micrograms/kg). Serial blood samples were collected over the next 72 h and analyzed for content of LH, follicle-stimulating hormone (FSH) and progesterone. At sacrifice, uteri and ovaries from these animals were examined for viability of the embryos and for signs of recent ovulation. Injection of hCG or LHRH into pregnant animals led to ovulation and to patterns of LH, FSH and progesterone secretion like those which precede ovulation in estrous rabbits. However, mating the pregnant does did not lead to ovulation or to any changes in the circulating hormones. To investigate whether the elevated levels of progesterone during pregnancy were responsible for the dissociation of coitus from ovulation, nonpregnant rabbits were injected with progesterone (2 mg/kg) and then mated or injected with hCG or LHRH. In virtually every respect, the numbers of ovulations and the patterns of hormone secretion in the progesterone-treated, nonpregnant rabbits mimicked those observed in the 8-day pregnant animals; injection of hCG or LHRH caused ovulation and hormonal surges while hCG caused ovulation only. Mating did not lead to ovulation or any change in blood levels of LH, FSH or progesterone. Taken together, the results show that the elevated circulating levels of progesterone, characteristic of pregnancy, are probably responsible for the dissociation of copulation from gonadotropin release in pregnant rabbits.     

Presence of follicular fluid in the porcine oviduct and its contribution to the acrosome reaction

Two experiments were conducted to measure the quantity of follicular fluid entering the porcine oviduct following ovulation and to establish its influence on the sperm acrosome reaction in vivo. Prepubertal gilts treated with pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) were used in both experiments. In experiment 1, each of 64 gilts was assigned at random to one of four treatment groups (n = 16 per group): I (preovulatory), surgery 38 hr post-hCG; II (ovulatory), (surgery 42 hr post-hCG; III (postovulatory), surgery 46 hr post-hCG; IV (ovulation blocked), surgery 46 hr post-hCG but also treated with indomethacin (INDO) at 24 hr. At surgery, both follicular and oviductal fluid were collected for determination of volume and progesterone (P4) concentration. In experiment 2, sperm were recovered surgically from the uterine horn, isthmus, and ampulla of gilts at 46 hr post-hCG either 1) inseminated and non-INDO-treated controls (n = 5) or 2) inseminated and INDO-treated at 24 hr (n = 4). Using P4 as a marker, it was calculated that only 0.51% +/- 0.10% of the available follicular fluid was present in the oviduct near the time of ovulation and that this amount had decreased 10-12-fold 4 hr later. Mean sperm concentration at 46 hr post-hCG was higher in the uterine horn than in the other two regions (P less than 0.05) but the percentage of acrosome-reacted sperm was greater in the ampulla (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian follicular fluid, progesterone and Ca2+ ion influences on sperm release from the fallopian tube reservoir.

As a means of determining whether ovarian follicular fluid reaches the functional sperm reservoir in the caudal isthmus of the Fallopian tube shortly after ovulation, 0.01-0.02 ml aliquots of whole or steroid-free follicular fluid were introduced into the distal extremity of the isthmus within 1 hr before ovulation. Eggs were recovered during a second intervention 4 hr 45 min-6 hr 10 min after treatment and examined by phase-contrast microscopy for the normality of fertilisation. In a separate experiment, 0.01-0.02 ml aliquots of 10 microM calcium ionophore solution were introduced into the same site in comparable animals. Sixty-nine fertilised eggs were recovered from 12 fallopian tubes treated with whole follicular fluid, of which 24 (34.8%) were polyspermic. The 12 contralateral control tubes (PBS-treated) yielded 47 fertilised eggs, of which only one (2.1%) was polyspermic (P < 0.001). Steroid-free aliquots of the same follicular fluid introduced bilaterally into eight fallopian tubes (4 animals) resulted in recovery of 59 fertilised eggs, of which only one (1.7%) was polyspermic. Treatment with ionophore solution yielded a 41.6% incidence of polyspermy (10 of 24 eggs from four tubes) compared with 3.8% polyspermy (1 egg) from the control tubes (P < 0.01). Dispermy was the principal form of polyspermy. The numbers of accessory spermatozoa on/in the zona pellucida were increased by the experimental treatment. Follicular fluid passing down the fallopian tube ampulla at ovulation was therefore considered not to be the physiological stimulus for an initial, tightly-controlled release of spermatozoa from epithelial binding in the caudal isthmus. Indeed, because such sperm activation commences shortly before ovulation, a locally transmitted ovarian programming with relatively high concentrations of follicular hormones remains the favoured model. Although pre-ovulatory progesterone is considered to be the coordinating steroid of increasing influence in these pre-fertilisation events, its effects are proposed to be modulated in the endosalpinx by mobilisation of Ca2+ ions into a discrete population of bound spermatozoa. Results of the steroid-free follicular fluid and calcium ionophore treatments stand in support.     

