Pregnancy Rates in Angus Cross Beef Cows Bred at Observed Oestrus With or Without Second GnRH Administration in Fixed‐Time Progesterone‐Supplemented Ovsynch and CO‐Synch Protocols

Crossbred cows (n = 1073) from five locations had oestrous cycles synchronized with 100 μg of GnRH IM and insertion of controlled internal drug release device (CIDR) on Day 0 followed by 25 mg of PGF_2α IM and CIDR removal on Day 7. Kamar^® patches were placed on all cows at CIDR removal. Cows were observed three times daily for oestrus after PGF_2α administration. In the Ovsynch‐CIDR group, cows detected in oestrus (n = 193) within 48 h after PGF_2α were inseminated using the AM–PM rule. Among these cows, 80 received and 113 did not receive a second GnRH at 48 h after PGF_2α. Cows (n = 345) not detected in oestrus received a second GnRH at 48 h after PGF_2α on Day 9, and fixed‐time AI 16 h after the GnRH on Day 10. In the CO‐Synch‐CIDR group, cows detected in oestrus (n = 224) within 48 h after PGF_2α were inseminated using the AM–PM rule. Among these cows, 79 received and 145 did not receive a second GnRH at 64 h after PGF_2α. Cows (n = 311) not detected in oestrus received a second GnRH on Day 10 at the time of AI, 64 h after PGF_2α. The AI pregnancy rates were not different between the Ovsynch‐CIDR and CO‐Synch‐CIDR groups (p = 0.48). There were no differences in the AI pregnancy rates for cows inseminated at a fixed time (p = 0.26) or at detected oestrus (p = 0.79) between the treatment groups. Among cows inseminated in oestrus, there were no differences in the AI pregnancy rates between cows that received or did not receive the second GnRH (p = 0.47). In conclusion, acceptable AI pregnancy rates can be achieved with or without inclusion of oestrus detection in the Ovsynch‐CIDR and CO‐Synch‐CIDR protocols. Among cows detected in oestrus, cows that received a second GnRH yielded similar pregnancy rates when compared with cows that did not receive the second GnRH.     

15-Ketodihydro-PGF2α, Progesterone and Uterine Involution in Primiparous Cows with Induced Retained Placenta and Post-partal Endometritis Treated with Oxytetracycline and Flunixin

Retention of the foetal membranes (RFM) and post‐partal endometritis are common problems in dairy cows. Among other things, the disease is characterized by a bacterial endometritis with aerobic as well as anaerobic bacteria. From an endocrine perspective, cows with RFM have high levels of 15‐ketodihydro‐PGF_2α (PG‐metabolite) immediately after parturition but these levels fall rapidly within 2 weeks post‐partum (early PG‐metabolite elevation). After this decline, the PG‐metabolite levels increase again and the levels (at this time of a lower magnitude) remain elevated during the period of uterine infection (late PG‐metabolite elevation). The aim of this study was to investigate the PG‐metabolite profiles in cows with retained placenta and post‐partal endometritis treated with the prostaglandin synthesis inhibitor flunixin (F), either alone or in combination with oxytetracycline (T). The study was accomplished over 2 years with 12 primiparous cows in each experiment. As a model for RFM, preterm parturition was induced in late‐pregnant heifers by injecting PGF_2α (25 mg i.m) twice with a 24 h interval. In each experiment, the cows were divided into four groups and treated with either T (10 mg/kg b.w. i.m. once daily), F (2.2 mg/kg b.w. p.o. twice per day), a combination of T and F (dosage, as above), or conservatively (0). The treatment periods lasted from day 11 to day 14 post‐partum (pp) in experiment 1 (after placental shedding, groups T1, F1, TF1 and 0) and from day 3 to day 6 pp in experiment 2 (before placental shedding, groups T2, F2, TF2 and 0). Jugular vein blood samples were collected for analyses of PG‐metabolite and flunixin. Uterine biopsies were collected twice weekly for investigation of endometrial microbiology. Rectal palpation and ultrasonographic examinations were performed three times per week for investigations of uterine and cervical involution and ovarian activity. No attempts were made to remove the placentas manually. The experiment lasted until day 56 pp. The induction of parturition was successful in all heifers and 22 of 24 animals had RFM. All RFM cows had bacterial endometritis, based on bacteriological examinations. Flunixin treatment (F1, TF1, F2 and TF2) suppressed PG‐metabolite levels significantly (p=0.006) during the period of treatment in both experiments. However, the early flunixin treatment only suppressed PG synthesis partially. Late oxytetracycline treatment (T1) did not influence the PG‐metabolite levels but oxytetracycline treatment (T2 and TF2) before placental shedding significantly altered the kinetics of the early PG‐metabolite elevation compared with other treatments. Late PG‐metabolite elevation was significantly correlated to duration of uterine infection and cervical involution. In conclusion, flunixin treatment of cows with retained placenta either before or after placental shedding suppresses prostaglandin synthesis. However, early treatment, when the release of prostaglandins is high, might need more intensive treatment in order to prevent the PG synthesis effectively. Oxytetracycline treatment during the period immediately after parturition before placental shedding might influence the PG‐metabolite profile and suggests a bacteriological contribution to the high levels of PG‐metabolite seen during the first 2 weeks pp in cows with retained placenta. In this study, a correlation between prostaglandin release, the final cervical involution and the end of infection was found. This suggests a link between uterine endocrinology, bacteriology and involution in cows with retained placenta.     

