PGF2α Administration Research Articles

Group IVA phospholipase A 2 activity may mediate prostaglandin F 2α-induced luteal regression in pseudopregnant rats

We investigated role(s) of luteal group IVA phospholipase A 2 (GIVA PLA 2) in prostaglandin (PG) F 2α-induced regression in pseudopregnant rats. Prostaglandin F 2α (PGF 2α) treatment of day 6 pseudopregnant rats stimulated luteal PLA 2 activity, which was sensitive to inhibitors and associated with increased GIVA PLA 2 immunoreactivity. Intra-bursal treatment with the enzyme inhibitor (AACOCF 3) prior to PGF 2α failed to prevent the initial decline in progesterone but induced subsequently a persistent rise that was significantly higher than that of vehicle-treated group. TUNEL-positive signals in luteal cells of control group were reduced by AACOCF 3 treatment. TUNEL-positive reaction induced in luteal cells in vitro by combined cytokines and agonistic anti-Fas were both reduced by AACOCF 3 and another inhibitor pyrrophenone. Overall data show that luteal GIVA PLA 2 activity and expression increased following PGF 2α administration and that acute chemical inhibition of this activity could reverse, at least partly, PGF 2α-induced functional regression and prevent apoptosis induced by PGF 2α in vivo and by cytokines in vitro.

Prostaglandin F2 alpha-induced changes in the sex organs of the male laboratory mouse.

Administration (3 mg/kg body weight/day, for 21 days) of prostaglandin F2 alpha (PGF2 alpha) caused marked suppression of spermatogenesis and significant reduction in the weights of the testis, epididymis and accessory sex glands. The seminiferous tubules were devoid of spermatozoa and contained only Sertoli cells, spermatogonia, spermatocytes and occasionally spermatids; several multinucleated giant cells were observed in the lumen of the tubules. The Leydig cells were atrophied. The levels of RNA, DNA and protein in the testis were, however, unaffected by drug therapy. In drug-treated mice the epididymal epithelium presented a degenerate appearance; the lumen was generally devoid of spermatozoa and contained mainly exfoliated immature germ cells and sperm debris; cauda epididymidal spermatozoa, when present, were immotile and fragmented. PGF2 alpha treatment also caused significant decrease in the levels of sialic acid in the caput and cauda epididymides and of fructose in the seminal vesicle. The results suggest that the regressive changes induced by PGF2 alpha in the reproductive organs of the mouse are due to the interference with the secretion of androgen. The alterations induced in the reproductive organs by administration of PGF2 alpha were reversible and 56 days after drug withdrawal the organs returned to their normal state.

Influence of the length of proestrus on fertility and endocrine function in female cattle

Previous research from our laboratory in beef cattle suggests that ovarian follicle maturity and subsequent fertility is influenced by length of proestrus across a range of follicle sizes. To test this hypothesis an animal model was used in which ovulation from similar sized follicles was induced following either a long (LPE; ∼2.25 days) or short (SPE; ∼1.25 days) proestrus (interval from PGF 2α administration to a GnRH-induced LH surge). Specific objectives were to compare pregnancy rates and luteal phase concentrations of progesterone (Experiment 1) and to characterize preovulatory concentrations of estradiol, the GnRH-induced LH surge, and concentrations of progesterone in the subsequent estrous cycle (Experiment 2) between the LPE and SPE treatments. In Experiment 1, ovulation from follicles that were previously synchronized using follicular aspiration was induced with GnRH (Day 0) after either 2.25 days (LPE; n = 40) or 1.25 days (SPE; n = 38) of proestrus. Lactating and non-lactating cows were inseminated 12 h following GnRH administration. Ovulatory follicle diameter was similar between treatments. Pregnancy rates to AI were greater ( P < 0.01) in the LPE (50.0%) compared to the SPE (2.6%) treatment. The proportion of cows having a short luteal phase in the subsequent estrous cycle was greater ( P < 0.01) in the SPE than LPE treatment. In cows with a luteal phase of normal length, timed-AI pregnancy rates and concentrations of progesterone in the subsequent luteal phase were greater ( P < 0.05) in the LPE than SPE treatment. In Experiment 2, a similar experimental approach was taken with non-lactating beef cows and ovulation was induced following either 2.2 days (LPE; n = 8) or 1.2 days (SPE; n = 8) of proestrus. Ovulatory follicle diameter was similar between treatments. Concentrations of estradiol during the proestrus period were greater ( P < 0.05) in the LPE than SPE treatment from Days −1.9 to Day 0 (GnRH administration). Concentration of LH during the GnRH-induced LH surge and concentrations of progesterone in the subsequent estrous cycle did not differ between treatments although there was a tendency ( P = 0.10) for increased incidence of short luteal phases in the SPE treatment. In conclusion, decreasing the length of proestrus before induction of ovulation of a large follicle resulted in lesser pregnancy rates and an increased incidence of short luteal phases. The impact of a shortened proestrus on concentrations of progesterone in cows with luteal phases of normal length varied among experiments. Decreased circulating concentrations of estradiol during the preovulatory period and/or reduced concentrations of progesterone during the subsequent estrous cycle in cows that do not experience a shortened luteal phase may represent the mechanism responsible for reduced fertility with the SPE treatment.

