In vitro and in vivo maturation of oocytes from gonadotrophin-treated brushtail possums

The time course of nuclear maturation of oocytes was examined in brushtail possums, Trichosurus vulpecula. Oocytes were recovered from ovarian follicles > 2 mm in diameter after pregnant mares' serum gonadotrophin/porcine luteinizing hormone (PMSG/LH) treatment (in vivo matured) or 72 hr after PMSG treatment (in vitro matured). Oocytes recovered from small (< 2 mm) and large (> 2 mm) follicles were also assessed for their ability to mature in vitro. Staining with the DNA-specific dye Hoechst 33342 was used to assess the stage of nuclear development by fluorescence microscopy. The process of nuclear maturation progressed rapidly in vivo, as oocytes collected at 20-27 hr post-LH all had a GV, but by 28-29.5 hr post-LH approximately a third of eggs were MII. By 30-hr post-LH, more than 70% of oocytes had reached MII stage and all ovulated eggs were MII. In vitro, all oocytes were at germinal vesicle stage at the start of culture. After 24 hr of culture, 67% of oocytes had progressed to metaphase I/anaphase I of meiosis. After 36 hr, 25% of oocytes had completed maturation to metaphase II, increasing to 52% after 48 hr. Maturation of oocytes after 48 hr in culture was unaffected by the presence or absence of granulosa cells, PMSG or LH/porcine follicle stimulating hormone (FSH). More oocytes from large follicles (55%) completed maturation by 48 hr than from small follicles (15%). The potential of oocytes to mature after 48 hr in culture was dependent on the follicle harvested having reaching a critical diameter of 1.5 mm.     

FSH-induced ovulation in intact and hypophysectomized mice

A single, ovulatory dose of 25 micrograms of a highly purified preparation of ovine FSH caused ovulation in 89% of hypophysectomized and 91% of intact female mice primed 48 h earlier with PMSG; the number of oocytes recovered (29.4 +/- 4.7 and 22 +/- 2.7/mouse ovulating, respectively) compared favourably with the 20.0 +/- 2.9 oocytes per ovulating female recovered from animals that received PMSG + hCG. After oFSH injection, 82% of oocytes released were fertilized and developed to blastocysts. That the trace contamination (less than 0.2%) of the oFSH with oLH was not responsible for the ovulation was shown by the markedly reduced number of oocytes collected from ovulating females that were injected with equivalent low levels of hCG (0.001 micrograms) or oLH (1 microgram) (9.0 +/- 3.3 and 8.0 +/- 3.1, respectively). These results demonstrate that oFSH is as effective as LH in inducing ovulation of competent oocytes in the mouse.     

Efficacy of hCG and GnRH for inducing ovulation in the jenny

Knowledge about management of ovulation in the donkey is limited compared to that in the horse. This experiment was designed to evaluate the efficacy of injecting single doses of lecirelin (a GnRH-analogue) or of hCG to induce ovulation in the jenny and to determine whether effects are dependent upon follicular diameter at time of injection. Ovarian activity and follicular growth were monitored by rectal ultrasonography. Jennies were randomly allotted to the following groups: Group GnRH, treated with 100 microg lecirelin; Group hCG, treated with 2500 IU hCG; Group C, untreated and monitored for spontaneous ovulation. Animals were also categorized into subgroups depending upon follicular diameter: 30-35 mm (GnRH-1, hCG-1 and C-1) or 36-40 mm (GnRH-2, hCG-2 and C-2). Jennies in the two hormone treatment groups did not differ significantly for time from treatment to ovulation, but there was a significant reduction in time to ovulation as follicle size at treatment increased. Jennies treated with either lecirelin or hCG had significantly smaller follicle size at ovulation than jennies in the Control groups that underwent spontaneous ovulation. Treatment groups did not differ significantly in the proportion of jennies that ovulated within 48 h of injection or between 25 and 48 h following injection. These results highlight the usefulness of lecirelin for induction and synchronization of ovulation in the jenny, particularly since it would avoid the risk of reduced hCG response in reproductive management programs in which that hormone was repeatedly used.     

