Injection Of PGF2 Alpha Research Articles

Comparison of Misoprostol and PGF2a for second trimester termination of Pregnancy

Aims: To compare the effectiveness of vaginally administered misoprostol with extra-amniotic prostaglandin F2alpha (PGF2alpha) injection for termination of pregnancy. Study Design: Interventional study. Setting: This study was conducted in gynae unit III Nishter hospital Multan over period f 15 months. Material & Methods: Patients admitted to labour ward with pregnancy of less than 20 weeks of gestation requiring termination of pregnancy due to fetal and maternal causes were included in this study. A detailed history was taken followed by clinical examination especially the cervical scoring was done. Sixty patients were randomly assigned to receive either tablet misoprostol (cytotec) intravaginally or extra-amniotic injection of prostaglandin F2a Induction-expulsion interval, need for evacuation and the incidence of side effects were analyzed for both groups. Results: Successful termination was achieved in 98% patients with misoprostol and 94% with PGF2a Induction expulsion interval was < 12 hrs in 80% patients in misoprostol group and 60% in PGF2a group (P value < 0.000). Evacuation rate was 23.3% with PGF2a and 10% with misoprostol group. The incidence of prostaglandin-associated pyrexia, vomiting and diarrhea were significantly increased in the PGF2alpha group (p<0.05). Conclusion: In this study, vaginal misoprostol has been found to be more effective and associated with a shorter induction to expulsion interval, fewer ERPOC for incomplete evacuation and less side effects when compared with extra-amniotic PGF2alpha. In view of its effectiveness, as shown in this study, vaginally administered misoprostol has the potential to provide a better option for medical abortion in many parts of the world.

Determining Proportion of Exfoliative Vaginal Cell during Various Stages of Estrus Cycle Using Vaginal Cytology Techniques in Aceh Cattle.

The aim of this study was to investigate the period of estrus cycle in aceh cattle, Indonesia, based on vaginal cytology techniques. Four healthy females of aceh cattle with average weight of 250–300 kg, age of 5–7 years, and body condition score of 3-4 were used. All cattle were subjected to ultrasonography analysis for the occurrence of corpus luteum before being synchronized using intramuscular injections of PGF2 alpha 25 mg. A vaginal swab was collected from aceh cattle, stained with Giemsa 10%, and observed microscopically. Period of estrus cycle was predicted from day 1 to day 24 after estrus synchronization was confirmed using ultrasonography analysis at the same day. The result showed that parabasal, intermediary, and superficial epithelium were found in the vaginal swabs collected from proestrus, metestrus, and diestrus aceh cattle. Proportions of these cells in the particular period of estrus cycle were 36.22, 32.62, and 31.16 (proestrus); 21.33, 32.58, and 46.09 (estrus); 40.75, 37.58, and 21.67 (metestrus); and 41.07, 37.38, and 21.67 (diestrus), respectively. In conclusion, dominant proportion of superficial cell that occurred in estrus period might be used as the base for determining optimal time for insemination.

Females with Reduced Fertility Have Excess Androstenedione in Follicular Fluid, Altered Theca Gene Expression and Increased VEGFA164b, Maternal Effect, and microRNA Processing mRNA Levels in Cumulus-Oocyte Complexes.

Ovarian dysfunction contributes significantly to female infertility. However, the intrinsic and exogenous factors that result in abnormal ovarian function are poorly defined. Thus, we have established a cow model of fertility to identify mechanisms regulating follicular growth, steroidogenesis and oocyte growth and maturation. Culling age due to failure to establish pregnancy was used to classify animals with low (>/= 2 years-of-age), moderate (> 2 - < 6 years-of-age), and high (>/= 6 years-of-age) fertility. Previous experiments using this model identified class-dependent changes in mural granulosa cell gene expression. The objective of this study was to identify the effect of ovarian and follicular environment on the oocyte molecular phenotype. To attain this objective, females from each fertility group were synchronized using a modified Co-Sync (CIDR) protocol and ovariectomies were performed 36 h after PGF2-alpha injection and CIDR removal. Surface antral follicle counts (AFC) for each ovary pair and the follicular fluid, theca cells, mural granulosa cells and cumulus-oocyte complex (COC) from each dominant follicle were collected. Dominant follicles were confirmed based on size (i.e. largest follicle on the ovaries) and an estrogen- to-progesterone ratio greater than 1.0 in the follicular fluid. Antral follicle counts were lower in low fertility (n = 14) than moderate (n = 17) or high fertility (n = 17) cows. Furthermore, androstenedione (A4) concentrations were 4 fold (P < 0.002) greater in follicular fluid from low compared to high fertility group cows. Given that increased androgens is a hallmark characteristic of the PCOS phenotype, gene expression in theca cells from the different fertility groups was compared. The affect of increased intrafollicular androgen levels on cumulus-oocyte gene expression in the dominant follicle was also examined. Quantitative, real-time RT-PCR demonstrated that mRNA abundance of maternal effect genes NLRP5 (8-fold P < 0.0001), Zar1 (2-fold; P < 0.0001) and Dnmt1 (P < 0.006) and miRNA processing genes DGCR8 (P < 0.005) and RNAsen (P < 0.005) were increased in low compared to high fertility cows. Increased mRNA abundance in low fertility animals is consistent with previous findings in PCOS oocytes and suggests that abnormal endocrine profiles of follicular fluid has a global effect on transcriptional and post-transcriptional regulation of mRNAs in the oocyte. Previous studies identified differences in the expression profile of vascular endothelial growth factor A (VEGFA) isoforms in mural granulosa cells between low, moderate, and high fertility group cows. In the current study, VEGFA_164B mRNA abundance was also increased in cumulus granulosa cells of low fertility cows (P < 0.005). Taken together, these data indicate that females with low fertility have intrinsic differences in ovarian morphology and steroidogenesis. Furthermore, the data suggest that increased androgen production in low fertility animals alters gene expression and/or mRNA stability during oocyte growth and maturation. These changes in oocyte gene expression may reduce the developmental competence of the oocyte and contribute to the reduced pregnancy rates in low fertility cows. USDA is an equal opportunity employer. (poster)

