Relaxin concentrations in endometrial, placental, and ovarian tissues and in sera from ewes during middle and late pregnancy

These studies were designed to determine the tissue source of ovine relaxin and to determine the feasibility of using the pregnant ewe for study of relaxin production and secretion. On Day 4 of gestation, ewes were laparotomized, the nonpregnant uterine horn was ligated, and the ovary not containing the corpus luteum was removed. During a second surgery at Day 45 (n = 8) or 140 (n = 9) of gestation, 10-ml blood samples were drawn from a uterine artery, the ovarian vein, and veins draining the pregnant and nonpregnant uterine horns. Endometrial, placental, and luteal tissues were obtained for immunocytochemistry and extraction. Relaxin was detected by a heterologous porcine radioimmunoassay (RIA) in 3 of 54 serum samples (701.3 +/- 25.4 pg/ml, mean +/- SEM). Relaxin was not detected in crude tissue extracts, but low quantities were detected by RIA following Sephadex G-50 column chromatography of tissue extracts. Total relaxin activity for all tissues was equivalent to 0.57 +/- 0.13 ng of porcine relaxin/g tissue (w.w.). Relaxin was not detected immunocytochemically by light or electron microscopy. These data indicate that low quantities of relaxin are present in tissues and sera of pregnant ewes.     

Systemic relaxin in pregnant pony mares grazed on endophyte-infected fescue: effects of fluphenazine treatment

Tall fescue is one of the most widely grown forage grasses for horses in the United States. However, it is frequently infected with the endophyte Neotyphodium coenophialum which produces ergot alkaloids that cause severe adverse effects in the pregnant mare. The objectives of this study were to determine the effects of fescue toxicosis and fluphenazine on circulating relaxin in pregnant pony mares and evaluate the usefulness of relaxin as a monitor of treatment efficacy. Twelve mares were maintained on endophyte-infected tall fescue pasture. Group TRT (n = 6), received 25 mg of fluphenazine decanoate (i.m.) on Day 320 of gestation while Group UTRT served as untreated controls. Daily blood samples were collected from Day 300 of gestation until Day 3 post partum and analyzed for plasma relaxin concentrations using a homologous equine radioimmunoassay. Mean gestation lengths were 330 +/- 0.7 and 336.5 +/- 3.2 days for TRT and UTRT mares, respectively (P = 0.07). Mean plasma relaxin concentrations in both groups of mares during the week before treatment (Day 313 to 319) were not different (UTRT, 53.4 +/- 11.3 ng/mL; TRT, 61.4 +/- 9.3 ng/mL). In the week after treatment (Day 320 to 326), mean plasma relaxin tended to be higher (P = 0.1) in TRT mares (66.7 +/- 6.2 ng/mL) when compared with UTRT mares (49.6 +/- 6.6 ng/mL), representing a 17.1 ng/mL difference in circulating relaxin between the two groups. Systemic relaxin during the last week before delivery (days relative to parturition) for UTRT and TRT mares was 45.7 +/- 6.7 and 64.7 +/- 6.4 ng/mL (P = 0.06), respectively. At Day -8 and Day -5 relative to parturition, systemic relaxin in TRT mares was significantly higher (P < 0.05) than in UTRT mares. Three of the six UTRT mares and one TRT mare showed clinical symptoms of fescue toxicosis. In the week before delivery, circulating relaxin in mares with problematic pregnancies (39.9 +/- 7.8 ng/mL) was significantly lower than concentrations measured in mares with normal pregnancies (63.4 +/- 5.4 ng/mL; P = 0.03). Clinical observations suggest that a one-time injection with fluphenazine improved pregnancy outcome by reducing the adverse effects of fescue toxicosis concomitant with a stabilization of plasma relaxin concentrations. These data support the hypothesis that systemic relaxin may be a useful biochemical means of monitoring placental function and treatment efficacy in the mare.     

Pregnancy rates and gravid uterine parameters in single, twin and triplet pregnancies in naturally bred ewes and ewes after transfer of in vitro produced embryos