Synchronization of ovulation in dairy cows using PGF2alpha and GnRH

This paper reports a new method for synchronizing the time of ovulation in cattle using GnRH and PGF(2alpha). In Experiments 1 and 2, lactating dairy cows (n=20) ranging from 36 to 280 d postpartum and dairy heifers (n=24) 14 to 16 mo old were treated with an intramuscular injection of 100 mug GnRH at a random stage of the estrous cycle. Seven d later the cattle received PGF(2alpha) to regress corpora lutea (CL). Lactating cows and heifers received a second injection of 100 mug GnRH 48 and 24 h later, respectively. Lactating cows were artificially inseminated 24 h after the second GnRH injection. Ovarian morphology was monitored daily by trans-rectal ultrasonography from 5 d prior to treatment until ovulation. In Experiment 3, the flexibility in the timing of hormonal injections with this synchronization protocol was evaluated by randomly assigning 66 lactating dairy cows to 3 different treatment groups. Lactating cows received the injection of PGF(2alpha) 48 (Group 1), 24 (Group 2), and 0 h (Group 3) prior to the second injection of GnRH, which was administered at the same time in each group to ensure the second injection of GnRH was given when follicles were at a similar stage of growth. In Experiments 1 and 2, the first injection of GnRH caused ovulation and formation of a new or accessory CL in 18 20 cows and 13 24 heifers. In addition, this injection of GnRH initiated or was coincident with initiation of a new follicular wave in 20 20 lactating cows and 18 24 heifers. Corpora lutea regressed after PGF(2alpha) in 20 20 cows and in 18 24 heifers. All cows and 18 24 heifers ovulated a newly formed dominant follicle between 24 and 32 h after the second injection of GnRH. Ten of 20 cows conceived to the timed artificial insemination. In Experiment 3, the conception rate in Groups 1 and 2 were greater than in Group 3, (55 and 46 % vs 11%, respectively). In summary, this protocol could have a major impact on managing reproduction in lactating dairy cows, because it allows for AI to occur at a known time of ovulation and eliminates the need for detection of estrus.     

The effect of pre-ovulatory anaesthesia on ovulation in laparoscopically inseminated domestic cats.

Laparoscopic intrauterine artificial insemination (AI) of electroejaculated spermatozoa was used to compare embryo development and conception rates in domestic cats inseminated either before or after ovulation. Females were given a single (100 iu) injection of pregnant mares' serum gonadotrophin (PMSG) followed by either 75 or 100 iu human chorionic gonadotrophin (hCG) 80 h later. Cats were anaesthetized (injectable ketamine HCl/acepromazine plus gaseous halothane) 25-50 h after administration of hCG for laparoscopic assessment of ovarian activity and for transabdominal AI into the proximal aspect of the uterine lumen. At the time of AI, 23 cats were pre-ovulatory (25-33 h after hCG injection) and 30 were post-ovulatory (31-50 h after hCG injection). Pre-ovulatory females produced 10.5 +/- 1.1 follicles and no corpora lutea compared with 1.9 +/- 0.5 follicles and 7.5 +/- 0.9 corpora lutea for the post-ovulatory group (P < 0.05). Six days later, the ovaries of nine pre-ovulatory and 12 post-ovulatory females were re-examined and the reproductive tracts flushed. On this day, pre-ovulatory cats produced fewer corpora lutea (2.8 +/- 1.5; P < 0.05) and embryos (0.4 +/- 0.3; P < 0.05) than post-ovulatory females (18.9 +/- 3.3 corpora lutea; 4.6 +/- 1.2 embryos). Two of the 14 cats (14.3%) inseminated before ovulation and not flushed became pregnant compared with 9 of 18 cats (50.0%) inseminated after ovulation and up to 41 h after hCG injection (P < 0.05). These results indicate that ovulation in cats is compromised by pre-ovulatory ketamine HCl/acepromazine/halothane or laparoscopy or by both and that electroejaculated spermatozoa deposited by laparoscopy in utero, after ovulation, result in a relatively high incidence of pregnancy. Because ovulation usually occurs 25-27 h after injection of hCG, the lifespan for fertilization of the ovulated ovum appears to be at least 14 h in vivo in cats.     

Characterization of the kinin system in the ovary during ovulation in the rat.