Comparison in Effect of Heatsynch with Heat Detection Aids and CIDR‐Heatsynch in Dairy Heifers

The objective of the present study was to determine whether oestrous detection with the help of oestrous detection aids during the Heatsynch without timed AI protocol is equally effective with the progesterone-combined protocol in dairy heifers. A total of 148 heifers were randomly assigned to one of the two groups. A group of heifers treated with Heatsynch with heat detection aids (n = 72) received GnRH on day 0, prostaglandin F(2alpha) (PGF(2alpha)) on day 7 and oestradiol benzoate (EB) on day 8, while in controlled internal drug release (CIDR)-Heatsynch group (n = 76), CIDR was included during a period from GnRH to PGF(2alpha). Heifers were checked for oestrus twice daily, i.e. from 09:00 to 10:00 hours and from 15:00 to 16:00 hours starting on day 2 for Heatsynch group and on day 8 in CIDR-Heatsynch group, and continued up to day 12. KAMAR heat mount detector (KAMAR Inc., Steamboat Springs, CO, USA) and ALL-WEATHER PAINTSTIK (LA-CO Industries Inc., Elk Grove Village, IL, USA) were used as heat detection aids. AI was conducted within 1 h after confirming oestrus in 72 heifers, while 19 animals were transferred with embryo 7 days after oestrus according to the request of the owners. Premature oestrus before PGF(2alpha) injection occurred in 18% of Heatsynch group. Of 13 heifers which showed premature oestrus, six were inseminated and two of them conceived. Oestrus detection rate within 12 days after initiation of the protocols did not differ between the two groups (94% vs 95%). There was no difference in the conception rate after first AI (including heifers that were inseminated before PGF(2alpha) injection) and embryo transfer between Heatsynch with heat detection aids and CIDR-Heatsynch groups (36% vs 44% and 70% vs 56%). It is concluded that the use of heat detection aids to monitor the occurrence of premature oestrus prior to PGF(2alpha) injection in Heatsynch protocol in dairy heifers was equally effective to the inclusion of CIDR.

Effect of dietary crude protein source on hormone and follicle characteristics in beef heifers1

Ground, raw soybeans (SB), or dried distillers grain plus solubles (DDGS) were utilized in heifer development diets to determine the effect of dietary fat and protein source on hormone and follicle characteristics and ADG. The experiment was conducted over 2 yr with 100 June-born heifers (199 +/- 2 kg initial BW, n = 50 per yr). The experimental periods were 157 and 207 d in yr 1 and 2, respectively. Heifers were provided a dietary supplement (DM basis) of 1.23 kg of SB and 0.40 kg of corn or 1.65 kg of DDGS between weaning and breeding. Estrus was synchronized with 2 injections of PGF(2alpha) 14 d apart. Dominant follicles were measured and aspirated via transvaginal ultrasonography 60 h after the second PGF(2alpha) injection. Heifers were exposed to bulls beginning 14 d after aspiration for 45 d. Heifer ADG was greater (P = 0.02) for DDGS heifers in yr 1, but was similar (P = 0.47) in yr 2. However, there was no difference (P = 0.35) in final BW in either year. There was no difference (P >or= 0.67) in follicle size, follicle hormone concentrations, or pregnancy rate (88%) between yr 1 and 2. Serum estrogen at 48 or 60 h after PGF(2alpha) injection were similar (P >or= 0.91); however, LH at 60 h in yr 2 tended to be greater (P = 0.07) for DDGS heifers. The percentage of heifers experiencing an LH surge 48 and 60 h after PGF(2alpha) injection was not affected (P >/= 0.40) by treatment. Calf production was not affected (P >or= 0.20) by developmental diet. In summary, DDGS and SB have similar effects on hormone and follicle characteristics at the inclusion rates used in these studies.