Effect of time interval between prostaglandin F 2α and GnRH treatments on occurrence of short estrous cycles in cyclic dairy heifers and cows

Prostaglandin F 2α (PGF 2α) and GnRH treatments, when administered 24 h apart during early diestrus, cause short estrous cycles in some dairy cows and heifers [J. Taponen, M. Kulcsar, T. Katila, L. Katai, G. Huszenicza, H. Rodriguez-Martinez, Short estrous cycles and estrous signs after premature ovulations induced with cloprostenol and gonadotropin-releasing hormone in cyclic dairy cows, Theriogenology 2002; 58, 1291–1302]. We investigated the effect of a time interval between PGF 2α and GnRH administration on the appearance of short cycles. Estrus was induced in heifers with dexcloprostenol. A second luteolysis was induced similarly on day 7 after ovulation, and either 0 (T0) or 24 h (T24) later an injection of GnRH (0.1 mg of gonadorelin) was administered. We monitored ovarian activity with progesterone analyses from blood plasma samples and with ultrasonography. Fourteen cases (12 in T0 and 2 in T24) were excluded due to either incomplete luteolysis (2 cases) or unresponsiveness to GnRH (10 in T0 and 2 in T24). Short estrous cycles (7 to 8 d) were detected in 11/11 and 8/17 heifers in groups T0 and T24, respectively, with a significant difference in the incidence of short cycles ( P < 0.01). In Experiment 2, estrus was induced in cows on day 8 (D8, n = 18), 9 (D9, n = 5), or 10 (D10, n = 3) with cloprostenol and gonadorelin administered simultaneously. Daily milk samples were collected for progesterone analysis until subsequent estrus was detected and ovarian ultrasound examinations were performed. Eight cases had to be excluded due to unresponsiveness to GnRH, leaving 18 cases eligible for the study. Short estrous cycles (7–12 d) were detected in 14/18 cows. In conclusion, shortening the time interval between PGF 2α and GnRH treatments increased the incidence of short estrous cycles and appeared to increase the proportion of females unresponsive to GnRH treatment.

Reproductive performance of donor mares subsequent to eFSH treatment in early vernal transition: Comparison between the first, second, and mid-season estrous cycles of the breeding season

The objective was to compare the reproductive performances associated with the first (Cycle-1), second (Cycle-2), and mid-season (MS-Cycle) ovulations of the breeding season in donor mares that were treated with equine-FSH (eFSH) in the early vernal transition. Mares ( n = 15) kept under ambient light were examined ultrasonographically per-rectum starting January 30. When an ovarian follicle ≥25 mm in diameter was detected, twice daily eFSH treatments were initiated. The eFSH treatments ceased when a follicle ≥35 mm was detected, and 36 h later hCG was administered. Thereafter, mares were artificially inseminated every 48 h until ovulation (Day 0). Trans-cervical embryo recovery attempts were performed on Day 8, and subsequently PGF2α was administered. Equine FSH was not administered in the subsequent estrous cycles. In Cycle-2 and in the MS-Cycle, hCG was administered when a follicle ≥35 mm was detected; breeding, embryo recovery, and PGF2α administration, were similar to Cycle-1. Mares had an untreated estrous cycle (no treatment or breeding) between Cycle-2 and the MS-Cycle. All mares developed follicle(s) ≥35 mm after 4.9 ± 0.6 days of eFSH treatment, and subsequently ovulations occurred; mean (95% CI) interval from treatment initiation to ovulation was 7.9 (6.5–9.3) days. The number of preovulatory follicles (≥30 mm) at the time of hCG administration (Cycle-1: 2.2 ± 0.3 compared with Cycle-2: 1.0 ± 0 compared with MS-Cycle: 1.1 ± 0.1 follicles), and the number of ovulations (2.5 ± 0.4 compared with 1.0 ± 0 compared with 1.1 ± 0.1 ovulations) were greater ( p < 0.05) in Cycle-1. Nevertheless, mean embryo numbers did not differ among cycles (0.8 ± 0.2 compared with 0.5 ± 0.1 compared with 0.5 ± 0.1 embryo/mare). On average, embryo morphology grade was less ( p < 0.05) in Cycle-1 as compared to non-eFSH cycles (combined Cycle-2 and MS-Cycle). This impaired embryo quality could be due to a seasonal effect, or negative effect of the eFSH treatment, which was possibly related to alterations in the hormonal environment (estradiol-17β and progesterone). A prolonged IOI (>21 days) was recorded in 7 of 15 mares following the Cycle-1 ovulation, but not subsequently. In conclusion, eFSH treatment of vernal transitional donor mares stimulated ovulation within only few days of treatment, and the following embryo recovery rate was at least as good as in the subsequent estrous cycles; however, on average, embryos were morphologically impaired. In subsequent estrous cycles in the breeding season, ovulations, embryo recovery rates, and embryo variables did not appear to be negatively affected; however, the first inter-ovulatory interval of the breeding season was prolonged in approximately half of the mares.