Ovulation has been noted for some time to bear a remarkable similarity to an inflammatory response. One of the principal components that is activated and helps mediate the events during an inflammatory response is the kinin system. Therefore, the purpose of the present study was to examine whether this system could be similarly activated and involved in the cascade of events that leads to ovulation. To answer this question, immature 23-day-old female rats were primed with eCG (10 IU) and ovulation was induced by administration of hCG (10 IU) 48 h later. Groups of rats were killed at 0 h, 10 h, 20 h, and 30 h after hCG for determination of ovulation, ovarian steroid levels, and changes in the levels of kinin system components. Plasma total kininogen levels did not change during the entire period studied. In contrast, ovarian total kininogen levels rose from 0 h to reach a peak at 10 h--a time immediately preceding the beginning of ovulation--after which the levels fell at 20 h, only to rise again at 30 h. Three species of kininogens, high molecular weight (HMW), low molecular weight (LMW), and T-kininogen, were shown to be present in the ovary. T-kininogen was the major kininogen present in the ovary, accounting for 60-92% of the total kininogen at any given time point during the ovulatory process. HMW kininogen levels accounted for only 1.2% of the total ovarian kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

RECOVERY OF PROLIFERATING HAEMOPOIETIC PROGENITOR CELLS AFTER KILLING BY HYDROXYUREA

Two doses of 1 mg/g of hydroxyurea (HU), injected 7 hr apart into irradiated mice in which CFU-S were proliferating during marrow regeneration, killed about 90% of CFU-S. This same dose regime injected into normal female mice, with non-proliferating CFU-S killed 92 % of CFU-C, 99 % of ESC and only 30 % of CFU-S. One day after the treatment CFU-S had decreased to 50 % and remained at about this level for a further day then returned to normal values. In spleen the increase in CFU-S was delayed by a day and showed a marked overshoot. During the period that CFU-S were decreased in number they were actively proliferating. Marrow CFU-C recovered in an exponential manner with a doubling time of 16 hr. Spleen CFU-C recovered 1 day later than marrow and showed a pronounced overshoot. ESC recovered very rapidly with doubling time of 5 hr. The changes in ⁵⁹Fe incorporation into RBC, and the peripheral blood picture, were a delayed reflection of the changes in ESC and CFU-C.     

Distribution of spermatozoa in the female reproductive tract of the domestic cat in relation to ovulation induced by natural mating

The purposes of this study were to demonstrate the localization of spermatozoa in the reproductive tract of female domestic cats before (30 min and 3 h after mating) and after ovulation (48 and 96 h after mating), and to evaluate the efficiency of two techniques for studying sperm distribution. Estrus was induced in twenty-four female cats using 100 IU eCG and the females were divided into four groups with six females per group. The same male cat was used for mating with all the females. One group of six females was mated once; the others were mated four times in 1 h. Ovariohysterectomy was performed at 30 min, 3 h, 48 h, and 96 h after mating and the excised reproductive tracts were divided into seven segments on each side: infundibulum, ampulla, isthmus, uterotubal junction (UTJ), cranial and caudal uterine horn, and uterine body. The vagina and the lumina of the segments from one side were flushed with 0.5 ml PBS. The flushed and the non-flushed segments from the contralateral side were then fixed in 3% neutral buffered formalin and processed for routine histology. The numbers of spermatozoa in the flushings and in 40 histological sections from each segment were counted. Before ovulation, the majority of spermatozoa was detected in the vagina and the uterine segments, whereas after ovulation, significantly higher numbers of spermatozoa were present in the uterine tubal segments. The decreasing gradient in sperm numbers at 30 min and 3 h after mating between the vagina, the uterine segments, including the UTJ, and the uterine tubal segments indicated that the cervix and the UTJ served as barriers for sperm transport in the cat. The UTJ and the uterine crypts acted as sperm reservoirs before ovulation whereas the isthmus was a sperm reservoir around the time of ovulation. There was no difference in sperm numbers in the tissue sections between flushed and non-flushed segments, implying that the flushing technique only recovered some intraluminal spermatozoa while most of the spermatozoa remained in the epithelial crypts. This was further supported by the finding that significantly higher numbers of spermatozoa were recovered in the flushings at 30 min and 3 h after mating, when more spermatozoa were free in the lumina, than at 48 and 96 h after mating, when the majority of the spermatozoa were entrapped in the uterine epithelial crypts.     

Fertilization of rat eggs in vitro at various times before and after ovulation with special reference to fertilization of ovarian oocytes matured in culture.