Comparison of short-term progestin-based protocols to synchronize estrus and ovulation in postpartum beef cows1

Two experiments evaluated short-term controlled internal drug release (CIDR) insert-based protocols to synchronize estrus and ovulation and compare differences in their potential to facilitate fixed-time AI (FTAI) in postpartum beef cows. Experiment 1 was designed to compare the 7- and 5-d Select Synch + CIDR protocols on the basis of timing and synchrony of estrus after treatment. Cows assigned to the 7-d protocol (n = 59) received GnRH [100 microg intramuscularly (i.m.)] and CIDR inserts (1.38 g of progesterone) on d 0 and PGF(2alpha) (25 mg i.m.) and CIDR removal on d 7. Cows assigned to the 5-d protocol (n = 58) received GnRH and CIDR inserts on d 2, PGF(2alpha) and CIDR removal on d 7, and a second injection of PGF(2alpha) 12 h after CIDR removal. Estrus detection and AI were performed for cows assigned to each protocol during the 144-h synchronized period. There was no difference in estrous response (P = 0.85), interval to estrus (P = 0.09), or variance for interval to estrus (P = 0.75) between treatments, nor were there differences in synchronized conception or pregnancy rates resulting from AI (P = 0.85, P = 0.91, respectively). Experiment 2 was designed to compare pregnancy rates resulting from FTAI after administration of the 7- and 5-d CO-Synch + CIDR protocols. Both treatments were administered the same as in Exp. 1; however, cows assigned to the 7-d protocol were inseminated 66 h after PGF(2alpha) and CIDR removal, and cows assigned to the 5-d protocol were inseminated 72 h after the first PGF(2alpha) injection. Cows assigned to both protocols were administered GnRH (100 microg i.m.) at AI. There was no effect of treatment (P = 0.85), technician (P = 0.20), or sire (P = 0.25) on pregnancy rates resulting from FTAI. Given these observations, the 5-d protocol provides an effective alternative to the 7-d protocol for use in facilitating FTAI; however, beef producers must carefully consider the increased labor and treatment costs associated with the 5-d protocol.

Periconceptional growth hormone treatment alters fetal growth and development in lambs1

Research in the area of fetal programming has focused on intrauterine growth restriction. Few studies have attempted to examine programming mechanisms that ultimately lead to lambs with a greater potential for postnatal growth. We previously demonstrated that treatment of ewes with GH at the time of breeding led to an increase in birth weight. Therefore, the objective of this study was to determine the effects of a single injection of sustained-release GH given during the periconceptional period on fetal growth and development and to determine if the GH axis would be altered in these offspring. Estrus was synchronized using 2 injections of PGF(2alpha); at the time of the second injection, ewes assigned to treatment were also given an injection of sustained-release GH. A maternal jugular vein sample was taken weekly to analyze IGF-I as a proxy for GH to estimate the duration of the treatment effect. In ewes treated with GH, IGF-I increased (P < 0.05) by wk 1 and remained elevated until wk 4 postinjection. Lambs were weighed, crown-rump length and abdominal girth were determined, and a plasma sample was collected. In a subset of male lambs, liver, heart, and brain weights were obtained, as well as left and right ventricular wall thicknesses. On postnatal d 100, a subset of ewe lambs were weighed and challenged with an intravenous injection of GHRH. Lambs from treated ewes had increased (P < 0.05) birth weight and abdominal girth compared with control lambs; however, there was no difference in crown-rump length. Expression of GH receptor and IGF-I were increased (P < 0.05) in lambs gestated by GH-treated ewes compared with control ewes. The left ventricular wall was thinner (P < 0.05) from lambs in the GH-treated group compared with control lambs. On postnatal d 100, those ewe lambs born to ewes treated with GH continued to be heavier (P < 0.05) and had no IGF-I response to GHRH challenge. In conclusion, treating ewes with a single injection of GH appeared to alter fetal growth and development. Lambs born to ewes treated with GH were larger at birth and had altered organ development, which may indicate that early maternal GH treatment may lead to permanent changes in the developing fetus. The ewe lambs maintained their growth performance to at least 100 d of postnatal life and appeared to have an altered GH axis, as demonstrated by the altered response to GHRH.

Prostaglandin F2.ALPHA. Differentially Affects mRNA Expression Relating to Angiogenesis, Vasoactivation and Prostaglandins in the Early and Mid Corpus Luteum in the Cow

Administration of prostaglandin (PG) F(2alpha) in cattle during the mid-luteal phase (Days 8-12 of the estrous cycle) drastically reduces the plasma progesterone concentrations and the volume of the corpus luteum (CL). However, PGF(2alpha) does not induce luteolysis during the early luteal phase (up to Day 5 of the estrous cycle). To characterize the possible distinct difference in acute response to a luteolytic dose of PGF(2alpha) administration, we determined various mRNA expressions in the early and mid CL relating to angiogenesis, vasoactivation and PG-related factors at 30 min after PGF(2alpha) injection in cyclic cows. The experiments were conducted on Day 4 (early CL) and Days 10-12 (mid CL). Cows were either injected with 500 microg PGF(2alpha) analogue or saline as the control (early CL control, n=5; early CL PGF(2alpha) treated, n=5; mid CL control, n=5; mid CL PGF(2alpha) treated, n=7). Thirty min after injection of PGF(2alpha) or saline, the cows were ovariectomized transvaginally, and the CL tissues were collected from regions designated as the periphery and center of the CL. Administration of PGF(2alpha) up-regulated the mRNA expressions of angiogenic-related factors such as vascular endothelial growth factors, vasohibin, fibroblast growth factor 2 and insulin-like growth factor-II in the early CL, whereas PGF(2alpha) down-regulated these mRNA expressions in the mid CL. In the vasoactive factors, PGF(2alpha) stimulated the mRNA expressions of endothelin-1, angiotensin converting enzyme, endothelial nitric oxide synthase (NOS) and inducible NOS in the periphery area of the mid CL, but not in the early CL. However, PGF(2alpha) drastically down-regulated PGF(2alpha) receptor mRNA expression in both regions of the early and mid CL. The results indicated a clear difference in the acute action of PGF(2alpha) depending not only on the luteal phase (immature vs. mature) but also the region (periphery vs. center) within the CL at 30 min after PGF(2alpha) injection in the cow.