292 EFFECT OF A SINGLE INJECTION OF FSH DISSOLVED IN POLYETHYLENE GLYCOL ON SUPEROVULATION RESPONSE IN KOREAN NATIVE COWS

Superovulation in bovine is usually achieved after several FSH injection in a time-consuming protocol, and the half-life of FSH is very short. This study was carried out to assess the efficiency of embryo production after induction of superovulation with a single injection of FSH (Folltropin-V, Vetrepharm, Canada) dissolved in polyethylene glycol (PEG, 8000 MW, Sigma-Aldrich Corp., St. Louis, MO, USA) in Korean Native cows. Eighty-eight cows were randomly assigned to one of four groups. In Group 1 (control), cows were intramuscularly treated with twice-daily administration of 50 mg FSH for 4 days. In Group 2 and 3, cows received a single subcutaneous injection of 400 mg and 200 mg FSH dissolved in 30% PEG, respectively. In Group 4, cows received a single subcutaneous injection of 200 mg FSH dissolved in 30% PEG given seven days after CIDR insertion. On days 10 to 12 after observed estrus, superovulation was started. Luteolysis was induced by administration of 25 mg of PGF2α (Pharmacia, Puurs, Belgium) IM 2 days after the start of the FSH injection. Donors were timed-inseminated with frozen–thawed semen at 48 and 60 h after the administration of PGF2α. Embryos were collected by non-surgical methods on 7 days after the second AI. The numbers of corpus lutea (CL), total ova, transferable embryos, and progesterone concentration were analyzed by General Linear Models Procedure with the Statistical Analysis System (SAS; SAS Institute, Inc., Cary, NC, USA) and means were compared with the Duncan’s multiple range test. Data on the pregnancy rate was analyzed by chi-square test. P values &lt;0.05 were considered to be significantly different. The number of CL in Group 2 was significantly (P &lt; 0.05) higher compared to Groups 1, 3 and 4 (18.5 ± 7.5 v. 11.2 ± 5.1, 13.1 ± 5.1 and 13.9 ± 6.4, respectively). However, the number of total ova (7.9 ± 6.0 to 10.4 ± 7.2) and transferable embryos (3.7 ± 3.3 to 4.7 ± 3.9) did not differ among treatment groups. In addition, blood progesterone concentrations at embryo recovery did not differ among all groups (10.0 ± 3.5 ng mL–1 to 15.7 ± 3.2 ng mL–1). No difference was observed among groups in pregnancy rate after transferring the recovered embryos (36.0 to 50.0%). In conclusion, our results show that a single injection of FSH dissolved in polyethylene glycol is an efficient and simple procedure for inducing superovulation in Korean native cows.

Differences in muscarinic-receptor agonist–, oxytocin-, and prostaglandin-induced uterine contractions

To investigate the contractile response of the perfused swine uterus to various receptor pathways (oxytocin, prostaglandins, and muscarine). An extracorporeal perfusion model of the swine uterus was used that keeps the uterus in a functional condition and is appropriate for the study of physiologic questions. Oxytocin-, prostaglandin-, and carbachol-induced uterine contractility and peristalsis were assessed using an intrauterine double-chip microcatheter. University hospital. Intrauterine pressure profiles. A dose-dependent increase in intrauterine pressure (IUP) in the isthmus uteri and corpus uteri was observed after the administration of prostaglandin F(2alpha) (PGF(2alpha)) and oxytocin, which reached a plateau after further stimulation. A dose-dependent increase in IUP in the isthmus uteri and corpus uteri was also observed after the administration of prostaglandin E(1) (PGE(1)) and prostaglandin E(2) (PGE(2)), with a plateau in IUP in the middle-concentration range and a decrease during the further course of stimulation. After administration of PGE(1), PGE(2), and PGF(2alpha), different directions of contraction waves were also observed. Carbachol also showed a unique contractility pattern, with isolated, very powerful, dose-dependent contractions with an IUP gradient, suggesting directed transport from the upper region to the lower region. This study demonstrated that oxytocin, the prostaglandins, and carbachol modulate contractility in nonpregnant swine uteri in a characteristic way, resulting in different contractility patterns.