Induction of ovulation in gilts with cloprostenol

Prepuberal gilts were treated with 750 IU pregnant mare serum gonadotropin (PMSG) followed 72 h later by 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. In this model, ovulation occurred at 42 +/- 2 h post hCG treatment. When 500 mug of cloprostenol was injected at 34 and of 36 h after hCG injection, 78% of the preovulatory follicles ovulated by 38 h compared with 0% in the control gilts. In addition, plasma progesterone concentrations were significantly higher in the cloprostenol-treated group than in the control group (P<0.01) at 38 h, indicating luteinization along with premature ovulation. These results suggest that prostaglandin F(2)alpha (PGF(2)alpha) or an analog can be used to advance, synchronize or induce ovulation in gilts.     

Effects of castration and estradiol treatment on the postovulatory secretion of follicle-stimulating hormone in the mated rabbit

The effects of castration on the postovulatory secretion of follicle-stimulating hormone (FSH) was measured in mated rabbits. When ovaries were removed at 12 or 18 h postcoitum, FSH increased within 24 h of surgery but without evidence of the previously observed pattern of FSH secretion in the postovulatory period. To prevent the postcastration rise in FSH, various doses of estradiol were injected into does castrated 12 h after mating. Two micrograms estradiol/kg, given daily, was found to prevent the postcastration rise of FSH but was not sufficient to suppress the postovulatory secretion of FSH in intact animals. The postovulatory pattern of FSH release was disrupted in does castrated at either 12 or 18 h postcoitum despite adequate estradiol replacement therapy. Furthermore, in chronically castrated does treated with estradiol (2 micrograms/kg per day), neither mating nor human chorionic gonadotropin (hCG) injection elicited any change in blood FSH levels even though both treatments have been previously found to cause a postovulatory FSH surge. The results of these studies indicate that the ovary, by way of some ovarian secretion, is required for the postovulatory secretion of FSH in the rabbit. The necessary ovarian factor does not appear to be estradiol.     

Pre-ovulatory arrest and peri-ovulatory redistribution of competent spermatozoa in the isthmus of the pig oviduct

Using the surgical approach of post-coital ligation and transection of the distal oviduct at different times relative to ovulation, together with subsequent recovery of the eggs, gilts mated at the onset of oestrus were studied for progression of viable spermatozoa within the isthmus. Results are derived from 76 animals and examination of 1047 eggs. Transection of the isthmus 1.5-2.0 cm proximal to the utero-tubal junction at intervals from 3 to 36 h after mating prevented fertilization in 269 of 270 eggs, whereas 98% of 223 eggs were fertilized in the control oviducts. Transection at 38 h (pre-ovulatory), 40 h (peri-ovulatory) and 42-44 h (post-ovulatory) after mating yielded, respectively, 5%, 40% and 100% fertilization. The mean number of spermatozoa associated with the zona pellucida increased in a parallel manner. These results, and those obtained with ligatures placed closer to the site of fertilization just after ovulation, indicate a pre-ovulatory arrest of viable spermatozoa in the caudal region of the isthmus for 36 h or more followed by an active ad-ovarian redistribution.     

Inhibition of ovulation in PMSG/hCG-treated immature rats by rotenone, a specific inhibitor of mitochondrial oxidation

Immature Wistar rats were induced to ovulate by treatment with PMSG and hCG. Control animals ovulated 43.5 +/- 0.36 ova/rat. Intraperitoneal injection of rotenone doses of 0.125, 0.25 and 0.50 mg/kg reduced the ovulation rate to 24.0 +/- 3.08, 8.0 +/- 0.88 and 1.5 +/- 0.44 ova/rat, respectively. The rotenone significantly reduced ovarian cytochrome oxidase activity and progesterone production, but not production of oestradiol or testosterone. Thyroxine treatment at a dose of 5 mg/kg s.c. reversed the rotenone inhibition of ovulation. The results suggest that an increase in mitochondrial respiration is an essential feature of the ovulation process in mammals.     