Oocytes recovered at various times from immature rats treated with PMSG and HCG were incubated with capacitated epididymal spermatozoa of mature rats. In the presence of follicular cells, sperm penetration was not observed 4 hr after incubation in the oocytes at stages from the intact germinal vesicle to the chromatin mass, but 7 to 55% of oocytes were penetrated at stages from the condensed germainal vesicle to metaphase II. After the removal of follicular cells, 15 to 72% of the oocytes at any stage were penetrated. After further incubation for 15 hr, the proportion of penetrated oocytes increased from 8 to 98% from early to late stages and that of penetrated oocytes with a male and female pronucleus increased from 9 to 100% as maturation progressed. Although the average number of spermatozoa/oocyte was not correlated with its maturation, transformation of the sperm head into a male pronucleus was retarded or failed, especially in the younger oocytes. Following incubation in a defined medium for 13 hr, 85% of oocytes at the intact germinal vesicle stage matured to the stage of the first polar body formation, but only 18 to 22% of these mature oocytes were penetrated by spermatozoa and only a few of the penetrated oocytes cleaved into normal two-cell eggs. When eggs recovered from oviducts 14 to 20 hr after ovulation were exposed to capacitated spermatozoa, the proportion of penetrated eggs (86 to 98%) and that of polyspermic eggs (11 to 27%) were not related to the ages of the eggs, but failure of transformation of the sperm head and the proportion of abnormal eggs increased 14 to 20 hr after ovulation.     

Effect of dose of GnRH analog on ovulation in mares

Proper timing of insemination for optimal conception is accomplished by frequent palpations per rectum, by ultrasonography of the preovulatory follicle and/or by treatment with hCG or GnRH. Sustained release of GnRH from implants has been shown to hasten ovulation. Therefore, 2 studies were conducted to evaluate the efficacy of a GnRH analog, deslorelin, for hastening ovulation in nonlactating cyclic mares. The GnRH implant was 2.3 x 3.7 mm and released deslorelin for 2 to 3 days. In Experiment 1, 60 nonlactating, cycling mares were assigned to 1 of 5 doses: 0, 1.2, 1.7, 2.2 and 2.7 mg per implant. Mares were assigned sequentially on the first day of estrus (Day 1). Ovaries were examined per rectum and with ultrasonography every 12 h until ovulation. Once the mares obtained a follicle >30 mm, they were injected subcutaneously with a GnRH implant. The mares were inseminated every other day during estrus with semen from 1 of 3 stallions. Pregnancy was determined with ultrasonography. Experiment 2, 40 nonlactating, cyclic mares were assigned to 1 of 5 treatments (same treatments as in Experiment 1). Data were obtained on interval to ovulation, duration of estrus and pregnancy rates at 12, 18 and 35 d after ovulation. Time to ovulation was shorter (P<0.05) in GnRH-treated mares than in control mares in the Experiment 1. Mean time to ovulation was 68, 49, 48, 47, 44 h in Experiment 1, and 91, 66, 58, 46, 58 h in Experiment 2 for mares given 0, 1.2, 1.7, 2.2 and 2.7 mg/mare in the 2 trials. Averaged for both experiments, the proportion of mares ovulating within 48 h of treatment was 40, 75, 85, 90 and 90% for 0, 1.2, 1.7, 2.2 and 2.7 mg/mare. For both experiments, there was no effect of GnRH on pregnancy rate. In summary, a subcutaneous implant containing GnRH analog induced ovulation in most mares by 48 h of injection, and there was no advantage of doses higher than 2.2 mg/mare.     

Effect of milk production on the incidence of double ovulation in dairy cows

To determine the effect of parity and milk production on the incidence of double ovulation, the synchronization of ovulation, using GnRH and prostaglandin F2 alpha followed by timed AI (Ovsynch), was initiated at a random stage of the estrous cycle in lactating Holstein cows (n = 237). Ovulatory response at 48 h after the second GnRH injection and conception rate at 28 d post AI were determined by transrectal ultrasonography. Ovulation was synchronized in 84% of cows receiving the Ovsynch protocol. Of the synchronized cows, 14.1% exhibited a double ovulation and 47.6% conceived. Conception rate tended to be greater (P = 0.08) for cows exhibiting double (64.0%) rather than single ovulation (45.2%). To determine the effect of milk production on the incidence of double ovulation, cows were classified into low (< or = 40 kg/d) or high (> 40 kg/d) milk production groups based on the average milk production of 40.5 +/- 0.8 kg/d collected 2 d before AI. Although the incidence of double ovulation tended to increase linearly (P = 0.09) with increasing parity, the incidence of double ovulation was nearly 3-fold greater (P < 0.05) for cows in the high (20.2%) than the low (6.9%) milk production group. Furthermore, the increase in the incidence of double ovulation with parity apparently occurred because, within a parity group, the proportion of cows with high milk production was greater for the older cows. Twinning rate of cows that calved (n = 58) was 5.2%. In a secondary objective, cows were retrospectively classified as cystic or normal based on ultrasonographic ovarian morphology at the time of the second GnRH injection. Incidence of ovarian cysts was 11%, and the synchronization and conception rate of cows classified as cystic was 73.1 and 36.8%, respectively, which did not differ from that of normal cows. We conclude that milk production is the primary factor affecting the incidence of double ovulation in lactating dairy cows and may explain the effect of parity on twinning rate. In addition, Ovsynch appears to be an effective method for establishing pregnancy in lactating dairy cows with ovarian cysts.     

