Influence of low plasma progesterone concentrations on the release of prostaglandin F2α during luteolysis and oestrus in heifers

A study was conducted to determine the effect of suprabasal plasma concentrations of progesterone on the release of prostaglandin F_2α (PGF_2α) at luteolysis and oestrus. Heifers received silicone implants containing 2.5 (n = 4), 5 (n = 4), 6 (n = 3), 7.5 (n = 3), 10 (n = 4), or 15 (n = 3) g of progesterone, or an empty implant (controls, n = 4) between Days 8 and 25 post ovulation. Blood was collected frequently between Days 14 and 28 and assayed for progesterone and 15-ketodihydroprostaglandin F_2α. Basal progesterone concentrations in control heifers did not differ from those in heifers with 2.5- or 5-g implants and remained around 0.4−0.5 nmol l⁻¹ until ovulation in all three groups. In the heifers treated with 6–15 g of progesterone, basal concentrations were maintained at higher (P < 0.05) levels compared with those in the controls, ranging from 0.8 to 1.6 nmol 1⁻¹. The effect of these elevated progesterone levels was to delay ovulation by prolonging the growth of the ovulatory follicle, which continued growing until the implant was removed. In all experimental groups, the first significant increase of the PGF_2α metabolite occurred between Days 15.3 and 16.3 (P > 0.05) and was associated with the onset of a decrease in progesterone concentrations, which had reached levels below 3 nmol 1⁻¹ by Days 17.4−19.1. PGF_2α metabolite peaks associated with luteolysis were frequent until Day 20. In the period from Day 20 until implant removal, sporadic peaks were observed, ranging in number from 1.0 ± 1.2 (mean ± SEM) in the control group to 3.0 ± 1.4 peaks in the heifers treated with 7.5 g of progesterone (P > 0.05). The number of PGF_2α metabolite peaks during that period was higher (P < 0.05) in heifers treated with 10 and 15 g than in controls. A positive correlation was found between the basal concentration of progesterone and the number of PGF_2α peaks after luteolysis (r = 0.54; P < 0.01). Plasma progesterone concentrations above approximately 1.4 nmol l⁻¹ were able to maintain the release of PGF_2α until the progesterone implants were removed and plasma levels decreased to basal values. These heifers had a preovulatory PGF_2α release pattern resembling that found in repeat breeder heifers.     

Corpus luteum function in the ewe: Effect of PGF2α and prostaglandin synthetase inhibitors

A series of experiments were conducted to evaluate the effects of mode and frequency of administration and estrous cycle stage on the response of the cycling ewe to PGF_2α. The effects of dexamethasone, arachadonic acid and prostaglandin synthetase inhibitors on estrous cycle length and plasma progesterone levels were also determined.Intramuscular administration of 5 or 10 mg of PGF_2α, on days 8 and 9 after estrus (5 ewes/group), significantly (p<.01) shortened the mean length of the estrous cycle and the interval from the end of treatment to estrus. Mean plasma progesterone levels, 24 hours after initial injection, were significantly (p<.01) lowered. When administered on day 8 only, these doses were considerably less effective in shortening estrous cycle length or lowering plasma progesterone levels. Intravaginal administration of PGF_2α, by polyurethane tampon, was also largely ineffective.Treatment of ewes with 10 mg of PGF_2α i.m., on days 3 and 4 of the estrous cycle, resulted in a return to estrus in 2 days in 25% of the treated animals. Plasma progesterone levels of PGF_2α-treated ewes were significantly lower than controls on the second, third and fourth days after the start of dosing. It would appear that PGF_2α exerts a retarding effect on developing CL functionality.The prostaglandin synthetase inhibitors, aspirin, flufenamic acid and 1-p-chlorobenzylidene-2-methyl-5-methoxy-3-indenylacetic acid, were administered orally or parenterally for 16 days beginning on day 8 of the estrous cycle. These compounds failed to prolong estrous cycle length. Parenteral administration of dexamethasone did not result in PGF_2α release in the cycling ewe, at least not in quantities sufficient to induce luteolysis. The prostaglandin precursor, arachadonic acid, also was not luteolytic when given parenterally to cycling ewes.     

Corpuscular oxidative stress in desert sheep naturally deficient in copper

Oxidative stress arises when there is an imbalance between radical-generating and radical-scavenging activity; it may therefore cause an increase in oxidation products and cell damage. This study aimed to determine antioxidant status, lipid peroxidation, and their relation to anemia of grazing sheep deficient in copper (Cu). For this purpose, 39 male lambs of native (Balady) breed, aged 6–7 months and reared in El-Dakhla oasis (in the western Egyptian desert), were divided according to plasma Cu (pCu) concentration into three groups, marginally deficient (MD, pCu = 4–8 μmol/l, n = 12), functionally deficient (FD, pCu < 3 μmol/l, n = 12) and control (pCu > 9 μmol/l, n = 15). Jugular blood was sampled for determination of red blood cell count (RBC), packed cell volume (PCV), hemoglobin concentration (Hb), plasma ceruloplasmin activity (pCp), antioxidant activities of erythrocytic superoxide dismutase (eSOD), catalase (eCAT), glutathione peroxidase (eGSH-Px), and levels of erythrocytic malondialdehyde (eMDA, as a biomarker of lipid peroxidation). The Cu-deficient lambs were characterized by microcytic hypochronic anemia accompanied by decreased pCp, eSOD, eCAT and eGSH-Px activities and increased eMDA level when compared to the controls. The indices of anemia, pCp and eSOD were lower and eMDA was higher in FD compared to MD lambs. The enhanced eMDA was strongly correlated (P < 0.01) with the inhibited activity of pCu (r = −0.79), pCp (r = −0.65) and eSOD (r = −0.71) and to a lesser extent (P < 0.05) with eGSH-Px (r = −0.38) and eCAT (r = −0.41). In addition, eMDA was negatively correlated (P < 0.01) with RBC (r = −0.75), PCV (r = −0.69) and Hb (r = −0.72). This study suggests that Cu-deficient lambs incur an erythrocytic oxidative damage secondary to impaired oxidant defenses, which may be one of the mechanisms underlying Cu deficiency-induced anemia in grazing sheep.     