Reproductive management of mares without detection of oestrus.

The use of photoperiod, progestagen, prostaglandin and hCG treatments was investigated to obtain mating of mares at predetermined times. The objectives were: (1) synchronization of oestrus at an early time of the year, (2) simplification of treatment schedules by use of vaginal sponges, and (3) use of several controlled cycles by successive synchronization. The following conclusions were reached. First, after a 16 h photoperiod was applied beginning on 25 November, hormonal synchronization of oestrus and ovulation followed by cyclicity were obtained on 1 February; i.e. 2 months of light are essential as hormonal synchronization of ovulation was not obtained by 10 January. Second onset of oestrus was well synchronized after vaginal application of progestagens (3.75 days +/- 0.98 s.d. after withdrawal) and in spite of vaginal irritation, fertility was high (71%, N = 24) after mating every 48 h of the induced oestrus. Third, for synchronization of return to oestrus in mated non-conceiving mares, oral progestagens were given from Days 7 to 21 after mating. Predetermined mating (Days 27 and 29) and hCG injection (day 28) for non-pregnant animals were decided after a progesterone assay of Day 21 blood plasma. After 3 controlled mating periods, the cumulative fertility was 88% (N = 24) in non-lactating mares and 58% (N = 19) in lactating mares. Programmed reproductive management is possible in the horse.     

Relation between mating or ovulation and the duration of gestation in dogs

The effects of day of mating and litter size on gestation length in dogs were studied in 36 beagle bitches (age 2-10 yr). The day that plasma progesterone concentrations exceeded 2 ng/mL was considered the day of ovulation; dogs were randomly assigned to be bred once, 1-5 days after the estimated day of ovulation. The interval from mating to parturition was negatively correlated with the number of days from estimated ovulation to mating (P < 0.01). Gestation length (interval from ovulation to parturition) was almost constant at 63.9 +/- 0.2 days (mean +/- S.E.M.), with no significant relationship between the number of fetuses and the duration of gestation.     

Effect of melatonin on postpartum anestrus in beef cows

The effect of melatonin treatment on intervals from calving to first postpartum estrus and ovulation was determined in Shorthorn cows which calved May 8 to June 14. Melatonin (500 mg in beef tallow) was injected subcutaneously (s.c.) into 20 cows on June 15 (4 to 38 d postpartum). Ovulation was determined from progesterone concentrations in jugular venous blood collected weekly from June to August. Mean intervals to first estrus and first ovulation were significantly longer in primiparous than in multiparous cows (85 +/- 4 vs 55 +/- 3 d and 83 +/- 4 vs 52 +/-3 d). Melatonin treatment caused a significant increase in the intervals to first postpartum estrus (68 +/- 4 vs 58 +/- 5d) and ovulation (68 +/- 4 vs 55 +/- 5 d). Mean plasma melatonin concentrations during the daytime were significantly higher in treated than in control cows one and two weeks after melatonin injection and were within the lower range of nighttime values reported previously for cows. Thus melatonin treatment raised daytime plasma concentrations of melatonin and delayed the onset of estrus and ovulation. These results support the possibility of a role of photoperiod through melatonin secretion in the onset of postpartum ovarian activity in cattle.     

孕马血清促性腺激素和氯地酚对猕猴超数排卵效果的观察

用孕马血清促性腺激素,总剂量为1550—2000单位,分4—7天处理猕猴,可促使其每侧卵巢出现滤泡超数发育,随后静脉注射入绒毛膜促性腺激素2500单位,在24小时内,猕猴即可出现超数排卵。     

Changes in the secretion of inhibin and steroid hormones during induced follicular atresia after hypophysectomy in the rat

Sequential changes in the function of antral follicles during the period of follicular atresia were investigated after hypophysectomy (Hypox) at 1100 hr on proestrus. Within 6 hours after Hypox, concentrations of progesterone (P), testosterone (T) and estradiol-17 beta (E) decreased abruptly in ovarian venous plasma (OVP) and follicles showed a reduced ability to ovulate. Six hours after Hypox, ovulation was still induced by human chorionic gonadotropin (hCG) in all animals but with significantly fewer number of oocytes compared to the group given hCG at 1100 hr on the day of proestrus. Nine hours after Hypox, several granulosa cells of all large follicles (greater than 400 microns in diameter) exhibited morphological signs of atresia. Twelve hours after Hypox, all large and medium sized (200-400 microns in diameter) follicles showed advanced stages of atresia and almost all follicles failed to ovulate in response to hCG. Inhibin activity in OVP declined more slowly compared to the profiles of steroid hormones and 53% of the initial inhibin activity was still maintained at 18 hours after the operation. Inhibin activity further decreased to 7% of the initial level at 24 hours and was undetectable by 48 hours after Hypox. These results suggest that fully developed Graafian follicles gradually lose their ability to secrete inhibin in contrast to the rapid decrease in secretion of steroid hormone during the process of atresia.     