The efficacy of different hCG dose rates and the effect of hCG treatment on ovarian activity: ovulation, multiple ovulation, pregnancy, multiple pregnancy, synchrony of multiple ovulation; in the mare

Despite the widespread use of hCG to advance ovulation in the mare there is little information on efficacy of dose rates and any contraindications of its use. This study aims to investigate the effect of dose of hCG on ovulation within 48h and the effect of hCG on: ovulation, multiple ovulation (MO), pregnancy, multiple pregnancy (MP) rates and synchrony of MO; additionally whether any seasonal effect is evident. Sequential ultrasonic scanning was used to monitor the occurrence of ovulation, within 48h of treatment, in 1291 Thoroughbred mares treated with either 750iu hCG or 1500iu hCG s.c. Ovulation rate, type (single ovulations (SO), MO, synchronous, asynchronous) and subsequent pregnancy were then monitored in 1239 Thoroughbred mares on a commercial stud over 3 years, 536 of which were treated with 750iu hCG at mating, all mares were also allocated into groups according to month of mating. No significant difference existed between the two dose levels of hCG and no significant difference existed between treated and untreated mares in overall ovulations (1.32 and 1.28 respectively), MO (31.7% and 27.7%), pregnancy (65.1% and 65.6%) or MP rates (10.8% and 11.8%). There was no significant association between month of year and pregnancy or MP rates for either treated or control mares, nor for MO for untreated mares. A significant (p<0.05) association was evident between month and MO in treated mares, MO being lowest in April (22.3%). 95.9% of treated mares multiple ovulated within 48h compared with 90.7% controls, a near significant difference. In conclusion this study demonstrates that: (i) hCG dose of 750iu s.c. is just as effective in inducing ovulation within 48h as 1500iu, (ii) 750iu hCG has no significant effect on ovulation, MO, pregnancy or MP rates; (iii) a significant (p<0.05) association exists between season and MO in hCG treated mares; (iv) a tighter synchrony (ovulation within 48h) of MO is evident in hCG treated compared with control mares (p=0.052).     

Efficacy of Heatsynch protocol for induction of estrus, synchronization of ovulation and timed artificial insemination in yaks (Poephagus grunniens L.)

The objective of this study was to test the efficacy of induction of estrus, synchronization of ovulation and timed artificial insemination in anestrous yaks using the Heatsynch protocol. In Experiment 1, 10 anestrous yaks were administered an analogue of gonadotropin releasing hormone (GnRH) followed by prostaglandin (PG)F2alpha 7 days later and then estradiol cyponate (ECP) 24 h after that. Ovulation was detected by rectal palpation at 2h intervals beginning at the initial signs of estrus. Blood samples were collected at 2h intervals beginning at the time of ECP injection up to 2h after the occurrence of ovulation for the determination of LH and progesterone. All the animals responded to the Heatsynch protocol with expression of estrus and synchronization of ovulation. The mean time interval from the ECP injection to ovulation was 59.4+/-2.62 h (range 50-72 h). The interval from the LH peak to ovulation was 30.2+/-2.3 h. The high degree of synchrony in ovulation could be attributed to the synchrony in the timing of LH peaks. In Experiment 2, 10 anestrous yaks were treated with the Heatsynch protocol (as in Experiment 1) and TAI was performed at 48 and 60 h after the ECP treatment. Concurrently, 16 cycling yaks were inseminated approximately 12 h after detection of spontaneous estrus. Pregnancy rates were similar in both groups, 40% for TAI and 43.75% for yaks inseminated following spontaneous estrus (p>0.05). From this study, two conclusions can be drawn. First, the Heatsynch protocol can be successfully used to induce and synchronize estrus in anestrous yaks and, second, ovulation following the Heatsynch protocol is synchronized adequately to permit the use of fixed time AI in this species.     