Comparison of aspiration and hormonal therapy for the treatment of ovarian cysts in cows

Despite the variety of hormonal treating regimes used, a proportion of cows with cystic ovarian disease (COD) fail to be cured. The hypothesis of this study was that cyst aspiration would improve the curing rate and/or accelerate the resumption of ovarian activity in affected cows. In four groups of cows the following treatments were administered: Group A (n = 18) only cyst aspiration, Group AGP (n = 19) cyst aspiration and a combination of GnRH and PGF 2alpha , Group GP (n = 25) only GnRH and PGF 2alpha , and Group C (n = 15) untreated control. Cysts were aspirated without ultrasonographic guidance, using a new device. All cows from Group AGP responded to treatment, while 5.5% from Group A and 16% from Group GP remained anoestrous (P < 0.05). These refractory cases were re-treated with the AGP protocol and exhibited oestrus within 12.4 +/- 1.1 days. The interval from PGF 2alpha injection to oestrus was significantly reduced in cows treated with cyst aspiration. Until day 80 post partum (pp), 11 of the 15 untreated cows (73.3%) retained the initial cyst and remained anoestrous. It is concluded that persisting cases of COD can be treated by combining aspiration with a hormonal regime. The technique presented here has no complication for the cow, is efficient, easy, safe for the operator, and requires low-cost equipment.

Local effects of the sphingosine 1‐phosphate on prostaglandin F2alpha‐induced luteolysis in the pregnant rat

Since the regression of the corpus luteum (CL) occurs via a tightly controlled apoptotic process, studies were designed to determine if local administration of the antiapoptotic agent sphingosine 1-phosphate (S1P) effectively blocks the luteolytic action of prostaglandin F-2alpha (PGF-2alpha). On day 19 of pregnancy, 2 hr before systemic PGF-2alpha administration, rats were injected intrabursa with either S1P or vehicle (control). The activity of four caspases, which contribute to the initial (caspase-2, -8, and -9) and final (caspase-3) events in apoptosis was measured in pooled CL from four individual ovaries at 0 and 4 hr after PGF-2alpha injection. The expression of the phosphorylated form of AKT (pAKT) and tumor necrosis factor-alpha (TNF-alpha) was analyzed by ELISA. In addition, cell death was evaluated by electronic microscopy (EM) in CL 4 and 36 hr after PGF-2alpha injection. The activity of caspase-2, -3, and -8 was significantly greater by 4 hr after PGF-2alpha, but not caspase-9 activity. In contrast, expression of pAKT and TNF-alpha decreased significantly. Administration of S1P suppressed (P < 0.05) these effects, decreasing caspase activities and increasing pAKT and TNF-alpha expression. The administration of S1P also significantly decreased the percentage of luteal apoptotic cells induced by PGF-2alpha. PGF-2alpha treatment increased the prevalence of luteal cells with advanced signs of apoptosis (i.e., multiple nuclear fragments, chromatin condensation, or apoptotic bodies). S1P treatment suppressed these changes and increased the blood vessel density. These results suggest that S1P blocks the luteolytic effect of the PGF-2alpha by decreasing caspase-2, -3, and -8 activities and increasing AKT phosphorylation and TNF-alpha expression.

Markers of Ovarian Antral Follicular Development in Sheep: Differences Between Final (FW) and Penultimate (PW) Wave Pre-Ovulatory Follicles.

Treatment of non-prolific Western white-faced (WWF) ewes with prostaglandin F2alpha (PGF2alpha) and medroxy progesterone acetate (MAP) increased ovulation rate. The increase in ovulation rate was achieved largely by the addition of the ovulation of follicles from the penultimate (PW) follicular wave of the cycle to the expected ovulation of follicles from the final (FW) wave of the cycle. However, the number of lambs born after this treatment was not enhanced, suggesting that the PW follicles ovulating were not functionally viable. We were interested to compare the functional viability of PW and FW follicles. The objective of this study was to evaluate the expression of markers of vascularization and/or angiogenesis: vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), endothelial nitric oxide synthase (eNOS) and Factor VIII; a marker of gap junctional communication, connexin (Cx) 43; and a marker of cellular proliferation, proliferating cell nuclear antigen (PCNA) in preovulatory follicles obtained from the PW and FW in WWF ewes treated as described above. On day 8 of the estrous cycle, 15 ewes were given a single injection of PGF2alpha (15 mg i.m) and an intravaginal MAP sponge, which remained in place for 6 days. Thirty six to forty eight hours after sponge removal, ewes were ovariectomized by mid-ventral laparotomy. Ovaries that contain FW and/PW follicles from 7 ewes were fixed in Carnoy's solution for immunohistochemical detection of selected proteins. Computerized image analysis was used for quantification of protein expression. Granulosa and theca were isolated from FW and PW follicles from the remaining 8 ewes, suspended in PBS and stored at -80°C until RNA extraction. Messenger RNA levels were quantified using real-time PCR. Prostaglandin F2alpha and MAP treatment gave rise to preovulatory follicles emerging both from the FW and PW of the cycle; PW follicles had a longer (P<0.05) lifespan compared to FW follicles (7.1 ± 0.2 vs 3.0 ± 0.2). Protein expression for VEGF, eNOS, Cx43, PCNA and Factor VIII was greater in FW than in PW preovulatory follicles (P<0.05). For granulosa cells, Cx43 mRNA expression was greater (P<0.05) in FW than in PW preovulatory follicles. For theca cells, VEGF mRNA expression was greater (P<0.05) in FW than PW preovulatory follicles. In conclusion, expression of some markers of vascularization/ angiogenesis, gap junctional communication and cell proliferation appeared to be decreased in PW compared to FW preovulatory follicles. This suggests that although the treatment with PGF2alpha and MAP enhanced ovulation rate by extending the lifespan of PW follicles, these follicles were not as functionally viable as FW follicles. Therefore, this treatment provides a useful model to study aged antral follicles. (poster)