The objectives of this study were to: (1) evaluate the pregnancy rates after transfer of embryos produced in the presence or absence of epidermal growth factor (EGF) during in vitro maturation, and (2) compare several variables of the gravid uterus on day 140 after fertilization in single, twin and triplet pregnancies in ewes (n = 12) bred naturally and in ewes (n = 18) after transfer of embryos produced in vitro. Oocytes collected from FSH-treated ewes (n = 18) were collected from all visible follicles and cultured in maturation medium with or without EGF. Oocytes were then fertilized in vitro by frozen-thawed semen. On day 5 after fertilization, embryos with > or = 16 cells were transferred to recipient ewes (n = 39). In addition 12 ewes were bred naturally. Pregnancy was verified by real-time ultrasonography on day 45 or later after embryo transfer (ET) or breeding. On day 140 of pregnancy, the reproductive tract was collected from all ewes and the following parameters were determined: the number, sex, weight and crown to rump length (CRL) of fetuses, weights of gravid uterus and fetal membranes, and weight and number of placentomes. Presence of EGF in maturation medium increased (P < 0.04) cleavage rates (78% versus 59%) and percentage of > or = 16 cell embryos on day 5 after fertilization (62% versus 40%). Pregnancy rates tended to be greater (P < 0.1) after transfer of embryos matured in the presence of EGF (52%) than in the absence of EGF (39%). EGF presence in maturation medium did not affect any variables of gravid uterus or fetal weight. For single pregnancies in naturally bred ewes and ewes after ET all uterine variables were similar. For twin pregnancies, weight of gravid uterus, weight of uterus plus fetal membranes, total weight of placentomes/ewe, mean weight of individual placentome, mean weight of fetus, total fetal weight/ewe and CRL were greater (P < 0.0001-0.04) for ewes after ET than for ewes bred naturally. The weights of gravid uterus, fluid, uterus plus fetal membranes, fetal membranes, total placentomes/ewe, mean weight of individual placentome and total fetal weight/ewe were greater (P < 0.0001-0.08) for triplet pregnancies in ewes after ET than single and twin pregnancies in ewes naturally bred or after ET. The number of placentomes/fetus was greatest (P < 0.0001-0.06) in single pregnancies in ewes bred naturally and after ET fewer in twin pregnancies in ewes bred naturally and after ET and fewest in triplet pregnancies in ewes after ET. The total number of placentomes/ewe was greatest (P < 0.0001-0.06) for twin pregnancies in ewes naturally bred, fewer in single pregnancies in ewes naturally bred and twin and triplet pregnancies after ET, and fewest in single pregnancies in ewes after ET. The mean weight of fetus was greater (P < 0.0001-0.07) in single pregnancies in ewes naturally bred or after ET than in twin or triplet pregnancies in ewes naturally bred or after ET. The CRL was the lowest (P < 0.01) in twin pregnancies in ewes bred naturally. For pregnancies after natural breeding and after ET, the number of fetuses/ewe was negatively correlated (P < 0.03-0.0001) with the weight of placentomes/fetus, the number of placentomes/fetus, the mean weight of the fetus and CRL, and was positively correlated (P < 0.0001-0.05) with weight of gravid uterus, the total number of placentomes/ewe and total fetal weight/ewe. These data demonstrate that the presence of EGF in maturation medium increases the rates of cleavage and early embryonic development, and has a tendency to enhance rates of pregnancy but does not affect variables of the gravid uteri in ewes after transfer of in vitro produced embryos. Transfer of embryos produced in vitro affected some uterine variables in twin but not single pregnancies to compare with pregnancies after natural breeding. In addition, culture conditions in the present experiment did not create large offspring syndrome. The low number of placentomes/fetus seen in triple pregnancies appears to be compensated for by the increase in the weight of each individual placentome.     

Determination of gestational age in sheep and goats using transrectal ultrasonographic measurement of placentomes

Three experiments were conducted to determine gestational age in the ewe and doe by measuring placentomes with a B-mode ultrasonograph and a 5 MHz transducer. Transrectal measurements were obtained by placing the female over a bale of hay. In Experiment 1, ewes (n = 12) and does (n = 15) were examined by transrectal ultrasonography every week from breeding to parturition to determine the growth pattern of placentomes during pregnancy. In Experiment 2, placentomes from 132 ewes and 169 does were measured between 30 and 90 d of gestation. A linear regression relationship between fetal age in days and placentome size in mm was calculated and adjusted for does (gestational age = 28.74 + 1.80PL + e, r(2) = 70.34) and for ewes (age = 47.98 + 0.62PL + e, r(2) = 15.59). In Experiment 3, the placentomes of 63 does were measured to validate this relationship by using linear regression. Gestational age was determined correctly in 66% of the does, with a range of +/- 7 d and in 96% with a margin of +/- 14 d. In conclusion, transrectal ultrasonography allowed for the measurement of placentome size, which increased rapidly during the first 70 to 90 d of gestation in ewes and does. In ewes, however, there was a poor correlation of placentome size with gestational age, while in goats, measurement of placentomes could be used along with pregnancy diagnosis by transrectal ultrasonography as an indication of gestation age.     

Effect of intra-ovarian infusion of oxytocin on plasma progesterone concentrations in pregnant ewes.

The function of oxytocin receptors in the corpus luteum of pregnant ewes was investigated by infusing saline or oxytocin (100 ng/min) into the utero-ovarian artery of pregnant ewes (62 +/- 5 days, n = 12). During a 4-h infusion, plasma oxytocin (OT) concentration increased to 268 +/- 80 pg OT/ml in the OT-infused group and remained unchanged at 2.5 +/- 1.5 pg OT/ml in the saline-infused group. Progesterone concentration in jugular venous plasma (17 +/- 9 ng/ml) rapidly decreased during oxytocin infusion to 59 +/- 10% and 26 +/- 9% of control at 1.5 and 2 h, respectively; the utero-ovarian venous concentration of 64 +/- 38 ng/ml decreased by a similar magnitude during oxytocin infusion. Electron microscopy of corpora lutea, removed at the end of the experiments, showed no indication of luteolytic changes following oxytocin infusion. It was concluded that oxytocin markedly and rapidly reduces progesterone secretion in pregnant ewes.     