Regulation of uterine function by cytokines in cows: Possible actions of tumor necrosis factor-α, interleukin-1α and interferon-τ

When animals do not become pregnant, regression of the corpus luteum (CL) is essential for normal cyclicity because it allows the development of a new ovulatory follicle. Luteal regression is caused by a pulsatile release of prostaglandin (PG) F_2α from the uterus in the late luteal phase in most mammals including cattle. Although it has been proposed in ruminants that pulsatile PGF_2α secretion is generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and uterine PGF_2α, the bovine endometrium may possess other mechanisms for initiation of luteolytic PGF_2α secretion. There is increasing evidence that several cytokines mainly produced by immune cells modulate CL and uterine function in many species. Tumor necrosis factor‐α (TNF‐α) stimulates PGF_2α output from bovine endometrium not only at the follicular phase but also at the late luteal phase. Administration of TNF‐α at a high concentration prolongs luteal lifespan, whereas administration of a low concentration of TNF‐α accelerates luteal regression in cows. The data obtained from the authors’ previous in vitro and in vivo studies strongly suggest that TNF‐α is a crucial factor in regulating luteolysis in cows. The authors’ recent study has shown that interleukin‐1α mediates PG secretion from bovine endometrium as a local regulator. Furthermore, interferon‐τ (IFN‐τ) suppresses the action of TNF‐α on PGF_2α synthesis by the bovine endometrium in vitro, suggesting that IFN‐τ plays a luteoprotective role by inhibiting TNF‐α‐induced PGF_2α production in early pregnancy. The purpose of the present review is to summarize current understanding of the endocrine mechanisms that regulate uterine function by cytokines during the estrous cycle and early pregnancy in cows.     

Temporal Relationships Between Oxytocin and 13,14-Dihydro-15-Keto-Prostaglandin F2α Pulses in Ovariectomized Ewes

The primary objective was to evaluate the role of non-ovarian oxytocin in the initiation of pulses of PGF_2α, as measured by peripheral concentrations of 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM). A 2 × 2 factorial arrangement of estradiol and progesterone treatments was administered to groups of five ewes after ovariectomy on Day 12. Progesterone (10 mg) was administered at 0700 and 1900 hr on Day 12, and then either progesterone or its vehicle was administered on Days 13 and 14. Silastic implants, either empty or containing estradiol, was administered at ovariectomy. Oxytocin and PGFM were measured in jugular blood samples withdrawn from an indwelling catheter at 5-min intervals for 8 hr on Day 15. Statistically significant pulses of oxytocin, presumably of posterior pituitary origin, were detected in all ewes. Approximately one-half of the oxytocin pulses preceded a pulse in PGFM concentrations by 10 min or less. These pulses tended (P = 0.09) to have a longer duration than those not linked to pulses of PGFM. The number of PGFM pulses that followed or did not follow an oxytocin pulse by 10 min or less was similar (P > 0.2). The amplitude and duration of oxytocin-linked PGFM pulses were greater (P = 0.05) than non-linked pulses. Although several explanations for the lower than anticipated temporal relationship between oxytocin and PGFM pulses are possible, the finding that oxytocin-related PGFM pulses are distinguishable from other pulses is consistent with the concept that oxytocin initiates robust pulses in PGF_2α secretion.     