Effects of circulating progesterone and insulin on early embryo development in beef heifers

The aims of this study were to determine the effect on early embryo development of feeding a diet formulated to enhance circulating insulin concentrations and secondly to investigate the association between early embryo development and maternal progesterone concentrations in beef heifers. The study was carried out in 32 Simmental x Holstein Friesian heifers 22-25 months of age weighing 506+/-7kg and in condition score 3.1+/-0.1. Animals were fed two diets that were isoenergetic and isonitrogenous, but that would encourage either propionate (diet A) or acetate (diet B) production in the rumen. The rationale was that propionate would induce a greater insulin release in response to feeding. Animals were fed a 50:50 mix of the two diets for 14 days at 0.8x maintenance, with straw provided ad libitum. Animals were then fed one of the experimental diets for 3 weeks prior to synchronisation of oestrus and insemination and for a further 16 days following mating. All heifers were blood sampled daily from oestrus synchronisation and eight animals on each diet underwent daily transrectal real-time ultrasonography to determine the day of ovulation. All heifers were slaughtered at Day 16 after mating. While feeding of diet A (propionic) caused a significant (P<0.05) increase in the plasma insulin to glucagons ratio differences in insulin were not significantly different. This is probably due to the fact that insulin concentrations were quite high as the heifers used in the present study were in good body condition making further increases in insulin difficult to achieve. Diet did not affect size of ovulatory follicle (DIET A: 15.1+/-0.7mm; diet B: 14.6+/-0.7mm), day of ovulation (diet A: 3.5+/-0.2 days; diet B: 3.4+/-0.2 days), mean plasma progesterone concentration (diet A: 4.7+/-0.4ng/ml; diet B: 5.2+/-0.3ng/ml), corpus luteum weight (diet A: 6.0+/-0.2g; diet B: 6.0+/-0.2g) or pregnancy rate (diet A: 81.3%; diet B: 81.3%). However, the proportion of well-elongated (>10cm) embryos on Day 16 was higher in animals fed diet A than in those fed diet B (84.6% versus 38.5%; P<0.05). While progesterone concentration did not differ between pregnant and non-pregnant heifers, progesterone did show an earlier post-ovulatory rise in heifers with well-elongated (>10cm) embryos with levels in these animals significantly higher on Days 4 and 5 than in heifers with small (<10cm) embryos at slaughter. This study demonstrated an enhancement in early embryo development in animals fed a diet generating an increased insulin:glucagon ratio that was not related to circulating maternal progesterone concentrations. However, across diets, enhanced embryo development was associated with elevated plasma progesterone on Days 4 and 5 following mating.     

Endocrine profiles and embryo quality in the PMSG-PGF2alpha treated cow

Plasma progesterone and LH concentrations around estrus were determined for both PMSG treated (experimental animals) and non-treated (control animals) dairy cows and heifers of the Holstein Friesian and Jersey breeds, and these hormone profiles were related to the embryo quality. Most experimental animals experienced an increase in progesterone concentrations following PMSG treatment and an abrupt decrease to values below 3 nmol/l after PG injection. The mean (+/-SE) intervals from prostaglandin treatment to estrus were 46.9+/-1.8 h and 64.5+/-4.8 h for experimental and control animals, respectively. At the onset of heat the progesterone concentration in experimental animals with optimal embryo quality (group I) was significantly lower (p<0.01) than in experimental animals which yielded unfertilized eggs (group II) (1.2+/-0.1 versus 3.9+/-0.8 nmol/l) and significantly higher than the level in the control group (0.6+/-0.1 nmol/l). Following estrus the progesterone profiles in all 3 groups were studied and the length of the superovulatory cycle was measured to 26.0+/-4.8 days. The preovulatory LH surge occurred sooner after prostaglandin injection in experimental (41 h) than in control animals (65 h). The LH surge in group I occurred within a narrow range and reached a higher average level than group II (24.2+/-2.2 ng/ml and 16.3+/-3.7 ng/ml, respectively). The control group attained an even higher LH surge (31.8+/-8.8 ng/ml) than did the experimental animals. The data presented in this experiment indicate that plasma levels of progesterone and LH in PMSG-PGF(2)alpha treated animals are related to embryo or egg quality.     