The effect of two different levels of progesterone priming on the response of ewes to superovulation

The use of either 1 or 3 controlled internal drug release (CIDR) devices for progesterone priming in ewes (n=11) superovulated with 1500 IU pregnant mare serum gonadotrophin (PMSG) at 28 hours prior to CIDR device withdrawal was investigated in relation to the stages of development and viability of the ova produced. Progesterone levels in the ewes (n=6) treated with 3 CIDR devices were significantly higher (P<0.01) during the 11 days of insertion than in those (n=5) treated with 1 CIDR device (7.3 vs 3.3 ng/ml) over the same period. However, following superovulation, the mean (+/-SEM) ovulation rates were similar for both groups (8.2 +/- 1.7 vs 10.2 +/- 1.5). The number of ova (M+/-SEM) recovered by laparoscopy 5 days after insemination was 4.2 +/- 1.0 for ewes treated with 3 CIDR devices and 7.0 +/- 1.1 for those treated with 1 CIDR device (P<0.10). The respective ovum recovery rates (M+/-SEM) were 55+/-9.8 and 74+/-13.2%. There was no effect of progesterone concentration in the priming phase on either the stages of development of the recovered ova or on their ability to develop during in vitro culture. It was concluded, therefore, that progesterone concentrations within the range 3.3 +/- 0.1 to 7.3 +/- 0.3 ng/ml during the priming phase and 2.4 +/- 0.3 to 6.5 +/- 0.2 ng/ml at the time of PMSG administration did not affect the ovulation rate or the viability of ova recovered from superovulated ewes.     

Effect of relaxin on facilitation of parturition in dairy heifers

Purified pig relaxin (3000 U/mg) was injected i.m. into pregnant Holstein dairy heifers on Day 276 or 277 to determine its effect on parturition and sequential measurements of the pelvic area, cervical dilatation, and peripheral blood-plasma concentrations of progesterone and relaxin. Treatments included phosphate-buffer saline (2 ml, Group C, N = 7), relaxin once (1 mg, Group 1R, N = 7), and twice (2 mg, 12 h apart; Group 2R, N = 7). Intervals (mean +/- s.e.) between the first injection of relaxin or PBS and calving were 64 +/- 17, 80 +/- 19 and 125 +/- 34 h for Groups 2R, 1R and C, respectively. The calving intervals were reduced in Groups 2R (P less than 0.01) and 1R (P less than 0.05) compared with Group C. The incidence of dystocia was 29% (2 of 7) in Group 2R and 43% (3 of 7) in Group 1R compared with 57% (4 of 7) in Group C (P less than 0.01). Body weights and ratios of males to females of the calves were similar (P greater than 0.05) between groups. Progesterone plasma concentrations decreased (P less than 0.01) earlier in Groups 1R and 2R compared with Group C, and this acute decrease began within 6 h of treatment. At 24 h after relaxin or PBS injection, progesterone concentrations were 2.7 +/- 1.1 ng/ml for Group 2R, 3.5 +/- 0.9 ng/ml for Group 1R, and 6.0 +/- 0.1 ng/ml for Group C. Relaxin reached peak blood-plasma levels of 19 +/- 2.2 ng/ml 1 h after injection of relaxin, but remained unchanged, 0.3 +/- 0.01 ng/ml, in Group C. Pelvic area was increased 26%, 22% and 14% and cervical dilatation was increased 109%, 76% and 53% 48 h after injection in Groups 2R, 1R and C, respectively, but these responses were similar among groups at the time of parturition. We conclude that two i.m. injections of relaxin facilitated earlier calving, acutely decreased progesterone secretion, increased cervical dilatation and pelvic area expansion, and decreased the incidence of dystocia in dairy heifers.     

Measurement of plasma and tissue relaxin concentrations in the pregnant hamster and fetus using a homologous radioimmunoassay