Strategies to improve fertility in postpartum multiparous Bos indicus cows submitted to a fixed-time insemination protocol with gonadotropin-releasing hormone and prostaglandin F2α1

In Exp. 1, we evaluated the effects of 2 lengths of progesterone exposure [CIDR (controlled intravaginal drug release); 7 vs. 14 d] before a modified CO-Synch protocol [50.0-microg injection of GnRH 6.5 d before a 25.0-mg injection of PGF(2alpha) followed by another injection of GnRH and fixed-time AI (TAI) 2 d after PGF(2alpha)], with or without temporary weaning (TW) before GnRH treatments, on fertility of suckled multiparous Bos indicus cows (n = 283) and on calf performance. Timed AI pregnancy rates for cows receiving 7 d CIDR + TW, 7 d CIDR, 14 d CIDR + TW, and 14 d CIDR were 53, 47, 46, and 41%, respectively (P > 0.10). Calves submitted to two 48-h TW 6 d apart had decreased mean BW at 240 d (187.9 +/- 2.7 vs. 195.5 +/- 2.7 kg; P < 0.05), but BW at 420 d was not affected by TW (240.1 +/- 5.1 kg). In Exp. 2, we evaluated the effect of no treatment and treatment with or without a CIDR insert between GnRH and PGF(2alpha) treatments of a modified CO-Synch protocol on pregnancy rate to TAI, and throughout a 90-d breeding season in suckled multiparous Bos indicus cows (n = 453). The inclusion of a CIDR between first GnRH and PGF(2alpha) treatments of a modified CO-Synch protocol did not improve pregnancy rate (29 and 33% for cows receiving CO-Synch + CIDR and CO-Synch protocol, respectively), and cycling cows had poorer TAI pregnancy rates than anestrous cows treated with either synchronization protocol (21.7 vs. 40.7%; P < 0.05). However, regardless of treatment with CIDR, cows submitted to TAI protocol had greater (P < 0.05) pregnancy rates at 30 (54.8 vs. 11.2%), 60 (72.1 vs. 38.8%), and 90 d (82.0 vs. 57.9%) of breeding season than untreated cows.

Expression and Localization of Gap Junctional Connexins 26 and 43 in Bovine Periovulatory Follicles and in Corpus Luteum During Different Functional Stages of Oestrous Cycle and Pregnancy

The aim of this study was to characterize the regulation of connexins (Cx26 and Cx43) in the bovine ovary (experiment 1-3). Experiment 1: ovaries containing preovulatory follicles or corpora lutea (CL) were collected at 0, 4, 10, 20, 25 (follicles) and 60 h (CL) relative to injection of GnRH. Experiment 2: CL were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16, >18 (after regression) of oestrous cycle and of early and late pregnancy (<4 and >4 months). Experiment 3: induced luteolysis, cows on days 8-12 were injected with PGF2alpha analogue (Cloprostenol), and CL were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after PGF2alpha injection. Real-time RT-PCR was applied to investigate mRNA expression and immunofluorescence was utilized for protein localization. Cx26 mRNA increased rapidly 4 h after GnRH injection (during LH surge) and decreased afterwards during the whole experimental period. Cx43 mRNA expression decreased continuously after GnRH application. Cx26 mRNA in CL increased significantly in the second part of oestrous cycle and after regression. In contrast, the highest mRNA expression for Cx43 in CL was detected during the early luteal phase. After induced luteolysis the mRNA expression of Cx26 increased significantly at 24 h. As shown by immunofluorescence, Cx26 was predominantly localized in the connective tissue and blood vessels of bovine CL, whereas Cx43 was present in the luteal cells and blood vessels. This resulted in a strong increase of Cx26 expression during the late luteal phase and after luteal regression. Subsequently, Cx43 expression was distinctly decreased after luteal regression. These data suggest that Cx26 and Cx43 are involved in the local cellular mechanisms participating in tissue remodelling during the critical time around periovulation as well as during CL formation (angiogenesis), function and regression in the bovine ovary.