USO DA PGF2? NO PUERPÉRIO PARA REDUZIR O ANESTRO PÓS-PARTO DE CABRAS EM ALEITAMENTO CONTÍNUO E CONTROLADO

This work aimed to study the effect of PGF2α administration on anestrous post-partum period in 80 goats, with continuos or controlled suckled, rised in semi-extensive system with water and mineral salt ad libitum . The females, with age between two and six years, were randomly distributed in three groups (GI, GII, GIII). The females of GI (n = 30) received 250µg of PGF2α in vulvar muscle on days 6 th and 12 th after delivery. The females of GII (n = 30) received the same treatment of Group I, but every day from days 6 th to 10 th after delivery. The GIII (n = 20) was the control group. The estrous detection was made by using teasers and the mating with bucks with confirmed fertility. The obtained data were analyzed by ANOVA and by analysis of standard errors of difference among proportions. Difference in the occurrence of estrous among the three experimental groups was not observed (P > 0.05), however the average time of post partum anestrous was significantly reduced (P 0.05) on pregnant percentages among the different groups. It may be concluded that the administration of PGF 2α in the beginning of puerperium in caprine is efficient to reduce the anestrous pos-partum period, specially in females controlled suckled; however do not influence the fertility in meat goats. KEY WORDS: Caprine, estrus, puerperium, prostaglandine.

Short Communication: Pregnancy Rates to Timed Artificial Insemination in Holstein Heifers Given Prostaglandin F2α Twenty-Four Hours Before or Concurrent with Removal of an Intravaginal Progesterone-Releasing Insert

The objective was to compare pregnancy rates in nulliparous Holstein heifers given PGF2α 24h before, or concurrent with, removal of an intravaginal progesterone-releasing (CIDR) insert in 3 timed artificial insemination (TAI) protocols. Heifers (from 2 herds) were assigned randomly, over 11 mo, to 1 of 3 modified Ovsynch protocols. On d 0 (without reference to the stage of the estrous cycle), all heifers were given 100μg of GnRH i.m. and a CIDR insert (containing 1.9g of progesterone). In the PG-7/P4-8 protocol (n=99), PGF2α was given on d 7, and CIDR inserts were removed on d 8. In the PG-7/P4-7 (n=98) and PG-8/P4-8 (n=102) protocols, PGF2α administration and CIDR removal occurred concurrently, on d 7 or 8, respectively. In all 3 protocols, a second GnRH treatment (100μg) was given 48h after PGF2α with TAI 16 to 20h later. Blood samples were collected (subset of 124 heifers) on d 0, 7, 10 or 11 (i.e., at TAI), and 17. Pregnancy rates (32 d after TAI) for protocols PG-7/P4-8, PG-7/P4-7, and PG-8/P4-8 were 61.8, 55.6, and 54.1%, respectively. Pregnancy rate was higher when synchronization was initiated during diestrus than when initiated at other stages (57.0 versus 34.8%). Although pregnancy rates were not affected by season, there was an interaction between protocol and season; pregnancy rates were significantly lower in summer in heifers subjected to PG-7/P4–7 and PG-8/P4–8, but season did not affect pregnancy rates in heifers subjected to PG-7/P4-8. In summary, giving PGF2α 24h before CIDR removal, followed by TAI (PG-7/P4-8 protocol), resulted in consistent pregnancy rates, regardless of season, relative to protocols involving PGF2α treatment concurrent with CIDR removal.

Role of PKCεin PGF2α-Stimulated MMP-2 Secretion from Human Ciliary Muscle Cells

Studies were designed to examine the roles of individual protein kinase C (PKC) isoforms in the prostaglandin F(2alpha) (PGF(2alpha))-induced matrix metalloproteinase-2 (MMP-2) secretion from human ciliary muscle cells. Studies utilized primary cultures of human ciliary muscle cells. Individual PKC isoforms were detected by Western blotting, using PKC-isoform-specific antibodies. To evaluate MMP-2 secretion, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 4 h and the media analyzed for MMP-2 by Western blotting. To assess ERK1/2 activation, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 5 min and cell lysates analyzed for ERK1/2 phosphorylation by Western blot analysis. To evaluate the roles of individual PKC isoforms, cells were pretreated with PKC inhibitors or siRNAs prior to the addition of PGF(2alpha). In cultured human ciliary muscle cells, the PKC isoforms exhibiting the highest level of expression were PKCalpha, epsilon, iota and lambda. The delta and eta isoforms exhibited moderate levels of expression and beta, gamma, and phi were not detected. The administration of PGF(2alpha) (1 micromol/L) primarily induced the translocation of PKCepsilon from cytosol to the membrane fraction, as well as increased MMP-2 secretion and ERK1/2 phosphorylation. The secretion of MMP-2 was inhibited by pretreatment with the broad-range PKC inhibitor, chelerythrine chloride; however, this response was not blocked by Go-6976, an inhibitor of conventional PKC isoforms. The PGF(2alpha)-induced secretion of MMP-2 was also blocked by pretreatment with the PKCepsilon-selective peptide translocation inhibitor, EAVSLKPT, or the transfection of siRNA-targeting PKCepsilon. The activation of ERK1/2 was inhibited by chelerythrine and the PKCepsilon translocation inhibitor. Human ciliary muscle cells express the alpha, epsilon, iota and lambda PKC isoforms. Stimulation of FP receptors in these cells activates PKCepsilon, resulting in ERK1/2 activation and an eventual increase in MMP-2 secretion. These data support the idea that the activation of FP receptors in vivo modulate uveoscleral outflow through the PKCepsilon-dependent secretion of MMPs.