Control of in vitro prostaglandin F2α and E2 synthesis by caruncular and allantochorionic tissues from cows that calved normally and those with retained fetal membranes

High concentrations of PGF_2α and PGE₂ are produced by the uterus during the early postpartum period in cows and may play an important role in both placental separation and uterine involution. In the present study, we have examined the hormonal and intracellular control mechanisms involved in PGF_2α and PGE₂ secretion by caruncular and allantochorionic tissue in vitro. Tissue explants, obtained about 6 hr postpartum from cows that delivered normally (NFM, n = 10) or cows with retained fetal membranes (RFM, n = 4), were incubated for 6 hr and PGF_2α and PGE₂ concentrations in the medium were determined by radioimmunoassay. Addition of oxytocin (100 μU/ml), platelet activating factor (PAF, 100 ng/ml) and epidermal growth factor (EGF, 100 ng/ml) had no effect on secretion of PGF_2α from the caruncle, but oxytocin and PAF did stimulate PGE₂. There was no difference between groups of cows. All three substances stimulated PGF_2α from the allantochorion of NFM, but not RFM, cows and stimulated PGE₂ secretion from the allantochorion of both groups of cows. Incubation of the tissues with cholera toxin (100 ng/ml), dibutyryl cyclic adenosine 3′,5′-monophosphate (dibutyryl cAMP, 1mM), calcium ionophore A23187 (5 μM) or phorbol ester 12-myristate-13 acetate (PMA, 100 nM) showed that PGF_2α secretion is essentially via the calcium-protein kinase C effector pathway. However, calcium-protein kinase C and cAMP second messenger systems appear to be involved in the secretion of PGE₂. Prostaglandin secretion was sensitive to cycloheximide in both caruncular and allantochorionic tissues, suggesting that protein synthesis may be involved. In conclusion, these data show that in vitro PGF_2α secretion can be modulated by the agonists used only in allantochorion and is essentially via the calcium-protein kinase C effector pathway. PGE₂ secretion can be modifified in both caruncular and allantochorion tissues and involves both inositol triphosphate-diacylglycerol and cAMP second messenger systems.     

Cellular mechanisms by which oxytocin mediates uterine prostaglandin F2α synthesis in bovine endometrium: role of calcium

The objective of these experiments was to determine the role of Ca²⁺ during oxytocin-stimulated prostaglandin (PG) F_2α release from bovine endometrial tissue in vitro. Uteri were collected from dairy cows on the day after spontaneous luteal regression. Caruncular endometrial explants were dissected and incubated in vitro to determine phospholipase C activity or PGF_2α release. A23187 (a calcium ionophore) and maitotoxin (an activator of voltage-gated L-type calcium channels) stimulated release of PGF_2α in a concentration-dependent manner (P < 0.05). Thapsigargin (induces accumulation of Ca²⁺ in the cytoplasm by inhibiting endoplasmic reticulum Ca²⁺/ATPase pumps) stimulated release of PGF_2α in a concentration-dependent manner as well (P < 0.13). Oxytocin (10⁻⁶ M), AlF₄⁻ (a nonspecific activator of G-proteins; 10⁻⁵ M), A23187 (10⁻⁵ M), and melittin (a stimulator of phospholipase A₂; 10⁻⁴ M) stimulated PGF_2α release when explants were incubated in Ca²⁺-free medium (P < 0.10); however, oxytocin, A23187, or melittin were unable to stimulate PGF_2α release when explants were incubated in Ca²⁺-free medium containing the calcium chelator EGTA (P < 0.10). This treatment did not prevent oxytocin or AlF₄⁻ from stimulating phospholipase C activity (P < 0.08). CoCl₂ (a nonspecific Ca²⁺ channel blocker) and methoxyverapamil (a specific voltage-gated L-type Ca²⁺ channel blocker) prevented oxytocin from stimulating PGF_2α release (P < 0.05). Our results suggest that both extracellular and intracellular Ca²⁺ may be required for oxytocin to stimulate PGF_2α secretion in bovine endometrial tissue.     