Effect of number of cows in estrus and confinement area on estrous behavior of beef cows1

Mature Angus and Angus x Hereford cows were used to determine the effects of confinement area and number of cows in estrus on estrous behavior. During each of 2 replicates, 32 nonpregnant, nonlactating cows were maintained in a drylot (60 x 100 m) or in a pasture (12 ha). Estrous cycles were synchronized with 2 injections of PGF(2alpha) 10 to 14 d apart at the initiation of the experiment. Thereafter, PGF(2alpha) was administered between d 6 and 18 of the estrous cycle so that 1, 2 to 3, 4 to 6, or > or = 7 cows were in estrus at the same time. Concentrations of progesterone were quantified in plasma at treatment to ensure cows had a normal response to PGF(2alpha). Duration of estrus and the number of mounts received during estrus were recorded by HeatWatch. If any part of estrus for a cow occurred at the same time as estrus for another cow, the cow was considered to have concurrent estrus with the other cow. Cows in the drylot had a shorter (P < 0.02; 61.8 +/- 3.1 h) interval to estrus after PGF(2alpha) compared with cows on pasture (72.8 +/- 3.3 h). The interval to estrus was longer (P < 0.07) when cows were treated with PGF(2alpha) on d 10 to 13 (76.7 +/- 3.3 h) of the estrous cycle than when treated on d 6 to 9 (62.3 +/- 4.7 h) or d 14 to 18 (62.9 +/- 3.6 h). Increasing the number of cows concurrently in estrus increased the number of mounts each cow received per estrus (P < 0.001) and the duration of estrus (P < 0.01). When only 1 cow was in estrus, she received 11.0 +/- 6.2 mounts during 11.6 +/- 1.5 h. When > or = 7 cows were in estrus at the same time, each cow received 50.4 +/- 3.2 mounts during 17.3 +/- 0.8 h. Cows in drylot were in estrus longer (P < 0.04; 16.4 +/- 0.8 h) than cows on pasture (14.2 +/- 0.7 h). Duration of the longest interval between mounts received decreased (P < 0.002) as the number of cows in estrus at one time increased (5.3 +/- 0.7 h for 1 estrous cow; 2.6 +/- 0.3 h when > or = 7 cows were in estrus). We conclude that increasing the number of beef cows in estrus at the same time will increase the number of times a cow is mounted and the duration of estrus. The increase in estrous behavior associated with more cows in estrus could increase the number of estrous cows detected with infrequent visual observation.

Acute Changes in Circulating Concentrations of Progesterone and Nitric Oxide and Partial Pressure of Oxygen During Prostaglandin F2.ALPHA.-induced Luteolysis in Cattle

To examine whether oxygen (O(2)) and nitric oxide (NO) are temporally associated with the acute changes in luteal function during luteolysis, we determined the real-time changes in the circulating concentrations of progesterone (P4) and nitrite/nitrate (the stable metabolites of NO) and the partial pressure of oxygen (pO(2)) during prostaglandin F(2alpha) (PGF(2alpha))-induced luteolysis in cattle. Catheters for frequent blood sample collection were inserted into the ovarian vein (OV), jugular vein (JV) and aorta abdominalis (AA) in 12 cows on Day 9 of the oestrous cycle (oestrus=Day 0). On Day 10, the cows were randomly divided into two groups and treated with a luteolytic dose of a PGF(2alpha) analogue or saline solution (control). Blood samples were collected at -2, -1, 0, 0.25, 0.5, 0.75, 1 and 2 h and then at 2-h intervals until 12 h after treatment (0 h). Injection of a PGF(2alpha) induced a significant decrease in the concentrations of P4 in OV plasma within 2 h. The decrease in P4 concentrations was preceded by an increase in the NO concentrations in the blood collected from OV, JV and AA. Basal pO(2) was significantly higher in OV blood than in JV blood (P<0.05). PGF(2alpha) injection increased pO(2) in OV blood between 0.5 and 2 h. These results demonstrate that PGF(2alpha) induced an acute increase in pO(2) and NO in the ovarian circulation and suggest that O(2) and NO are involved in the early events of CL regression, including inhibition of P4 secretion and output, in cattle.

Ultrasonographic and Endocrine Evaluation of Three Regimes for Oestrus and Ovulation Synchronization for Sheep in the Subtropics

This study aimed to evaluate three regimes for oestrus and ovulation synchronization in Farafra ewes in the subtropics. During autumn, 43 ewes were assigned to (i) controlled internal drug releasing (CIDR)-eCG group, treated with CIDR for 12 days and eCG at insert withdrawal, n=13; (ii) PGF2alpha-PGF2alpha group, treated with two PGF2alpha injections at 11 days interval, n=14; and (iii) GnRH-PGF2alpha-GnRH group, treated with GnRH, followed 5 days later with PGF2alpha and 24 h later with a second GnRH, n=16. Oestrus-mating detection was carried out at 4 h intervals starting on day 0 [the day of CIDR withdrawal (CIDR-eCG group), the day of second PGF2alpha treatment (PGF2alpha-PGF2alpha group) and the day of PGF2alpha treatment (GnRH-PGF2alpha-GnRH group)]. Ovarian dynamics was monitored by ultrasound every 12 h beginning on day 0 and continued for 4 days. Blood samples were obtained daily for progesterone (P4) and oestradiol 17beta (E2) estimation starting on day 0 and continued for 4 days. The obtained results showed that, oestrus expression, ovulation and conception were greater (p<0.05) in CIDR-eCG and PGF2alpha-PGF2alpha groups than in GnRH-PGF2alpha-GnRH group. All ewes of PGF2alpha-PGF2alpha group presented, on day of second PGF2alpha injection with mature CL (P4>2.0 ng/ml), compared to 42.9% in GnRH-PGF2alpha-GnRH group (p=0.01). The peak of oestrus occurred 32-52, 48-60 and 28-96 h after the end of treatment in CIDR-eCG, PGF2alpha-PGF2alpha and GnRH-PGF2alpha-GnRH groups, respectively. Ovulation started 48 h after treatment in all groups and extended for 24, 36 and 48 h for CIDR-eCG, PGF2alpha-PGF2alpha and GnRH-PGF2alpha-GnRH groups, respectively. Results demonstrated that oestrus and ovulation synchronization could be efficiently achieved in Farafra ewes using either CIDR-eCG or PGF2alpha-PGF2alpha regimes; however, the GnRH-PGF2alpha-GnRH treatment induced a more spread oestrus and ovulation that may make the protocol inadequate for timed artificial insemination.