Sperm transport and reservoirs in the pig oviduct in relation to the time of ovulation

The rate of establishment of a population of viable spermatozoa in the oviducts was studied using a technique of post-coital transection in conjunction with subsequent examination of the proportion of eggs fertilized. Gilts were mated early in oestrus (before ovulation) or on the 2nd day of oestrus (after ovulation), and 30, 45 or 60 min later the reproductive tract was sectioned just above or below the utero-tubal junction in a total of 48 animals; these were slaughtered 1 or 2 days after the operation. Some fertilized eggs were recovered from 40 animals, and 72.3% of the 679 eggs examined were fertilized. Mean percentage fertilization increased overall (a) with the time elapsing from mating to transection, (b) with transection below the utero-tubal junction compared with in the caudal isthmus, and (c) with a post-ovulatory versus pre-ovulatory mating. In a further 6 gilts, the results of transection in the lower third of the oviduct 3 h after mating at the onset of oestrus indicated that spermatozoa were initially sequestered in the caudal portion of the isthmus. It is concluded that a population of spermatozoa sufficient to give maximum fertilization is established in the oviducts within 1--2 h of mating, thereby affording protection from the uterine invasion of polymorphonuclear leucocytes.     

Embryo recovery and pregnancy rates after the delay of ovulation and fixed time insemination in superstimulated beef cows

The aim of this study was to evaluate the effect of delaying ovulation subsequent to superstimulation of follicular growth in beef cows (Bos indicus) on embryo recovery rates and the capacity of embryos to establish pregnancies. Ovulation was delayed by three treatments using either progesterone (CIDR-B) or a GnRH agonist (deslorelin). Multiparous Nelore cows (n = 24) received three of four superstimulation treatments in an incomplete block design (n = 18 per group). Cows in Groups CTRL, P48 and P60 were treated with a CIDR-B device plus estradiol benzoate (EB, 4 mg, i.m.) on Day-5, while cows in Group D60 were implanted with deslorelin on Day-7. Cows were superstimulated with FSH (Folltropin-V, 200 mg), from Day 0 to 3, using twice daily injections in decreasing amounts. All cows were treated with a luteolytic dose of prostaglandin on Day 2 (08:00 h). CIDR-B devices were removed as follows: Group CTRL, Day 2 (20:00 h); Group P48, Day 4 (08:00 h); Group P60, Day 4 (20:00 h). Cows in Group CTRL were inseminated at 10, 20 and 30 h after first detected estrus. Ovulation was induced for cows in Group P48 (Day 4, 08:00 h) and Groups P60 and D60 (Day 4, 20:00 h) by injection of LH (Lutropin, 25 mg, i.m.), and these cows were inseminated 10 and 20 h after treatment with LH. Embryos were recovered on Days 11 or 12, graded and transferred to synchronized recipients. Pregnancies were determined by ultrasonography around Day 100. Data were analyzed by mixed procedure, Kruskal-Wallis and Chi-square tests. The number of ova/embryos, transferable embryos (mean +/- SEM) and pregnancy rates (%) were as follows, respectively: Group CTRL (10.8+/-1.8, 6.1+/-1.3, 51.5), P48 (12.6+/-1.9, 7.1+/-1.0, 52.3), P60 (10.5+/-1.6, 5.7+/-1.3, 40.0) and D60 (10.3+/-1.7, 5.0+/-1.2, 50.0). There were no significant differences among the groups (P > 0.05). It was concluded that fixed time AI in association with induced ovulation did not influence embryo recovery. Furthermore, pregnancy rates in embryos recovered from cows with delayed ovulation were similar to those in embryos obtained from cows treated with a conventional superstimulation protocol.     