A homologous hamster relaxin RIA was developed to evaluate plasma and tissue concentrations of relaxin in the latter half of pregnancy in this species. Relaxin protein and mRNA were localized using antibodies developed to synthetic hamster relaxin and gene-specific molecular probes, respectively. Molecular weight and isoelectric point of the synthetic and native hormones were identical by electrophoretic methods, and synthetic hamster relaxin was active in the mouse interpubic ligament bioassay. Synthetic hormone was used as tracer and standard with rabbit antiserum to the synthetic hormone in the RIA. Relaxin was assayed in blood samples recovered from the retro-orbital plexus on Days 6, 8, 10, 12, 14, 15, and 16 of gestation and on Days 1 and 5 postpartum. Relaxin was first detected on Day 8 of gestation (3.7 +/- 0.6 ng/ml), increased to reach a maximum in the evening of Day 15 (826.0 +/- 124.0 ng/ml), and decreased by Day 16 (day of parturition). Relaxin concentrations were assayed in aqueous extracts of implantation sites (Days 6, 8, and 10) and chorioallantoic placentae (Days 12, 14, and 15). Concentrations were low on Day 6 (0.02 +/- 0.001 microg/g tissue), increased to Day 15 (6.96 +/- 0.86 microg/g tissue), and subsequently declined by the evening of Day 15. Relaxin protein and mRNA were localized to primary and secondary giant trophoblast cells in the chorioallantoic placental trophospongium. However, relaxin protein was not localized in ovaries of pregnant animals or oviductal tissues of cycling animals. Significant quantities of relaxin were detected in the serum of fetal hamsters recovered on Day 15.     

Relaxin regulates oxytocin secretion in late-pregnant beef heifers

The effects of porcine relaxin (3000 units/mg) on oxytocin (OT) and progesterone secretion were studied in beef heifers on Day 274 (10 days before expected parturition). Heifers (n = 11) were randomly assigned to three treatments: relaxin iv infusions combined with im injection (RLX-INF, 9000 units), relaxin im injection (RLX-im, 6000 units), and phosphate-buffered saline-treated controls (PBS). RLX-INF heifers received infusions of PBS and 1000 units of relaxin for 165 min, followed by 2000 units of relaxin im and finally 2000 units of relaxin infusion followed by 4000 units of relaxin im. Endogenous relaxin (immunoreactive) in the PBS-treated group was 0.2-0.9 ng/ml peripheral plasma. For the RLX-im group, peak relaxin was 81 +/- 12 ng/ml (+/- SE) at 45 min after treatment. There were two peaks of relaxin, 18 +/- 5.3 ng/ml and 74 +/- 7.5 ng/ml, 3.5-4.5 hr apart in the RLX-INF group. Significant peak releases of OT were evident in the relaxin-treated heifers. For the RLX-im group, an OT peak (42 +/- 16 pg/ml) occurred within 30 min after relaxin treatment. For the RLX-INF heifers, 2000 and 4000 units of relaxin were associated with major peaks of 14 +/- 0.5 and 43 +/- 1.7 pg/ml OT, respectively. Basal OT plasma levels in the PBS group were 2.5-3.1 pg/ml. Mean plasma progesterone for all heifers was 6.2 +/- 2.11 ng/ml before treatment. There was a significant decrease in progesterone (-2.5 ng/ml) in the RLX-im group within 60 min after relaxin treatment and 45 min after peak OT secretion. The maximum decrease in progesterone (-3.2 +/- 0.68 ng/ml) occurred 135 min after treatment in the RLX-im group. In the RLX-INF group, 2000 units of relaxin infusion combined with 4000 units of relaxin im significantly decreased progesterone (-3.2 +/- 1.59 ng/ml) in peripheral plasma. These results clearly indicate that relaxin causes an acute peak release of oxytocin within 30 min, followed by a marked decrease in plasma progesterone concentration in late-pregnancy cattle.     

Myometrial activity and plasma progesterone and oxytocin concentrations in cycling and early-pregnant ewes

Myometrial activity and plasma progesterone (P) and oxytocin (OT) were measured in early pregnant (n = 5) and cycling (n = 5) ewes. Electromyography (EMG) leads and jugular and inferior vena cava (IVC) catheters were surgically placed in ewes about 1 wk before data collection. When ewes returned to estrus, they were bred to either an intact or vasectomized ram. Continuous EMG data were collected, and blood samples were collected twice daily from day of estrus (Day 0) until Day 18. Ewes bred with an intact ram were checked surgically for pregnancy on Day 20. Computerized, quantitative analysis of EMG events showed no difference in signal from the right to left uterine horns, and no differences between pregnant and cycling ewes (p less than 0.05) until Days 14-18 when nonpregnant ewes returned to estrus and had increased EMG activity. The mean number of EMG events 180-900 s in length decreased in pregnant ewes, but this difference was not significant (p less than 0.05). Jugular plasma progesterone (P) levels confirmed corpus luteum (CL) formation in all ewes, and no differences in P between pregnant and nonpregnant ewes were measured until Days 14-18, when cycling ewes underwent luteolysis and pregnant ewes maintained CL. IVC plasma oxytocin concentrations were increased in pregnant ewes compared to concentrations in nonpregnant ewes on Days 5-13 (p less than 0.05), and the difference was largest at Day 6 (means +/- SEM pg/ml: pregnant = 68.7 +/- 13.9, nonpregnant = 30.9 +/- 19.9).(ABSTRACT TRUNCATED AT 250 WORDS)     