Direct Effects of Luteal Regression on Anterior Pituitary Response To GnRH

The preovulatory period of the ewe is marked by a dramatic decrease in concentrations of progesterone in serum during the late luteal phase, followed by elevated luteinizing hormone (LH) secretion, final follicular maturation and ovulation. This experiment was designed to ascertain the extent to which removal of endogenous progesterone negative feedback at the anterior pituitary gland, independent of effects at the hypothalamus, promotes increased secretion of LH in the hours immediately after induction of luteolysis. Estrus was synchronized in ovary-intact ewes with two injections of prostaglandin F₂α (PGF₂α) analog given 10 d apart (Day 0 = second day after the second PGF₂α injection). Ewes were subjected to hypothalamic-pituitary disconnection (HPD; n = 6) on Day 3 and were pulsed with gonadotropin-releasing hormone (GnRH). Ewes were used during the estrous cycle or received approximately 400 IU pregnant mare serum gonadotropin (PMSG) on Day 2 to stimulate ovulation; there was no difference (P < 0.10) in ovulation rate or progesterone production between these two groups. Luteal regression was induced by injection of PGF₂α analog on approximately Day 10 of the estrous cycle. Blood samples were collected around exogenous GnRH pulses before and at 2- or 4-hr intervals after PGF₂α administration and concentrations of LH and progesterone determined. At 4, 12 and 24 hr after PGF₂α administration, mean serum progesterone levels in all ewes had decreased by 54.7%, 66.2% and 89.4%, respectively (P < 0.05) from pre-injection levels. The decrease in progesterone was associated with an increase (P < 0.01) in LH pulse amplitude with means at 4-hr post-PGF₂α ranging from 190% to 288% of pre-PGF₂α values. Mean serum LH levels were also increased (P < 0.01) within 4 hr of PGF₂α administration and remained elevated at all but the 24-hr time point. The timing of this increase (within 4 hr) indicates that it is independent of changes in serum estradiol concentrations, which do not increase for at least 16 hr after induction of luteolysis. Thus, removal of endogenous progesterone negative feedback at the anterior pituitary gland in the hours immediately after induction of luteolysis seems to play a role in facilitating LH release independently of hypothalamic action.     

Timing of Ovulation and Fertility of Heifers After Synchronization of Oestrus with GnRH and Prostaglandin F2α

Synchronization of oestrus and/or ovulation can reduce workload in heifer reproductive management. The objective of this study was to compare two protocols to synchronize oestrus and/or ovulation using GnRH and prostaglandin F_2α (PGF_2α) in dairy heifers concerning their effect on follicular dynamics and reproductive performance. Four trials were carried out. In trial 1, 282 heifers were treated with GnRH and PGF_2α 7 days apart (GP protocol). One group was inseminated on detection of oestrus (IDO 1), and the other group received two timed artificial inseminations (AI) 48 and 72 h after PGF_2α administration (TAI 1). In trial 2, 98 heifers were synchronized with the same GP protocol. Heifers in IDO 2 were treated as in IDO 1, heifers in TAI 2 received two TAI 48 and 78 h after PGF_2α administration. In trial 3, heifers in IDO 3 (n = 71) were again treated as in IDO 1. Heifers in TAI 3 (n = 166) received a second dose of GnRH 48 h after PGF_2α (GPG protocol) and TAI together with this treatment and 24 h later. Trial 4 compared the timing of ovulation after the GP and the GPG protocol, using a subgroup of the heifers from trials 1 to 3. The ovaries of the heifers were scanned via ultrasound at 48, 56, 72, 80, 96 and 104 h after PGF_2α administration. Timing of ovulation and size of the ovulatory follicles were compared between the two groups. In trials 1 to 3, conception rates to first service were between 49 and 66%. They did not differ significantly between IDO and TAI groups within or between trials. Pregnancy rates per synchronization were numerically higher in the TAI groups, but the difference was not significant. Conception rates to breeding on spontaneous oestrus in heifers returning to oestrus were higher than that after synchronized oestrus. In trial 4, more heifers ovulated before the end of the observation period in GPG than in GP (96.5% vs 74.7%; p < 0.001). Overall, ovulatory follicles were smaller in GPG (13.1 ± 1.9 mm vs 14.3 ± 1.9 mm; p < 0.001).     

Effects of arachidonic acid and oxytocin on equine endometrial PGF2α during normal cycles and pseudopregnancy

The effect of excess arachidonic acid or oxytocin on equine endometrial prostaglandin F_2α(PGF_2α) synthesis was measured in vitro under physiologic and pathophysiologic conditions. Endometrial tissues obtained by uterine biopsy at 5, 10, 12, 14, 16 and 20 days post-ovulation from cycling mares, after 0, 2, 4, 6, 8, 10, 12, 14 or 16 days of progesterone (P₄) in ovariectomized mares and at 30 days postovulation in mares undergoing spontaneously prolonged corpus luteum (SPCL) activity were incubated in vitro with and without added arachidonic acid or oxytocin. Endometrial PGF_2α content and synthetic capacities were determined by radioimmunoassay. PGF_2α production increased significantly at Days 12–16. Arachidonic acid did not alter this effect. Oxytocin stimulated additional PGF_2α production on Days 5, 16, and 20. SPCL tissues had minimal PGF_2α production which was increased significantly by arachidonic acid but not oxytocin. PGF_2α synthesis in ovariectomized P₄ treated mares was minimal and did not vary with length of progesterone exposure or addition of arachidonic acid. These results suggest that a) oxytocin may play a role in luteolysis in the equine, b) although arachidonic acid appears not to be limiting to PGF₂α production under normal physiological conditions, its absence may play a role in pathophysiological conditions, c) factors in addition to progesterone and arachidonic acid are required to initiate PGF_2α synthesis in the mare.     