Influence of a controlled internal drug release after fixed-time artificial insemination on pregnancy rates and returns to estrus of nonpregnant cows1

We determined whether an ovulatory estrus could be resynchronized in previously synchronized, AI nonpregnant cows without compromising pregnancy from the previous synchronized ovulation or to those inseminated at the resynchronized estrus. Ovulation was synchronized in 937 suckled beef cows at 6 locations using a CO-Synch + progesterone insert (controlled internal drug release; CIDR) protocol [a 100-microg injection of GnRH at the time of progesterone insert, followed in 7 d by a 25-mg injection of PGF(2alpha) at insert removal; at 60 h after PGF(2alpha), cows received a fixed-time AI (TAI) plus a second injection of GnRH]. After initial TAI, the cows were assigned randomly to 1 of 4 treatments: 1) untreated (control; n = 237); 2) progesterone insert at 5 d after TAI and removed 14 d after TAI (CIDR5-14; n = 234); 3) progesterone insert placed at 14 d after TAI and removed 21 d after TAI (CIDR14-21; n = 232); or 4) progesterone insert at 5 d after TAI and removed 14 d after TAI and then a new CIDR inserted at 14 d and removed 21 d after TAI (CIDR5-21; n = 234). After TAI, cows were observed twice daily until 25 d after TAI for estrus and inseminated according to the AM-PM rule. Pregnancy was determined at 30 and 60 d after TAI to determine conception to the first and second AI. Pregnancy rates to TAI were similar for control (55%), CIDR5-14 (53%), CIDR14-21 (48%), and CIDR5-21 (53%). A greater (P < 0.05) proportion of nonpregnant cows was detected in estrus in the CIDR5-21 (76/110, 69%) and CIDR14-21 (77/120, 64%) treatments than in controls (44/106, 42%) and CIDR5-14 (39/109, 36%) cows. Although overall pregnancy rates after second AI service were similar, combined conception rates of treatments without a CIDR from d 14 to 21 [68.7% (57/83); control and CIDR5-14 treatments] were greater (P = 0.03) than those with a CIDR during that same interval [53.5% (82/153); CIDR5-21 and CIDR14-21 treatments]. We conclude that placement of a progesterone insert 5 d after a TAI did not compromise or enhance pregnancy rates to TAI; however, conception rates of nonpregnant cows inseminated after a detected estrus were compromised when resynchronized with a CIDR from d 5 or 14 until 21 d after TAI.

Effects of standing estrus and supplemental estradiol on changes in uterine pH during a fixed-time artificial insemination protocol1

Cows that exhibit estrus within 24 h of fixed-time AI have elevated concentrations of estradiol and greater pregnancy rates compared with cows not in estrus. Our objective was to determine whether estradiol, estrus, or both had an effect on uterine pH during a fixed-time AI protocol. Beef cows were treated with the CO-Synch protocol (100 mircog of GnRH on d -9; 25 mg of PGF(2alpha) on d -2; and 100 mircog of GnRH on d 0). One-half of the cows received an injection of estradiol cypionate (ECP; 1 mg) 12 h after PGF(2alpha). Cows detected in standing estrus within 24 h of the second GnRH injection were considered to be in standing estrus. Uterine pH was determined in all animals 12, 24, and 48 h after the PGF(2alpha) injection. For Exp. 1, pH was also determined 72 and 96 h after the PGF(2alpha) injection; in Exp. 2, pH was also determined at 54, 60, 66, 72, 78, 84, 90, and 96 h after the PGF(2alpha) injection or until ovulation. A treatment x time interaction (P < 0.01) influenced concentrations of estradiol. All cows had similar (P > 0.15) concentrations of estradiol at the time of ECP administration, but after ECP treatment all cows treated with ECP and control cows that exhibited estrus had greater (P < 0.01) concentrations of estradiol compared with nontreated cows that did not exhibit estrus. In all animals, estradiol diminished 48 h after the PGF(2alpha) (time of the second GnRH injection), but ECP-treated cows, regardless of estrus, had elevated (P < 0.02) concentrations of estradiol compared with control cows. There was a treatment x time interaction (P < 0.001) on uterine pH. All cows had similar uterine pH (P > 0.19) 24 h after the PGF(2alpha) injection. Control cows that did not exhibit estrus had a greater uterine pH compared with control cows that exhibited estrus (P < 0.01) and ECP cows that exhibited estrus (P = 0.05) 48 h after the PGF(2alpha) injection (7.0 +/- 0.1 vs. 6.7 +/- 0.1 and 6.8 +/- 0.1, respectively). Estradiol cypionate-treated cows not exhibiting estrus were intermediate (6.8 +/- 0.1; P > 0.05). All cows had similar uterine pH 72 h after the PGF(2alpha) injection through ovulation (P > 0.06). In summary, uterine pH was similar among all animals that exhibited estrus, regardless of treatment with ECP.