Administration of pig relaxin to beef heifers 4 or 7 days pre partum

Crossbred beef heifers (N = 36) were assigned to one of three treatment groups: untreated controls (C; N = 15); Group R4, treated with pig relaxin (1.0 mg i.m.) 4 days pre partum (N = 11); or Group R7, treated with pig relaxin (1.0 mg i.m.) 7 days pre partum (N = 10). Bioactivity of the pig relaxin (UMC-R-P8) was determined by the mouse interpubic ligament assay to be greater than or equal to 3000 U/mg, both before and after the experiment was conducted. Peripheral serum immunoreactive relaxin values were 7.5, 3.4, 2.5, and 1.5 ng/ml at 2, 4, 6 and 8 h after injection of relaxin, respectively. Gestation lengths were 282.9 +/- 1.1, 285.5 +/- 1.3 and 285.6 +/- 1.5 days for Groups C, R4 and R7 (C vs R4 + R7; P congruent to 0.08). Calving difficulty score (1 to 4) tended to be greater (P congruent to 0.08) for Group R4 and R7 heifers (C vs R4 + R7; 1.3 +/- 0.24 vs 1.75 +/- 0.28 + 2.04 +/- 0.32), but the incidences of dystocia and retained placentae were not influenced by treatment (P greater than or equal to 0.10). The mean concentration and concentration profile of daily serum progesterone, oestradiol-17 beta, dihydroprostaglandin F-2 alpha and relaxin were not affected by treatment from 6 days pre partum through 2 days post partum. Cervical diameter, cervical softness score, pelvic measurements, and vulva opening length during the periparturient period were not affected (P greater than or equal to 0.10) by treatment, but all of these characteristics changed over time (P less than or equal to 0.01), relative to calving. We conclude that i.m. administration of pig relaxin (greater than or equal to 3000 U) does not effectively alter periparturient characteristics of beef heifers. Discrepancies between these results and those reported for intracervical administration cannot be readily explained.     

Synchronization of follicular wave emergence in the seasonally anestrous ewe: the effects of estradiol with or without medroxyprogesterone acetate

Fertility is often lower in anestrous compared to cyclic ewes, after conventional estrus synchronization. We hypothesized that synchronization of ovarian follicular waves and ovulation could improve fertility at controlled breeding in anestrous ewes. Estradiol-17beta synchronizes follicular waves in cattle. The objectives of the present experiments were to study the effect of an estradiol injection, with or without a 12-d medroxyprogesterone acetate (MAP) sponge treatment, on synchronization of follicular waves and ovulation in anestrous ewes. Twenty ewes received sesame oil (n=8) or estradiol-17beta (350 microg; n=12). Eleven ewes received MAP sponges for 12d and were treated with oil (n=5) or estradiol-17beta (n=6) 6d before sponge removal. Saline (n=6) or eCG (n=6) was subsequently given to separate groups of ewes at sponge removal in the MAP/estradiol-17beta protocol. Estradiol treatment alone produced a peak in serum FSH concentrations (4.73+/-0.53 vs. 2.36+/-0.39 ng/mL for treatment vs. control; mean+/-S.E.M.) after a short-lived (6 h) suppression. Six of twelve ewes given estradiol missed a follicular wave around the time of estradiol injection. Medroxyprogesterone acetate-treated ewes given estradiol had more prolonged suppression of serum FSH concentrations (6-18 h) and a delay in the induced FSH peak (32.3+/-3.3 vs. 17.5+/-0.5 h). Wave emergence was delayed (5.7+/-0.3 vs. 1.4+/-0.7d from the time of estradiol injection), synchronized, and occurred at a predictable time (5-7 vs. 0-4d) compared to ewes given MAP alone. All ewes given eCG ovulated 3-4d after injection; this predictable time of ovulation may be efficacious for AI and embryo transfer.     

Reproductive responses following royal jelly treatment administered orally or intramuscularly into progesterone-treated Awassi ewes