Prostaglandin F2α involves heparan sulphate sugar chains and FGFRs to modulate osteoblast growth and differentiation

The present investigation extends our previous studies on PGF2alpha-mediated signalling in osteoblast metabolism. In particular, the role of PGF2alpha as modulator of heparan sulphate proteoglycans (HSPGs), fibroblast growth factor 2 (FGF-2) and fibroblast growth factor receptors (FGFRs) was evaluated. We hereby reported the novel observation that PGF2alpha was able to promote the formation of HSPGs/FGF-2/FGFRs complexes. Moreover, our data suggested that PGF2alpha could induce new synthesis of heparan sulphate (HS) chains on osteoblasts by a mechanism involving a modulation of MAPK signalling and that HS is required for the regulation of FGF-2 induced by PGF2alpha. Indeed, a proteolytic cleavage of HSPGs with heparinase III (Hep III) prior to PGF2alpha administration down-regulated the basal expression of phospho-p44/42, likely inhibiting FGFRs tyrosine kinase activity. Interestingly, MAPK signalling influenced syntheses and subcellular localization of FGF-2, its specific receptor and HS. In addition, the proteolytic cleavage by Hep III and the MAPK kinase inhibition by PD-98059 also revealed that PGF2alpha induced cell proliferation is dependent on HSPGs and FGF-2 specific receptor, respectively. Of further relevance of this study, we demonstrated, by using a specific siRNA for FGFR1, that PGF2alpha modulates Runx2 expression by FGFR1 and HS.

Effect of Prostaglandin F2α (PGF2α) Administration on the Luteotropic and Angiogenic Factors During Functional Luteolysis in the Bovine Corpus Luteum.

The essential role of endometrium PGF2α in induction of the corpus luteum (CL) regression (luteolysis) is well documented in the cow. However, the following regulatory cascade of functional luteolysis is still not fully elucidated. The aim of the present study was therefore to determine the regulation patterns of local luteotropic factors (progesterone, oxytocin and insulin-like growth factor (IGF) family members) and angiogenic factors (vascular endothelial growth factor-A (VEGF) and angiopoietin (ANPT) family members) during functional luteolysis (first 12 h after PGF2α injection) in time-defined CL classes. Cows (n=4-5 per group) in the mid-luteal phase (days 8-12) were injected with the PGF2α-analogue (cloprostenol), and CLs were collected by transvaginal ovariectomy at 0 h, 0.5 h, 2 h, 4 h and 12 h after PGF2α injection. The mRNA expression was analyzed by a quantitative real-time PCR (Rotor-Gene 3000), and the protein concentration was evaluated by enzyme immunoassay (EIA) or radio immunoassay (RIA). Progesterone concentration in blood serum and in CL tissue as well as mRNA expression of progesterone receptor in CL tissue, were significantly downregulated at 12 h after PGF2α. Oxytocin and IGF-1 peptide concentrations rapidly declined after 0.5 h and remained at low level afterwards. The IGF binding protein (BP)-1 mRNA was strongly upregulated with the maximal level at 4 h after PGF2α. This may cause further reduction of bioactive free IGF-1 in tissue. The VEGF protein decreased also at 0.5 h, with a synchronous acute and temporal increase of angiopoietin (ANPT)-2 peptide concentrations (0.5 h and 2 h after PGF2α). The similar tendency of upregulation of ANPT-2 was found for mRNA expression level, whereas the level of ANPT-1 mRNA did not change. The results suggest that the acute decrease of the local luteotropic factors and of VEGF may play a key role during the early step of functional luteolysis. The increase of ANPT-2 after PGF2α suggests a change in vascular stability to enhance the cascade of functional luteolysis.

Effect of prostaglandin F2α at the time of AI on progesterone levels and pregnancy rate in synchronized Italian Mediterranean buffaloes

The aims of this study were to evaluate the effects of an intravenous or intramuscular PGF2alpha analogue administration on the day of estrus on progesterone concentration and pregnancy rate in buffaloes undergoing artificial insemination (AI). To this end, two experiments were carried out. The first study was performed on 72 Mediterranean buffaloes synchronized by the Ovsynch-TAI Program. On the day of estrus only animals considered in heat were divided into four groups: Groups IVC and IMC received, respectively, an intravenous or intramuscular injection of cloprostenol (0.524 mg), whereas control Groups IVS and IMS received the same injections of saline. Milk samples were collected daily from each animal to assess progesterone concentration in the whey by RIA method. In addition on alternate days, buffaloes underwent transrectal ultrasound analysis. The second study was carried out on 385 buffaloes synchronized by the Ovsynch-TAI Program. On the day of AI, animals were divided in four groups, as described in experiment 1. Pregnancy rate was evaluated either on day 26 or day 45 and embryonic mortality rate was recorded. Statistical analysis was performed by ANOVA and chi2 test. A higher (P<0.05) progesterone concentration was recorded on day 11 (Day 0=estrus day) in Groups IVC and IMC compared to Groups IVS and IMS (351.6+/-129.7 and 355.8+/-112.2 pg ml(-1) vs. 239.8+/-81.1 and 243.6+/-90.5 pg ml(-1), respectively). Furthermore, a larger CL was recorded on the same day in treated vs. control groups (1.25+/-0.15 and 1.27+/-0.17 cm, respectively, in Groups IVC and IMC vs. 1.08+/-0.14 and 1.05+/-0.13 cm in IVS and IMS). In the second study, a higher pregnancy rate was observed in treated (IVC+IMC) vs. control (IVS+IMS) groups (46.7% vs. 30.7%; P<0.01), while no differences were recorded between treated groups. From these data, it can be concluded that either intravenous or intramuscular administration of PGF2alpha at the time of AI can enhance progesterone levels and pregnancy rate in buffaloes.