Efficacy of Acupuncture Sites for Delivery of Agents to Control Estrus and Ovulation in Mares

This study was designed to determine if prostaglandin F₂α (PGF₂α) when administered on d 6 post-ovulation in a low dose in the lumbosacral space (LSS) would induce luteolysis while minimizing side effects usually associated with intramuscular administration of this analogue in mares. A second objective was to determine if human chorionic gonadotropin (hCG) injected into the LSS would reduce time to ovulation in the mare. Ten normally cycling mares served as their own controls in a crossover design, receiving intramuscular injections of PGF₂α(10 mg), intravenous injections of hCG (3000 IU) and injections of PGF and hCG at the acupuncture site (2 mg and 3000 IU, respectively), as well as sham injections of saline. Beginning 12 h after injection, mean progesterone concentrations were less (P<0.05) in PGF₂α-treated mares than in mares receiving saline. Moreover, progesterone concentrations were similar (P<0.001) between both groups of mares receiving PGF₂α. In addition, there was no difference (P>0.1) between mares receiving the acupuncture injection of PGF₂α and the intramuscular injection in days to ovulation. However, duration and severity of side effects associated with PGF₂α administration were dramatically decreased (P<0.01) when PGF₂α was delivered to the acupuncture site compared to intramuscular delivery. The time to ovulation was similar (P>0.1) for mares receiving shams, or hCG. These data indicate that delivery of 2 mg of PGF₂α in the LSS induces luteolysis and reduces the sweating and muscle cramping associated with PGF₂α administration. There was no advantage to the delivery of hCG in the LSS.     

Untersuchungen zur Brunstsynchronisation und Samenübertragung bei Schafen verschiedener Rassen

Inhalt Bei insgesamt 500 Schafen (260 Merino, 120 Dagliç, 120 Ramliç) in drei Schafzuchtbetrieben in der Türkei wurde durch Verabreichung von Gestagen-Vaginal-schwämmchen + PMSG oder PGF_2α (ein- bzw. zweimal) die Brunst induziert. 4 bis 6 Stunden nach der Brunstbeobachtung mittels Suchbock erfolgte die zervikale Besamung mit tiefgefrorenem (TG) oder frisch gewonnenem Sperma von Böcken der jeweils selben Rassen. Mit Hilfe von Gestagen+PMSG wurde bei allen Rassen die höchste Östrusinduktionsrate (96, 7 bis 98,3%) erzielt. Einmalige PGF_2α Verabreichung rief bei 60%, zweimalige PGF_2α Injektion im Abstand von 9 Tagen bei 88,3% der Schafe Brunst hervor. PGF_2α hatte den besten Synchronisationseffekt (80,6 bis 88,2%) 48 Stunden nach der Applikation. Die Non Return Raten 35 bis 40 Tage nach Besamung betrugen unabhängig vom Synchronisationsverfahren und von der Rasse 21,4 bis 41,9% bzw. 55,6 bis 84,2% nach Verwendung von TG- bzw. Frischsamen. Die besten Non Return Ergebnisse wurden bei den drei Rassen nach Brunstinduktion mit Gestagen+PMSG beobachtet. Rasseabhängige Unterschiede bezüglich der Brunstinduktion-und -synchronisation ließen sich statistisch nicht absichern. Contents: Investigations upon oestrus synchronization and artificial insemination in ewes of different breeds In a total of 500 ewes (260 Merino, 120 Dagliç, 120 Ramliç) of three sheep breeding farms in Turkey oestrus induction was performed by application of progestagen imprepated vaginal sponges+PMSG or of PGF_2α (given once or twice). 4 to 6 hours after oestrus detection by a teaser ram the ewes were inseminated cervically with frozen-thawed or fresh semen of rams of the same breeds. In all breeds the highest rate of oestrus induction (96.7 to 98.3%) was obtained by means of progestagen+PMSG. A single injection of PGF_2α provoced heat in 60%, while two injections with an interval of 9 days induced oestrus in 88.3% of the treated ewes. PGF_2α had the best synchronizing effect (80.6 to 88.2%) at 48 hours after application. Regardless of the breed and the synchronization method, the non return rates 35 to 40 days after insemination were 21.4 to 41.9% and 55.6 to 84.2% for frozen-thawed and fresh semen resp. In all breeds the best non return results were observed after oestrus induction with progestagen+PMSG. With regard to induction and synchronization of oestrus breed dependent differences could not be statistically confirmed.     