An experiment was conducted to determine whether natural royal jelly (RJ) paste administered orally or intramuscularly (i.m.) in conjunction with exogenous progesterone is associated with improved reproductive responses in ewes. Thirty 3-6-year-old Awassi ewes were randomly allocated into three (RJ-capsule, RJC; RJ-injection, RJI and control, CON) groups of 10 ewes each. All ewes were treated with intravaginal progesterone sponges for 12 days. Ewes in the RJC and RJI were administered orally or i.m. with a total of 3g of RJ given in 12 equal doses of 250 mg per ewe per day starting at the time of sponge insertion. At the time of sponge withdrawal (day 0, 0 h), ewes were exposed to three rams and checked for breeding marks at 6-h intervals for 3 days. Blood samples were collected from all ewes for analysis of progesterone concentrations. Pretreatment progesterone levels were <0.5 ng x ml(-1) in 16/30 and >1.3 ng x ml(-1) in the remaining ewes indicating luteal function and cyclicity. Similar reproductive responses and progesterone levels occurred in ewes of the RJC and RJI; therefore, data of the two groups were pooled. Following sponge insertion, progesterone levels increased rapidly and reached maximum values of 5.8+/-0.2 ng x ml(-1) within 2 days among ewes of the three groups, and then declined gradually to day 0 values of 1.6+/-0.1 and 1.9+/-0.1 ng x ml(-1) for the RJ-treated and CON ewes, respectively. The rate of progesterone decline was greater (P<0.001) in RJ-treated than in CON. Mean progesterone levels during the 12-day period were lower (P<0.001) in RJ-treated than in CON (2.8+/-0.2 ng x ml(-1) versus 3.3+/-0.2 ng x ml(-1)). Treatment with RJ resulted in greater (P<0.05) incidence of oestrus and shorter (P<0.05) intervals to onset of oestrus than CON. Based upon progesterone levels, ovulation occurred following day 0 in all ewes. Progesterone increased on day 3 in RJ-treated and on day 4 in CON ewes. Progesterone remained elevated through day 18 in 8/20 RJ-treated and 1/10 CON ewes (P=0.09). All pregnant ewes exhibited oestrus 14 h earlier (P<0.02), ovulated approximately 1 day earlier and had higher (P<0.001) luteal phase progesterone levels than non-pregnant ewes. Non-pregnant had higher (P<0.04) body weights than pregnant ewes. In conclusion, results demonstrate that both RJ treatments in conjunction with exogenous progesterone were equally capable of improving oestrus response and pregnancy rate.     

Induction of parturition, progesterone secretion, and delivery of placenta in beef heifers given relaxin with cloprostenol or dexamethasone

Sixty primiparous beef heifers from a crossbreeding study were used to examine the effects of inducing parturition with relaxin (3,000 U/mg) combined with cloprostenol (500 micrograms, i.m., n = 30) or dexamethasone (20 mg, i.m., n = 30) at Day 273, 10 +/- 1 days before expected parturition (Day 283). Heifers were assigned at random within cloprostenol and dexamethasone groups to receive relaxin (1 mg, n = 5/treatment), i.m. or in the cervical os (OS), at 0 h (the same time as cloprostenol and dexamethasone) or 24 h later. Eleven and six first-calving heifers and sixteen and nine second-calving cows also received cloprostenol + relaxin and cloprostenol + phosphate-buffered saline, respectively. Radioimmunoassay of daily plasma samples indicated an abrupt decrease in progesterone with time (p less than 0.001), from 7.5 +/- 0.50 to 1.0 +/- 0.30 ng/ml (mean +/- SE) within 48 h for all groups. The mean rate of progesterone decrease (ng/ml in 24 h) was accelerated (p less than 0.01) in relaxin-treated heifers (5.3 +/- 0.36), in contrast to dexamethasone- and cloprostenol-treated control heifers (2.8 +/- 0.40). Relaxin combined with cloprostenol or dexamethasone shortened the calving period in these heifers by reducing the interval between treatment and calving (33 vs. 56 h; p less than 0.01). The incidence and duration of retained placenta were reduced by 22 vs. 75% and 14 vs. 34 h for relaxin combined with cloprostenol or dexamethasone as compared with cloprostenol- or dexamethasone-treated controls, respectively (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of morphology on placentome size, vascularity, and vasoreactivity in late pregnant sheep

Ovine placentomes vary in shape, with type A placentomes being concave, type D convex, and types B and C intermediate in morphology. It has been speculated that as placentomes advance in type they differ in vascularity and nutrient transport capacity. Our objective was to determine cellularity and vascularity measurements, angiogenic factor expression, and arterial vasoactivity within different morphologic types of placentomes. On Day 130 of gestation, placentomes were collected from multiparous ewes (n = 38) and were evaluated for size, cellularity estimates, angiogenic factor mRNA expression, capillary vascularity (capillary size, capillary surface density [CSD], capillary number density [CND], and capillary area density [CAD]), and vasoreactivity to potassium chloride and angiotensin II. The average weight and size of type A and B placentomes were less (P < 0.01) than those of type C and D placentomes. Placentome morphology did not affect (P > or = 0.24) cotyledonary or caruncular cellularity estimates or percentage of cellular proliferation. Placentome morphology affected (P > or = 0.41) neither caruncular CAD, CND, CSD, or capillary size nor cotyledonary CND, CSD, or capillary size. Cotyledonary CAD was increased (P < 0.01) in type B and D placentomes compared with type A placentomes. Furthermore, placentome type did not affect (P > or = 0.06) angiogenic factor gene expression in the cotyledon or the caruncle. Size, but not morphologic type of placentome, was associated with greater caruncular artery contractility to potassium chloride and angiotensin II (P < 0.01 for both). Placentome size, but not morphologic type, may be important for vascularity and nutrient transfer in the placenta of the pregnant ewe.     