Effects of passive immunization against inhibin on ovulation rate and embryo recovery in holstein heifers

The effects of acute neutralization of endogenous inhibin on ovulation rate and circulating FSH levels were investigated. Nine or ten days after estrus, 5 heifers were given a single injection of 75 ml iv inhibin antiserum produced in a castrated male goat, while another 5 were given the same amount of a castrated male goat serum. All heifers were given injections of PGF2alpha im at 48 h and 60 h after the serum injection. Those exhibiting an estrus were artificially inseminated with frozen-thawed semen. Seven or eight days after the insemination, ova or embryos were collected using a non-surgical method. Administration of inhibin antiserum resulted in a significant increase in the number of medium-sized follicles compared with the number in the control animals. The number of large follicles in the inhibin-neutralized animals was 4.8 +/- 2.4 (mean +/- SEM; n = 5) on the day of estrus, while there was a single large follicles in the ovaries of control animals. Seven or eight days after estrus, 3 to 16 ova or embryos were recovered from 4 of 5 animals, and 64 % of the total ova/embryos were transferable. Administration of inhibin antiserum produced a significant increase in the concentrations of plasma FSH from 12 to 72 h after the serum injection compared with the levels in the control animals (P < 0.05). After the onset of estrus, preovulatory LH and FSH surges were noted in inhibin-neutralized animals and magnitude of the rise in each hormone was similar to the control animals. The present study demonstrates that a single injection of the inhibin antiserum induces multiple ovulations probably by enhancing FSH secretion, and that recovery of embryos is equal to that observation after an ordinary FSH treatment.     

Timing of the onset and duration of ovulation in superovulated beef heifers

Thirty-two beef heifers were induced to superovulate by the administration of follicle stimulating hormone-porcine (FSH-P). All heifers received 32 mg FSH-P (total dose) which was injected twice daily in decreasing amounts for 4 d commencing on Days 8 to 10 of the estrous cycle. Cloprostenol was administered at 60 and 72 h after the first injection of FSH-P. Heifers were observed for estrus every 6 h and were slaughtered at known times between 48 to 100 h after the first cloprostenol treatment. The populations of ovulated and nonovulated follicles in the ovaries were quantified immediately after slaughter. Blood samples were taken at 2-h intervals from six heifers from 24 h after cloprostenol treatment until slaughter and the plasma was assayed for luteinizing hormone (LH) concentrations. The interval from cloprostenol injection to the onset of estrus was 41.3 +/- 1.25 h (n = 20). The interval from cloprostenol injection to the preovulatory peak of LH was 43.3 +/- 1.69 h (n = 6). No ovulations were observed in animals slaughtered prior to 64.5 h after cloprostenol (n = 12). After 64.5 h, ovulation had commenced in all animals except in one animal slaughtered at 65.5 h. The ovulation rate varied from 4 to 50 ovulations. Approximately 80% of large follicles (> 10 mm diameter) had ovulated within 12 h of the onset of ovulation. Onset of ovulation was followed by a dramatic decrease in the number of large follicles (> 10 mm) and an increase in the number of small follicles (相似文献   

Superovulation in ewes by a single injection of pFSH dissolved in polyvinylpyrrolidone (PVP): effects of PVP molecular weight, concentration and schedule of treatment

Three experiments were carried out to evaluate induction in ewes of superovulation and embryo production by a single injection of a porcine pituitary extract (pFSH) dissolved in polyvinylpyrrolidone (PVP), investigating the effects of PVP molecular weight and its concentration (Experiment I), time and method of treatment (Experiments II and III). All ewes were synchronized for estrus by vaginal sponges impregnated with fluorogestone acetate (FGA; 30 mg, 9 days) plus PGF(2alpha) (Cloprostenol, 50 microg, 48h before sponge removal - s.r.), and superovulated by 250 IU pFSH. In Experiment I, 60 Gentile di Puglia ewes were subdivided into five experimental groups (n = 12): Group A, the control, received six decreasing intramuscular (i.m.) doses of pFSH, 12 h apart, beginning 48h before s.r.; Groups B and C were given 48 h before s.r. a single i.m. injection of pFSH dissolved in PVP with MW = 10,000, respectively, at concentrations of 15 and 30% w/v; Groups D and E received the same treatments as for B and C using PVP with MW = 40,000. None of the pFSH-PVP treatments were effective in inducing superovulation. In Experiment II, 22 Leccese ewes were subdivided into two groups (n = 11): Group A, control received i.m. four decreasing doses of pFSH, beginning 24 h before s.r., 12h apart; Group B was given a single i.m. injection of pFSH dissolved in PVP (MW = 40,000 at 30% w/v), 24 h before s.r. The pFSH-PVP treatment provided an ovulation rate similar to the control and tended to enhance embryo yield (4.4 versus 2.4, P>0.05). In Experiment III, 60 Leccese ewes were subdivided into six treatment groups (n = 10). Groups A and D served as controls and received i.m. 12 h apart, six doses (from 48 h before s.r.) and four doses (from 24h before s.r.) of pFSH, respectively. Groups B and C were treated by a single injection of pFSH in PVP (MW = 10,000; 30% w/v) 48 h before s.r., respectively by i.m. or subcutaneous (s.c.) administration. Groups E and F received the same treatments as for B and C 24 h before s.r. Intramuscular pFSH-PVP administration 24 h before s.r. provided an ovulation rate (8.1), mean numbers of ova recovered (5.6) and fertilized (4.2) comparable to the six or four dose treatments and significantly higher (P <0.01) compared to the pFSH-PVP treatment carried out i.m. 48 h before s.r.These results show that a single injection of pFSH dissolved in PVP at 30% w/v, performed i.m. 24 h before s.r., is able to induce a superovulatory response comparable to that following multiple injection treatment, regardless of PVP molecular weight.     