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) PGF₂α‐PGF₂α group, treated with two PGF₂α injections at 11 days interval, n = 14; and (iii) GnRH‐PGF₂α‐GnRH group, treated with GnRH, followed 5 days later with PGF₂α 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 PGF₂α treatment (PGF₂α‐PGF₂α group) and the day of PGF₂α treatment (GnRH‐PGF₂α‐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 17β (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 PGF₂α‐PGF₂α groups than in GnRH‐PGF₂α‐GnRH group. All ewes of PGF₂α‐PGF₂α group presented, on day of second PGF₂α injection with mature CL (P₄ > 2.0 ng/ml), compared to 42.9% in GnRH‐PGF₂α‐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, PGF₂α‐PGF₂α and GnRH‐PGF₂α‐GnRH groups, respectively. Ovulation started 48 h after treatment in all groups and extended for 24, 36 and 48 h for CIDR‐eCG, PGF₂α‐PGF₂α and GnRH‐PGF₂α‐GnRH groups, respectively. Results demonstrated that oestrus and ovulation synchronization could be efficiently achieved in Farafra ewes using either CIDR‐eCG or PGF₂α‐PGF₂α regimes; however, the GnRH‐PGF₂α‐GnRH treatment induced a more spread oestrus and ovulation that may make the protocol inadequate for timed artificial insemination.     

Effects of Ram Introduction After the Second Prostaglandin F2α Injection on Day 11 on the LH Surge Characteristics in Fat-Tailed Ewes

The aim of this study was to evaluate the effects of ram introduction after the second prostaglandin F_2α (PG F_2α) injection on day 11 on the secretion characteristics of pre‐ovulatory LH surge of fat‐tailed ewes. Multiparous Morkaraman ewes (n=12) were divided into three groups by balancing the groups for liveweight (BW) and body condition score (BCS). On the day of second PGF_2α injection (0 h), performance tested rams (n=2) were either introduced to the ewes at 0 h (ram 0 group, n=4) or at 18 h (ram 18 group, n=4) or were not introduced (control group, n=4). Blood samples were collected at 6, 18, 42, 48, 56, 62, 66, 70, 74, 78 and 90 h for the determination of pre‐ovulatory LH surge. BCS and BW during the experimental period were 2.2 ± 0.2 units and 50.9 ± 2.3 kg, 2.4 ± 0.4 units and 49.2 ± 6.2 kg, 2.1 ± 0.3 units and 45.9 ± 4.4 kg, respectively for the ram 0, ram 18 and control groups (p > 0.05). No significant difference was observed in LH surge characteristics for the experimental groups. Peak LH concentrations were also not different between groups (p > 0.05) and they were 12.2 ± 8.3, 29.1 ± 9.9 and 15.8 ± 9.5 μg/l for the ram 0, ram 18 and control groups, respectively. There was, however, a significant correlation between peak LH concentrations and BCS (p < 0.05, R²=0.373). In conclusion, it appears that, compared with ram introduction, variability in body condition of the ewe has much pronounced effect on the amount of LH secreted after the usage of two PGF_2α injections (11 days apart) as a tool for oestrus synchronization.     

The role of phosphoinositide-derived second messengers in oxytocin-stimulated prostaglandin F2α release from endometrium of pigs

The mechanism for prostaglandin (PG) F_2α release from pig endometrium after oxytocin (OT) treatment is unknown. OT may rapidly stimulate inositol (1,4,5)-trisphosphate (IP₃) and diacylglycerol (DAG) formation, consistent with the concept of rapid activation of a second-messenger system. In support of this hypothesis, endometrial IP₃ levels were increased (P < 0.05) within 0.5 min after treatment with 0.1 μM OT. In contrast, increased DAG formation was not detected after treatment with OT. However, similar to the stimulation of endometrial PGF_2α secretion observed after OT treatment (P < 0.001), PGF_2α release was increased (P < 0.01) after treatment with phorbol-12myristate-l3-acetate (PMA), which mimics DAG activation of protein kinase C. Further, stimulation of endometrial PGF_2α secretion did not result from cell death induced by PMA or OT because lactate dehydrogenase, a cytosolic marker of cellular integrity, did not leak into the medium after PMA or OT treatment. In contrast, 0.5% saponin (positive control for cell death and concomitant release of lactate dehydrogenase) increased PGF_2α secretion (P < 0.05) and lactate dehydrogenase release (P < 0.001). These results indicate that OT induces endometrial IP₃ production in a rapid manner indicative of a second-messenger system. The finding that increased DAG was not also detected after OT treatment may reflect rapid metabolism or compartmentalized production of DAG involved in the second-messenger stimulation of phospholipase C. The high background of DAG used in the biosynthesis of cellular lipids would obscure the rather small spatially localized changes in DAG levels resulting from the activation of phospholipase C. The finding that DAG was present at approximately 10 to 20-fold higher levels than IP₃ in resting cells was consistent with this conclusion.     