Evaluation of human chorionic gonadotropin as a replacement for gonadotropin-releasing hormone in ovulation-synchronization protocols before fixed timed artificial insemination in beef cattle1

Two experiments were conducted during 2 yr to evaluate differences in ovulation potential and fertility in response to GnRH or hCG. In Exp. 1, 46 beef cows were given 100 microg of GnRH or 500, 1,000, 2,000, or 3,000 IU of hCG. Ovulation incidence was not different between GnRH and any of the hCG doses, indicating that ovulatory capacity of at least 500 IU of hCG was equivalent to GnRH. In Exp. 2, beef cows (n = 676) at 6 locations were assigned randomly to a 2 x 3 factorial arrangement of treatments. Main effects were: 1) pre-timed AI (TAI) treatment (GnRH or hCG) and 2) post-TAI treatment (saline, GnRH, or hCG) to initiate resynchronization of ovulation in previously inseminated cattle. Blood samples were collected (d -21 and -10) to determine progesterone concentrations and assess cyclicity. Cattle were treated with a progesterone insert on d -10 and with 100 microg of GnRH or 1,000 IU of hCG. A PGF(2alpha) injection was given at insert removal on d -3. Cows were inseminated 62 h (d 0) after insert removal. On d 26 after first TAI, cows of unknown pregnancy status were treated with saline, GnRH, or hCG to initiate a CO-Synch protocol. Pregnancy was diagnosed 33 d after first TAI to determine pregnancies per AI (P/AI). Nonpregnant cows at 6 locations in yr 1 and 1 location in yr 2 were given PGF(2alpha) and inseminated 56 h later, concurrent with a GnRH injection. Five weeks later, pregnancy diagnosis was conducted to determine pregnancy loss after first TAI and pregnancy outcome of the second TAI. Injection of pre-TAI hCG reduced (P < 0.001) P/AI compared with GnRH, with a greater reduction in cycling cows. Post-TAI treatments had no negative effect on P/AI resulting from the first TAI. Serum progesterone was greater (P = 0.06) 7 d after pre-TAI hCG than after GnRH and greater (P < 0.05) after post-TAI hCG on d 26 compared with saline 7 d after treatment in association with greater frequency of multiple corpora lutea. Compared with saline, injections of post-TAI GnRH and hCG did not increase second insemination P/AI, and inconsistent results were detected among locations. Use of hCG in lieu of GnRH is contraindicated in a CO-Synch + progesterone insert protocol. Compared with a breeding season having only 1 TAI and longer exposure to cleanup bulls, total breeding season pregnancy rate was reduced by one-third, subsequent calving distribution was altered, and 50% more AI-sired calves were obtained by applying 2 TAI during the breeding season.

Local Effects of the Sphingosine 1-phosphate on PGF2alpha-induced Luteolysis in the Pregnant Rat.

Since apoptosis is associated with regression of the corpus luteum (CL) in many species, studies were designed to determine if local administration of the antiapoptotic agent sphingosine-1-phosphate (S1P) effectively blocks the luteolytic action of PGF-2alpha. On day 19 of pregnancy, 2 h before systemic PGF-2alpha administration, rats were injected with either S1P or vehicle (control) into the bursa of both ovaries. The activity of four caspases, which contribute to the initial (caspase-2, -8 and -9) and final (caspase-3) events in apoptosis, was measured in pooled CL from 4 individual ovaries at 0 h (pretreatment) and 4 h after PGF-2alpha injection in both control and S1P groups. The expression of the phosphorylated form of AKT was analyzed in CL using an ELISA. In addition, cell death was evaluated by electronic microscopy (EM) in CL 36 h after PGF-2alpha injection. The activity of caspase-2, -3 and -8 was significantly greater by 4h after PGF-2alpha administration (0h vs 4h: 8491±1406 vs 36702±3155; 45049±5679 vs 155053±7391; 1895±283 vs 9888±585 RFU respectively, p<0.05), but not caspase-9 activity. In contrast, expression of the phosphorylated form of AKT decreased (0h vs 4h: 22.5±0.5 vs 19.5±0.2 U/ml; p<0.05). Administration with S1P suppressed these effects, decreasing caspase activities and increasing the phosphorylated form of AKT (p<0.05). EM studies revealed ultrastructural indices of apoptotic and non-apoptotic cell death in the CL at day 19 of pregnancy. PGF-2alpha treatment increased luteal cells with advanced signs of apoptosis (i.e. containing multiple nuclear fragments, chromatin condensation or apoptotic bodies) by 36h post-injection. S1P treatment suppressed these changes and increased the blood vessel density. These results suggest that S1P can block the luteolytic effect of the PGF-2alpha by decreasing caspase-2, -3 and -8 activities and increasing the phosphorylation of AKT. Supported by: ANPCYT (BID 1201 OC-AR PICT 99:05-06384), NIH-FIRCA RO3-TW007041 and NIH-NCCR RR00163.