The relationship between ovarian progesterone and proteolytic enzyme activity during ovulation in the gonadotropin-treated immature rat.

To clarify the mechanisms by which progesterone acts as a mediator in the ovulatory process, ovulation rate and proteolytic enzyme activities were investigated in immature 22-day-old rats treated with PMSG/hCG, RU486 (10 mg/kg), synthetic antiprogesterone, and RU486 (10 mg/kg) + progesterone (10 mg/kg). The number of ova was significantly decreased when RU486 (10 mg/kg) was given from 2 h before to 4 h after the hCG injection. In addition, its inhibitory action on ovulation was reversed by exogenous progesterone (10 mg/kg) at 2 or 4 h after the hCG injection. Serum progesterone and estradiol concentrations in the rats treated with RU486 did not show any significant differences compared to controls. The proteolytic enzyme activities were measured by using the synthetic substrates alpha-N-benzoyl-DL-Arg-beta naphthylamide (BANA) and dinitrophenyl peptide (DNP). Activities were significantly increased after hCG injection in the control group during 8-9 h for BANA hydrolase and 7-10 h for DNP peptidase. The preovulatory increase of these activities was totally suppressed by RU486 with hCG. After administration of progesterone (10 mg/kg) following hCG and RU486 injection, the elevation of proteolytic, enzyme activities in the preovulatory phase was effectively reversed, and levels became similar to those in the control group. These results suggest that progesterone plays an indispensable role in the first 4 h of the ovulatory process by regulating proteolytic enzyme activities.     

Reproductive traits in the spur-thighed tortoise (Testudo graeca terrestris): new tools for the enhancement of reproductive success and survivorship

The Mediterranean tortoise (Testudo graeca) is listed as "Vulnerable" on the IUCN Red List. Reproductive characteristics and means to increase offspring production were studied in T. graeca terrestris in a semi-natural environment. Courtship and mating occurred during early spring for about 4 weeks, followed by a laying season of approximately 2 months, with a second, shorter mating period in the fall. During the first mating, calcified eggs were already present in the uterus; we inferred that sperm from both mating seasons were stored in the oviduct for fertilization of eggs of the second laying cycle and of the following year. Average egg production was 3.8+/-0.3 eggs/year. Most females laid all of their eggs in a single clutch, but 18% laid in a second clutch, 11-21 days later. X-ray radiography revealed calcified eggs in the uterus about 4 weeks before oviposition. All eggs in the uterus were calcified simultaneously and were laid in a single clutch; if a second clutch developed, those eggs were also calcified simultaneously. Based on endoscopic examinations, ovaries were active throughout the entire year. Plasma progesterone concentrations in females were very low and were detected only soon after oviposition ( 440 +/-141 pg/ml). Plasma estradiol concentrations in females varied from 4.1 +/-1.5 pg/ml to 70.2 +/-29.4 pg/ml, with no clear seasonal pattern. Maintaining tortoises at a low environmental temperature (9 +/-1 degrees C versus 28 +/- 1 degrees C) reduced plasma estradiol concentrations. Giving 2mg/kg tamoxifen (TAM) increased plasma estradiol to 220 +/-33 pg/ml when treatment was given in September but not in late October, winter or spring. Treatment with TAM increased the number of eggs laid during the following laying season to 7.3 +/- 1.0 eggs/year, laid in one to three clutches. In males, plasma testosterone concentrations had a seasonal pattern with the onset of a rise in July from 2 to >4ng/ml, a continued increase to a peak of 12.8+/-5.3 ng/ml during November and a decline thereafter. Artificial incubation in sand at 29 +/-1 degrees C shortened the natural incubation time of 103+/-3.1 days to 83.5 +/- 1.3 days, increased hatching rate from 28 to 53%, and increased survival rate from 51 to 71% at 40 weeks of age. In summary, this study provides options for increasing reproductive performance, hatchability and offspring survival in captive Mediterranean tortoises, and may offer new tools for conservation of animals that are on the verge of extinction.