Luteolytic Effect of Prostaglandin F2α on Bovine Corpus Luteum Depends on Cell Composition and Contact

Prostaglandin F_2α (PGF_2α) is a main luteolytic factor in vivo; however, its direct luteolytic influence on steroidogenic cells of bovine corpus luteum (CL) is controversial and not fully understood. The aim of the study was to clarify PGF_2α action on bovine CL in different in vivo and in vitro conditions and to examine whether the contact among all main types of CL cells is necessary for luteolytic PGF_2α action. In experiment 1, the bovine CL (day 15 of the oestrous cycle) was perfused using in vivo microdialysis system with dinoprost (an analogue of PGF_2α) for 0.5 h. Dinoprost caused a short‐time increase in progesterone (P4), whose concentration decreased thereafter (at 6‐, 10‐, 12‐ and 24‐h after treatment). In experiment 2, the direct effect of PGF_2α on P4 accumulation in CL steroidogenic cells cultured in monolayer (day 15 of the cycle) was determined. PGF_2α after 24 h of incubation increased P4 accumulation in steroidogenic CL cells. In experiment 3 steroidogenic, endothelial CL and immune cells (day 15 of the cycle) were incubated with PGF_2α in cocultures for 24 h in glass tubes and the levels of P4, stable metabolites of nitric oxide (NO) and leukotriene (LT) C₄ were determined. Although PGF_2α treatment increased P4 secretion in homogeneous steroidogenic CL cell culture, the decrease in P4 secretion in cocultures of all types of CL cells was observed. The secretion of NO and LTC₄ increased after the treatment of PGF_2α both in pure cultures of CL cells and in cocultures. The interactions between endothelial and immune cells with steroidogenic CL cells are needed for luteolytic PGF_2α action within the bovine CL. Our results indicate that the cell coculture model, including the main types of CL cells, is the most approximate to study PGF_2α role in vitro.     

Conversion of Cortisone to Cortisol and Prostaglandin F2α Production by the Reproductive Tract of Cows at the Late Luteal Stage In Vivo

Previous in vitro studies demonstrated that bovine endometrium has the capacity to convert inactive cortisone to biologically active cortisol (Cr) and that Cr inhibits cytokine‐stimulated prostaglandin F_2α (PGF) production. This study was carried out to test the hypothesis that bovine reproductive tract has the capacity to convert cortisone to Cr in vivo and to evaluate the effects of intravaginal application of exogenous cortisone on uterine PGF secretion during the late luteal stage. The temporal relationships between PGF and Cr levels in uterine plasma were also determined. Catheters were inserted into jugular vein (JV), uterine vein (UV), vena cava caudalis (VCC) and aorta abdominalis (AA) of six cows on Day 15 of the oestrous cycle (ovulation = Day 0) for frequent blood collection. On Day 16, the cows were divided randomly into two groups and infused intravaginally with vaseline gel (10 ml; control; n = 3) or cortisone dissolved in vaseline gel (100 mg; n = 3). Blood samples were collected at ?2, ?1, ?0.5, 0, 0.5, 1, 1.5, 2, 3, 4, 5 and 6 h after treatments (0 h). Intravaginal application of cortisone increased plasma concentrations of Cr between 0.5 and 1.5 h in UV, at 0.5 h in VCC, at 1 h in JV and at 1.5 h in AA. The plasma concentrations of PGF in UV and of PGF metabolite in JV were greater at 0.5 and 1 h in the cortisone‐treated animals than in control animals. The levels of PGF in UV blood plasma decreased after Cr reached its highest levels. The overall findings suggest that the female reproductive tract has the capacity to convert cortisone to Cr in vivo. Based on the temporal changes of PGF and Cr levels in the uterine plasma, a biphasic response in PGF secretion was found to be associated to the Cr increase induced by the cortisone treatment at the late luteal stage in non‐pregnant cows.     