Seasonal changes in LH secretion in normal ewes and ewes which grazed oestrogenic clover

Plasma luteinizing hormone (LH) concentrations were measured in normal (control) Corriedale X Merino (comeback) ewes and in clover-infertile comeback ewes which had grazed oestrogenic Yarloop clover (Trifolium subterraneum L. cv. Yarloop) for more than 4 years. Plasma LH concentrations were measured in samples taken at 20-min intervals for 6 h during the dioestrous stage of the oestrous cycle in the breeding season (BS) and during the anoestrous season (AS). In the control ewes during BS, transitory elevation in plasma LH concentration (pulses) occurred, reflecting secretory episodes, with a frequency of one per 5.2 h. This frequency fell to one per 16.5 h during the anoestrous season. In clover-infertile ewes, LH pulses occurred with a frequency of one per 4.5 h during BS and one per 4.9 h during AS (difference not significant). In the controls, plasma LH levels were higher (P less than 0.05) during BS (mean +/- s.d. = 1.2 +/- 0.4 ng/ml, n = 9) than in AS (0.7 +/- 0.3 ng/ml, n = 5). In the clover-infertile ewes, plasma LH levels in BS (1.3 +/- 0.6 ng/ml, n = 12) were similar to those of controls. During AS, plasma LH levels in the clover-infertile ewes (1.0 +/- 0.6 ng/ml, n = 10) remained similar to their BS levels, being significantly (P less than 0.05) higher than LH levels in the controls at this time. These studies indicate that the higher plasma concentrations of LH which have been reported in clover-infertile ewes arise from more frequent LH pulses. Furthermore, in contrast to normal ewes, average plasma LH, reflecting pulse frequency, is not reduced in AS. This supports the view that ingestion of phytooestrogens affects neural centres involved in regulating LH secretion.     

Serum thyroid hormones and performance of offspring in ewes receiving propylthiouracil with or without melatonin

Two experiments were conducted during mid-gestation to examine effects in ewes of propylthiouracil (PTU) treatment alone or with melatonin on serum thyroid hormones, postpartum reproduction, and lamb performance. In the first experiment, beginning on day 0 (first day of treatment when all animals were 72.2+/-0.9 days of gestation), ewes received daily treatments (gavage) consisting of either 0mg (n=6) or 40 mg (n=6) PTU/kg body weight/day for 15 days. After 15 days, the 40 mg dosage was decreased to 20mg/kg body weight for an additional 20 days (35 days of PTU). Serum thyroxine (T4) did not differ (P>0.10) between groups through day 4; but on day 5, control females had a serum value of 67 ng/ml compared with 46 (+/-5)ng/ml for PTU-treated ewes (P=0.02). On the last day that 40 mg of PTU was administered, serum T4 averaged 67 and 7 (+/-5)ng/ml (P<0.001) in the two respective groups. Serum T4 remained low and was 80 and 1 ng/ml (P<0.001) in control and treated ewes on day 34. Serum T4 rose gradually after PTU but remained different from that observed in control ewes through day 48. Lambs from control and treated ewes had similar (P=0.46) T4 values at birth but lambs from PTU-treated ewes had lower (P=0.03) birth weights than did those from control ewes. Serum progesterone (P4) after parturition indicated a lack of cyclicity in all ewes. In the second experiment, beginning on day 0 (76.8+/-4.7 days of gestation), ewes received PTU as in Experiment 1. In addition, after 15 days of PTU, melatonin was given (i.m. injections at 5mg/day) for 30 days. Propylthiouracil decreased (P0.60) for lambs born to control and treated ewes. Female offspring of PTU+melatonin-treated dams reached puberty, became anestrus, and returned to cyclicity at similar (P>0.10) times to contemporary ewe lambs. Results indicate that 40/20mg PTU alone or with melatonin does not induce cyclicity after lambing in spring lambing ewes and has little effect on offspring performance.     

Effect of prostaglandin F2 alpha (PGF2 alpha) on sources of progesterone and pregnancy in intact, ovariectomized and hysterectomized 90-100 day pregnant ewes.

A single dose of 8 or 16 mg of PGF2 alpha per 58 kg body weight was injected intramuscular into intact, ovariectomized or hysterectomized 90-100 day pregnant sheep in three separate experiments. Both doses of PGF2 alpha decreased the weights of the corpora lutea (P less than or equal to 0.05) and the concentration of progesterone in ovarian venous plasma at 72 hr (P less than or equal to 0.05) compared to the 0 hr sample within treatment groups and to control ewes at 72 hr in intact and hysterectomized pregnant ewes. In hysterectomized pregnant ewes, progesterone in jugular plasma declined (P less than or equal to 0.05) from 0 to 72 hr but never fell below 4 mg/ml and this decrease in progesterone after 8 or 16 mg PGF2 alpha was greater than in control hysterectomized ewes (P less than or equal to 0.05). There was a significant decrease in progesterone over time in jugular or uterine venous plasma in the presence of absence of the ovaries in 90-100 day pregnant ewes (P less than or equal to 0.05) but the profiles of progesterone were not different between vehicle and PGF2 alpha-treated ewes (P greater than or equal to 0.05). Uterine venous progesterone never declined below 30 ng/ml in the presence or absence of the ovaries and there was a significant quadratic increase (P less than or equal to 0.05) in uterine venous progesterone toward the end of the 72 hr sampling period indicating an increase in steroidogenic activity of the placenta. PGF2 alpha did not affect the number of abortions in intact or ovariectomized pregnant ewes (P greater than 0.05). Thus, the corpus luteum of sheep at 90-100 days of pregnancy is functional and responsive to PGF2 alpha, placentomes are functional but do not appear to be responsive to the doses of PGF2 alpha tested and PGF2 alpha was not an abortifacient over the 72 hr treatment period.     