Superovulation treatments and embryo transfer in Angora goats

A high incidence of early luteal regression after PMSG superovulation was associated with low recovery of embryos from reproductive tracts of Angora goats flushed later than Day 5 after onset of oestrus. Embryos were successfully recovered (mean 7.9/female) by flushing on Days 2-5. Mean ovulation rate after an FSH regimen (16.1 +/- 0.8) was significantly higher than that after a single injection of PMSG (10.8 +/- 1.2). Fertilization rate and survival of embryos following transfer to naturally synchronized recipient feral goats did not differ between the two gonadotrophin regimens: the mean number of kids born to 47 donors treated with FSH (7.5 +/- 0.6) was significantly greater than that to 28 donors treated with PMSG (4.8 +/- 0.6). Irrespective of hormonal treatment, the numbers of embryos recovered and of kids born were correlated with ovulation rate (r = 0.82, P less than 0.001 for both). Embryo survival was influenced by ovulation rate in recipients, with 52%, 63% and 75% of transferred embryos being carried to term by recipients with 1,2 and 3 CL, respectively (P less than 0.01). More embryos survived (65%) when 2 embryos were transferred to each recipient than when 1 (51%) or 3 (48%) were transferred. In recipients receiving 2 embryos, survival was significantly improved by transfer of both embryos to the same oviduct (70%) than when one was transferred to each oviduct (62%). The percentage survival of embryos was optimal when oestrus of recipients was synchronized within +/- 1 day of oestrus in donors.     

Effects of luteinizing hormone, progesterone, testosterone, estradiol and corticosterone on ovulation and luteinizing hormone release in hens treated with aminoglutethimide

Aminoglutethimide (AG), an inhibitor of steroidogenesis, was administered s.c. to 5 groups of laying hens at a dose of 200 mg AG/kg body weight 9 h before expected midsequence ovulation. This dose has previously been demonstrated to consistently block ovulation. The injection of AG was followed by s.c. injections of: Group 1, 1.0 mg progesterone; Group 2, 0.1 mg estradiol-17 beta; Group 3, 1.5 mg corticosterone, all at 6 h prior to expected ovulation; Group 4, 1.0 mg testosterone at both 8 h and 5 h before expected ovulation; and Group 5, 25 micrograms of ovine luteinizing hormone (LH) at 8 and 50 micrograms ovine LH at 6 h before expected ovulation. For each group, 4 control hens were injected with AG and the appropriate vehicle. Blood samples were taken at 1- or 2-h intervals from the time of AG injection to the expected time of ovulation. The hens were killed 4 h after expected ovulation and examined for the occurrence of ovulation. In all hens injected with vehicle, ovulation and the preovulatory surges of progesterone, testosterone, estradiol-17 beta and LH were inhibited. The plasma concentration of corticosterone was not reduced following an injection of AG. Four of 6 hens ovulated in response to injection of ovine LH, although neither endogenous LH nor progesterone were released. Thus, LH appears to play a direct role in follicular rupture and extrusion of the ovum. The administration of progesterone induced a significant and prolonged rise in LH, restoring AG-blocked ovulation in all hens treated (n = 6). Injections of testosterone restored LH release in all hens and ovulation in 2 of 7 hens treated. Three of 7 hens ovulated in response to the corticosterone injection. A preovulatory rise in LH was not observed, indicating that corticosterone may exert its ovulation-inducing effect directly on the mature follicle. Estradiol-17 beta did not restore LH release or ovulation in any of the hens treated with AG.