The effect of two different routes of administration of oxytocin on peripheral plasma prostaglandin F(2α) metabolite levels in early post-partum dairy cows

Various parenteral treatment forms of oxytocin, as often used under praxis circumstances, may act differently on contractility of the uterus during the first days of the puerperium. Various patterns of such induced uterotonic responses may lead to alterations in the emptying characteristics of the uterine lumen, thus influencing, as a late consequence, the process of involution. Therefore, this study was designed to test whether two different parenteral administration forms of oxytocin induce changes in peripheral plasma concentrations of 15-ketodihydro-prostaglandin F(2α) (PGF(2α) metabolite) in early post-partum cows. Between 13 and 15 h after uncomplicated calving, healthy dairy cows without retained foetal membranes were treated with 50 IU oxytocin, either intramuscularly (OT-IM group; n = 15) or intravenously (OT-IV group; n = 16). Saline solution was administered intramuscularly as controls (CON group; n = 15). Jugular blood samples were taken at 10-min intervals from 1 h before to 2 h after treatment. Plasma PGF(2α) metabolite levels were measured by radioimmunoassay. No significant differences in peripheral plasma PGF(2α) metabolite concentrations occurred in the OT-IM and CON groups, but mean values significantly increased in the OT-IV group, peaking at 20 min after treatment and reaching pre-treatment baseline values again at 120 min. Although the source of prostaglandins was not investigated in this study, our results suggest that exogenous oxytocin may enhance secretion of prostaglandins by the uterus during the first day after normal calving. These prostaglandins might contribute, by an endocrine or paracrine route, to the stimulation of myometrial contractility when exogenous oxytocin is given during this early post-partum stage.     

Combined GnRH and PGF2α Application in Cows with Endometritis Puerperalis Treated with Antibiotics

The investigations were carried out on a total of 70 cows with puerperal endometritis. In addition to intrauterine antibiotic treatment, 30 experimental animals were administered 20 μg GnRH analogue, buserelin, between days 10 and 12 post‐partum followed by 500 μg PGF_2α analogue, cloprostenol, 10 days later. Forty control cows were treated only with intrauterine antibiotics. Blood samples for progesterone determination were collected from the tail vein twice weekly until day 70 post‐partum. The first rise in progesterone level above 3.18 nmol/l occurred significantly earlier in the experimental than in control cows (21.6 ± 9.2 versus 27.8 ± 12.3 days; p ≤ 0.05). The duration of the first cycle post‐partum was 15.0 ± 4.3 days in experimental and 19.7 ± 7.3 days in control animals (p ≤ 0.05). However, no significant differences were observed in the occurrence of first oestrus post‐partum. The involution of the uterus was improved after hormone treatment. At day 42 post‐partum, completion of uterine involution was found in 93.3% of hormone‐treated cows and in 82.5% of those treated with antibiotic only (p ≤ 0.05). Clinical recovery was 96.6% in the experimental and 82.5% in the control group (p ≤ 0.05). First service pregnancy rate was significantly better in hormone‐treated than control cows (51.7 versus 36.4%; p ≤ 0.05). Total pregnancy rate and insemination index values were not significantly improved following GnRH and PGF_2α treatment. The average service period was 89.8 ± 21.2 days in cows after hormone treatment, and 112.6 ± 24.5 days in control cows. The difference was statistically significant (p ≤ 0.05). These results indicate, that the sequential GnRH and PGF_2α application in cows with puerperal endometritis positively affected ovarian function and uterine involution, resulting in improved fertility performance.     

The uterine motility in cattle during late pregnancy, labor and puerperium. II. Drug modification

The uterine effects of oxytocin, the prostaglandins dinoprost and cloprostenol as well as clenbuterol, ergometrin, xylazine and Utrorale were investigated in 8 cows during late pregnancy, parturition and early puerperium (until 4th day p. p.). Uterine motility was measured by means of pressure microsensors and electrodes which were surgically implanted 3 to 4 weeks before parturition. Hysterograms were characterized by means of pressure amplitude, frequency and duration of uterine contractions and also by electromyography. Oxytocin (2-5 IE) given intravenously always provoked strong uterine contractions until the 4th day p.p. From the prostaglandins examined during early puerperium only dinoprost (15 mg i.v.) produced uterotonic effects, while the synthetic analogue cloprostenol (0.25 mg i.v.) had a weak stimulatory activity only on day 1 p.p. Both prostaglandins were ineffective when injected intramuscularly. Clenbuterol (0.3 mg i.v.) a beta 2-mimetic compound effectively induced long lasting tocolysis during parturition, which could be abolished by oxytocin. Xylazine (10 mg i.v.) was able to significantly increase uterine motility during late gestation. Following intravenous administration of ergometrin (1 and 10 mg), bunitrolol (1-16 mg) and Utrorale (0.1-4 ml) including its compounds oleum sabinae, oleum terebinthinae, balsamum copaivale and Styrax no uterokinetic activity was recorded at any time.