Embryo recovery from exercised mares

The effect of exercise on mare reproductive efficiency was evaluated by comparing rates of embryo recovery from mares assigned to either an exercise regimen or a non-exercise (control) regimen. Exercised mares were worked daily for 30 min under average ambient conditions of >30 °C and >50% humidity. Mares were inseminated during estrus and subjected to uterine flush for embryo recovery on d 7 after ovulation for two consecutive cycles. After this, mares were allocated to the opposite group and allowed an estrous cycle without reproductive manipulation; then insemination and uterine flushing were conducted on two more consecutive cycles. Prostaglandin F 2α was administered on the day of uterine flush. Mare rectal temperature increased during exercise from a mean of 38 °C to a mean of 39.9 °C. Mares had ovulations from smaller follicles when exercised than they did under control conditions (39.8 ± 0.5 compared with 41.5 ± 0.5 mm diameter; P < 0.05), and had an increased time from PGF 2α administration to subsequent ovulation (8.47 ± 0.337 compared with 9.27 ± 0.294 d; P < 0.05). Embryo recovery from control mares was 22 of 35 (63%). Fewer embryos were recovered from exercised mares (11 of 32, 34%; P < 0.05). The proportion of embryos classified as Grade 1 tended to be less in exercised than in non-exercised mares (4 of 11, 36% compared with 16 of 22, 73%; P = 0.051). These data indicate that exercising mares in a hot and humid environment are associated with changes in ovarian follicle development and ovulation, and a reduction in embryo recovery.

Follicle and systemic hormone interrelationships during spontaneous and ablation-induced ovulatory waves in mares

The characteristics of ovulatory follicular waves were studied for spontaneous waves and waves induced during the next estrous cycle by ovarian follicle ablations and administration of PGF2α 10 days after ovulation in 21 mares. In the induced group, both the days of the FSH surge and day of deviation were more synchronized, LH concentrations were greater before and after deviation, estradiol concentrations were greater after deviation, and the ovulatory follicle grew at a faster rate (3.4 ± 0.2 compared with 2.7 ± 0.1 mm/day). The frequency of two dominant follicles/wave was not different between induced waves (7 of 21) and spontaneous waves (9 of 21), but both dominant follicles ovulated more frequently in induced waves (6 of 7 waves compared with 0 of 9).

Effect of Norgestomet Treatment on Plasminogen Activator Activity in the Cervical Mucus and the Endometrium in Dairy Cows

The aim of this study was to determine the effect of norgestomet treatment, in the absence or the presence of a functional corpus luteum (CL), on plasminogen activators activity (PAA) in the cervical mucus and the endometrium in dairy cows. Eleven days after oestrus (Day 0 = oestrus), 38 cows were randomly assigned to one untreated control group (n = 9) and three treatment groups (S(1), S(2) and S(3)). Animals of S(1) group (n = 9) received an implantation of norgestomet on the outer surface of the ear for 8 days, simultaneous injection of oestradiol valerate 5 mg and norgestomet 3 mg, i.m., and on Day 19 an injection of ECG 500 IU, i.m. Animals of S(2) group (n = 11) received the treatment of S(1) group, plus an administration of PGF(2)alpha on Day 10 for the regression of CL. Animals of S(3) group received the treatment of S(2) group, plus two additional norgestomet implants inserted on Day 16 for 36 h. Both types of plasminogen activators [the tissue-type (t-PA) and the urokinase-type (u-PA)] were detected in the cervical mucus and the endometrium of the cows. Plasminogen activators activity in the cervical mucus was higher in control group than in S(1), S(2) and S(3) groups (P < 0.001). In contrast, endometrial PAA did not differ among groups (P > 0.05). Oestradiol-17beta concentrations on Day 21 were higher in S(2) group than in control group (P < 0.01) and S(3) group (P < 0.05). Progesterone concentrations did not differ among groups (P > 0.05). Oestradiol-17beta concentrations could positively affect cervical mucus PAA in control group (P < 0.1), but not in other groups (P > 0.05). These findings suggest that control of estrous cycle by norgestomet administration, in dairy cows, exerts a suppressive effect on plasminogen activators synthesis and/or secretion in the cervical mucus, regardless of the absence or the presence of the CL. On the contrary, endometrial PAA is not affected by norgestomet treatment.