Insulin-like growth factor-I and binding proteins 1, 2, and 3 in bovine nuclear transfer pregnancies

In cloned pregnancies, placental deficiencies, including increased placentome size, reduced placentome number, and increased accumulation of allantoic fluid, have been associated with low cloning efficiency. To assess differences in paracrine and endocrine growth regulation in cloned versus normal bovine placentomes and pregnancies, we have examined the expression of insulin-like growth factor (IGF)-I and -II and their binding proteins (IGFBP)-1 through -3 in placentomes of artificially inseminated (AI), in vitro-produced (IVP), and nuclear transfer (NT) pregnancies at Days 50, 100, and 150 of gestation. Fetal, maternal, and binucleate cell counts in representative placentomes were performed on Days 50-150 of gestation in all three groups. Increased numbers of fetal, maternal, and binucleate cells were present in NT placentomes at all stages of gestation examined. Immunolocalization studies showed that spatial and temporal patterns of expression of IGFBP-2 and -3 were markedly altered in the placentomes of NT pregnancies compared to AI/IVP controls. Concentrations of IGF-I in fetal plasma, as determined by RIA, were significantly higher (P = 0.001) in NT pregnancies (mean +/- SEM, 30.3 +/- 2.3 ng/ml) compared with AI (19.1 +/- 5.5 ng/ml) or IVP (24.2 +/- 2.5 ng/ml) pregnancies on Day 150 of gestation. Allantoic fluid levels of IGFBP-1 were also increased in NT pregnancies. These findings suggest that endocrine and paracrine perturbations of the IGF axis may modulate placental dysfunction in NT pregnancies. Furthermore, increased cell numbers in NT placentomes likely have significant implications for fetomaternal communication and may contribute to the placental overgrowth observed in the NT placentomes.     

Modification of prostaglandin F-2 alpha synthesis and release in the ewe during the initial establishment of pregnancy

Pregnant (N = 10) and non-pregnant (N = 10) ewes were bled every 2 h from Days 12 to 17 after oestrus (oestrus = Day 0). Plasma concentrations of progesterone, 15-keto-13,14-dihydro-PGF-2 alpha and 11-ketotetranor-PGF metabolites were determined in all samples. The number of PGF-2 alpha pulses in non-pregnant ewes was 8.2 +/- 0.4 (mean +/- s.e.m.) with an interpulse interval of 10.7 +/- 0.7 h. Two or 3 pulses of low frequency (interpulse interval = 13.4 +/- 1.6 h) occurred in most non-pregnant ewes before the onset of luteolysis; the interpulse interval then decreased to 7.9 +/- 0.4 h for the 6.0 +/- 0.3 pulses temporally associated with luteolysis. In contrast, the number of PGF-2 alpha pulses in pregnant ewes was lower (2.5 +/- 0.7, 0-8) and the interpulse intervals longer (18.9 +/- 6.1 h). Most pulses occurred on Days 14 and 15 in the pregnant and non-pregnant ewes. The mean concentrations of both PGF-2 alpha metabolites in non-pregnant ewes were highest on Day 15 while basal levels of both metabolites remained constant at all times. In pregnant ewes, the mean concentrations of both metabolites were highest on Day 14; basal concentrations of both metabolites were also highest on Day 14. The mean concentrations of 15-keto-13,14-dihydro-PGF-2 alpha were higher in pregnant than in non-pregnant ewes on Days 13 and 14 (P less than 0.05) and higher in non-pregnant than pregnant ewes on Day 15 (P less than 0.05). The basal concentrations of the 15-keto metabolite were higher in pregnant than non-pregnant ewes at Days 13, 14, 15, 16 and 17 (P less than 0.05). Both the mean and the basal concentrations of 11-ketotetranor-PGF metabolites were higher in pregnant than in non-pregnant ewes on Day 14 (P less than 0.05). It is concluded that uterine production of PGF-2 alpha peaks at Days 14-15 after oestrus in pregnant and non-pregnant ewes. Patterns of release differ, however, in that non-pregnant ewes have a pulsatile PGF-2 alpha pattern superimposed on a constant baseline, while pregnant ewes have an increasing basal secretory pattern which is more nearly continuous, i.e. not pulsatile in form. Modification of pulsatile PGF-2 alpha synthesis and release is therefore a key aspect of prolongation of luteal function at the beginning of pregnancy in the ewe.