Activity and expression of different members of the caspase family in the rat corpus luteum during pregnancy and postpartum

Studies were designed to examine the expression and activity of four caspases that contribute to the initial (caspases-2, -8, and -9) and final (caspase-3) events in apoptosis in the rat corpus luteum (CL) during pregnancy (days 7, 17, 19, and 21 of gestation), postpartum (days 1 and 4), and after injection (0, 8, 16, 24, and 36 h) of the physiological luteolysin PGF2alpha. In addition, the temporal relationship of caspase expression/activity relative to steroid production and luteal regression was evaluated. During pregnancy, the activity of all four caspases was significantly greater on day 19, before a decline in CL progesterone (P) and CYP11A1 levels at day 21 of gestation. The levels of the caspase-3 active fragment (p17, measured by Western blot) also increased at days 19 and 21 of pregnancy. Immunohistochemical analyses detected specific staining for the caspases in luteal cells (large and small) as well as in endothelial cells. However, the percentage of apoptotic cells did not increase in the CL until postpartum. Following PGF2alpha injection, there was a significant decrease in CL P by 24 h, although the activity of all four caspases did not increase until 36 h posttreatment. The active p17 fragment of caspase-3 also significantly increased at 36 h post-PGF2alpha. These results suggest that an increase in the activity of caspases-2, -8, -9, and -3 is associated with the early events of natural luteolysis at the end of pregnancy. Also, the exogenous administration of the luteolysin PGF2alpha may regulate members of the caspase family.

17 INFLUENCE OF DIFFERENT CONCENTRATIONS OF PROGESTERONE DURING FIXED-TIME ARTIFICIAL INSEMINATION PROTOCOLS ON OVULATION AND PREGNANCY RATES IN NELORE HEIFERS

Previous reports indicate that a high concentration of serum progesterone (P4) decreases LH pulse frequency (Burke et al. 1996 Anim. Reprod. Sci. 45, 13–28) and, consequently, follicular growth. This may reduce the efficiency of fixed-time artificial insemination (FTAI) protocols that utilize P4 in Bos indicus heifers (Marques et al. 2005 Acta Sci. Vet.). The objective of the present work was to evaluate the influence of different P4 concentrations on the ovulation and pregnancy rates of zebu heifers subjected to hormonal protocols for FTAI. Four hormonal protocols with different P4 concentrations were used to induce synchronization of ovulation in Nelore (Bos indicus) heifers (n = 292). Before the beginning of the treatments, the animals were subjected to ovary evaluations by ultrasonography (Aloka SSD 500, 5-MHz probe; Aloka, Tokyo, Japan) twice in an interval of 10 days. Only heifers with a corpus luteum (CL) in at least one of the ultrasonographic evaluations were utilized. At a random stage of the estrous cycle, the selected heifers received an intravaginal progesterone-releasing device containing 0.558 g of P4 [Triu�, Biogenesis, Buenos Aires, Argentina; Treatment 1 (T1); n = 76], or 1 g of P4 [Cronipress�, Biogenesis; Treatment 2 (T2); n = 70], or 0.558 g of P4 and prostaglandin F2α (PGF2α) [150 �g d-cloprostenol, IM, Croniben�, Biogenesis; Treatment 3 (T3), n = 75], or 1 g of P4 and PGF2α [Treatment 4 (T4), n = 71]. At the time of intravaginal device insertion (Day 0 = D0), all animals received 2 mg of estradiol benzoate (EB, Bioestrogen�, IM, Biogenesis), and PGF2α was administered only in animals from groups T3 and T4. Eight days later (D8), PGF2α (0.150 g d-cloprostenol) was administered to all heifers and the P4 source was removed. Twenty-four h after removal of the intravaginal device, the heifers were treated with EB (1 mg, IM), and 30 to 36 h later all animals were fixed-time inseminated (FTAI, D10), without estrus detection. In a subset of heifers from each group (T1 = 28; T2 = 34; T3 = 33; T4 = 31), ovarian ultrasonography was performed on D9 (at 8:00 A.M.) and D11 (at 14:00 P.M.) in order to determine ovulation rate after each treatment. The data were analyzed by logistic regression (GENPROC, SAS; SAS Institute, Inc., Cary, NC, USA). Ovulation rates after treatments T1, T2, T3, and T4, were, respectively, 71.4% (20/28), 76.5% (26/34), 84.9% (28/33), and 70.9% (22/31), and there was no significant difference among groups (P &gt; 0.05). Pregnancy rates, ascertained by ultrasonography 40 days after AI, were 30.2% (23/76), 25.7% (18/70), 33.3% (25/75), and 28.2% (20/71), respectively, for T1, T2, T3, and T4, and likewise there was no significant difference among groups (P &gt; 0.05). It is concluded that reduction of progesterone concentration in the intravaginal device (1 g v. 0.558 g) associated or not with administration of PGF2α at the beginning of treatments did not significantly influence ovulation and pregnancy rates in Nelore heifers submitted to a FTAI protocol. Additional experiments are underway to confirm or not the present results. This work was supported by FAPESP (Brazil) and BIOGENESIS (Argentina).

Is slow follicular growth the cause of silent estrus in water buffaloes?

The present experiment was conducted to study the growth profile of the ovulatory follicle in relation to the expression of estrus following administration of PGF 2α to subestrus buffaloes. After detection of a mature corpus luteum by examination per rectum, confirmed by ultrasound scanning, subestrus buffaloes ( n = 20) were treated (Day 0) with single dose of Dinoprost tromethamin (25 mg, i.m.). Blood samples were collected at 0, 24 and 48 h after treatment for estimation of plasma progesterone concentration. Growth profile of the ovulatory follicle was monitored daily through ultrasound scanning starting from Day 0 until ovulation and the regression profile of CL was monitored at 0, 24 and 48 h of treatment. Estrus was detected by exposure to a fertile buffalo bull three times a day until expression of overt estrus or ovulation. Behavioral estrus was recorded in 14 animals and 6 animals ovulated silently. Sixteen animals including six animals with silent estrus ovulated from the dominant follicle present at treatment (Group A) and remaining four animals ovulated from the dominant follicle of succeeding follicular wave (Group B). The intervals from treatment to estrus (6.5 ± 0.25 versus 3.2 ± 0.27 days, P < 0.001) and treatment to ovulation (7.5 ± 0.25 versus 5.4 ± 0.46 days, P < 0.005) were significantly longer in animals of Group B compared with animals of Group A. Significant differences were observed in growth profile of the ovulatory follicle between animals of Groups A and B with respect to size of the follicle on Day 0 (9.8 ± 0.7 versus 5.3 ± 0.45 mm, P < 0.001), daily growth rate (0.97 ± 0.07 versus 1.6 ± 0.2 mm/day, P < 0.01) and increase in diameter (4.1 ± 0.6 versus 7.8 ± 0.7 mm, P < 0.01). The animals with silent estrus (subgroup A-2) had significantly smaller diameter of the ovulatory follicle on Day 0 (7.7 ± 0.4 versus 11.0 ± 0.7 mm, P < 0.005), its daily growth rate was significantly slower (0.7 ± 0.02 versus 1.1 ± 0.1 mm/day, P < 0.01) and they recorded significantly longer interval from treatment to ovulation (7.3 ± 0.56 versus 4.2 ± 0.27 days, P < 0.001) compared with the animals that showed overt estrus (subgroup A-1). The corpus luteum area (CL area) and plasma progesterone (P 4) concentration declined continuously from 0 to 48 h after PGF 2α treatment in the animals of both the Groups A and B. Non-significant differences were observed in mean CL area and plasma P 4 concentration at 0, 24 and 48 h post-treatment between animals of Groups A and B and also between animals of subgroups A-1 and A-2. The small size and the slow growth rate of the ovulatory follicle were identified as the possible cause of silent estrus in subestrus buffaloes after PGF 2α treatment.

Using “Uterine Index” to diagnose embryonic death in mares

Embryonic death plays an important role in infertility in mares, and most of embryonic mortality occurs at an early stage (before day 40) of pregnancy. Previous investigations focused on B-mode sonographic images to determine abnormal pregnancy; however, no study has provided a quantitative and objective method to diagnose embryonic death. Here we developed a new index, “Uterine Index,” using color Doppler sonography to describe alterations of uterine blood flow in physiological and pathological gestations during early gestation. Six Trotter mares were used in this study. The pregnancies were interrupted with a prostaglandin F2α (PGF 2α) analog (tiaprost) at days 25, 30, and 40. Three gestations were investigated in each stage. After the PGF 2α administration, these mares were examined every 8 hours until the embryonic vesicles were not visible on ultrasound. In the control group, three pregnancies were examined daily from days 12 to 60. The embryo size was measured in B-mode sonography, and the blood flow in uterine arteries was examined in Doppler ultrasound. The resistance index (RI) and uterine index (UI) were calculated. In the control group, the RI values of uterine artery were lower in pregnant side compared with that of the non-pregnant side from day 15 on, and the difference became greater as the pregnancy advanced ( P < .05). The UI was close to 0 before week 3 and increased to approximately 10 after week 4. In the PGF 2α-treated group, the UI values reduced from pregnancy level to approximately 0. As early as 32 hours before embryonic death, we observed a significant difference of UI values between control and treated mares. On the contrary, the embryo size of treated mares was not different from that of normal mares. Through the analysis of all recorded UI values, we suggest that the UI values 10 and 5 are good values for distinguishing normal pregnancy (UI >10) from embryonic death (UI < 5). If the UI values are located between 10 and 5, a reexamination at intervals of 1 to 3 days is recommended. Our findings demonstrate that the UI is a useful method to diagnose and predict embryonic death in mares by providing reliable and objective information.