Differential luteolytic function between the physiological breeding season,autumn transition and persistent winter cyclicity in the mare

There is a well-documented increase in luteolytic failure, resulting in spontaneously prolonged corpus luteum (SPCL) function, during estrous cycles of horses in autumn. The cause of this phenomenon may be due to seasonal alterations in PGF_2α and/or in prolactin (PRL) secretion around luteolysis. To investigate this, progesterone (P4), 13, 14-dihydro, 15-keto PGF_2α (PGFM) and PRL concentrations were compared between summer and autumn estrous cycles during natural luteolysis and luteolysis induced by benign uterine stimulation. A single estrous cycle from mares in June–July (n = 12) was compared to multiple estrous cycles from these 12 mares plus 8 additional mares in September through December. Reproductive behavior was monitored by bringing a stallion in close proximity to the mare and ovarian events by ultrasonography. Blood was collected via jugular cannula every 6 h from d 13 to 17 post-ovulation in untreated control mares (n = 8 summer, n = 9 autumn). In treated mares, blood collection occurred at 0, 15, 30, 45, 60, 90, 120, 180 and 240 min followed by 6 h intervals for a total of 5 d following intrauterine saline infusion on d 7 (n = 4 summer, n = 11 autumn). Mares failing to return to estrus for 30 d received intrauterine saline and the described intensive blood sampling protocol on d 30. Progesterone and PRL were determined on daily samples and PGFM on frequent plasma collections by RIA. Duration of ovarian luteal and follicular phases, P4 and PRL concentrations and PGFM secretion around luteolysis were compared between treatments and seasons by ANOVA. Mean P4 declined from June to December in all groups. Pulses of PGFM were detected on d 13–17 in controls and d 7–11 in saline-infused mares. Pulse patterns were not different between groups. The incidence of SPCL increased during autumn in the control group. PGFM pulses were absent on d 13–17 in mares with SPCL, but PGFM pulses could be induced in these mares by saline infusion at d 30. Autumn PGFM profiles were unchanged during spontaneous or saline-induced luteolysis compared with summer. Circulating PRL increased around natural or induced luteolysis. These results provide evidence that changes in luteal function during the autumn transition are not the result of alterations in the ability of the uterus to produce PGF_2α nor due to changed CL sensitivity to PGF_2α. We conclude that seasonal changes in luteolytic function are caused by an alteration in the signal for PGF_2α release.     

Root health evaluation of three alfalfa varieties in Kerquin sandy land

Without a robust and healthy root system, establishment, productivity, and persistence are compromised. Consequently, research on alfalfa root morphology and health is very important in development of technology for efficient improvement and production of alfalfa. The objectives of this study were to evaluate the root morphology and health of three alfalfa varieties, Algonquin, Golden Queen, and Yellow Flower and to determine relationships among root morphology traits and root health. Yields from these varieties ranged from 5.83 to 43.93 t/ha, total root length ranged from 215.17 to 708.89 mm, root surface area from 124.95 to 468.37 cm², volume from 3.24 to 57.72 cm³, and forks from 1.25 × 10³ to 10.54 × 10³, and tips from 0.65 × 10³ to 3.17 × 10³. Root infestation score was negatively correlated with yield (r = ?0.997, P < 0.01), and was positively correlated with all root morphology traits (r = 0.466–0.997, P < 0.01), and yield was negatively associated with root morphology traits (r = ?0.755 to ?0.998, p < 0.01) with the exception of root tips (r = 0.448, P < 0.01). Results from these analyses indicated that root infestation score was the lowest averaged over age of alfalfa stand in Algonquin. Yield in 2-year old stands was greater in Golden Queen compared to the other two cultivars.     

Time related changes in luteal prostaglandin synthesis and steroidogenic capacity during pregnancy, normal and antiprogestin induced luteolysis in the bitch

In nonpregnant and pregnant dogs the corpora lutea (CL) are the only source of progesterone (P4) which shows an almost identical secretion pattern until the rapid decrease of P4 prior to parturition. For the nonpregnant dog clear evidence has been obtained that physiological luteal regression is devoid of a functional role of the PGF2α-system and seems to depend on the provision of StAR. Yet in pregnant dogs the rapid prepartal luteal regression, coinciding with an increase of PGF2α, may be indicative for different regulatory mechanisms. To assess this situation and by applying semi-quantitative Real Time (Taq Man) RT-PCR, expression patterns were determined for the following factors in CL of pregnant and prepartal dogs and of mid-pregnant dogs treated with the antiprogestin Aglepristone: cyclooxygenase 2 (Cox2), prostaglandin E2 synthase (PGES), prostaglandin F2α synthase (PGFS), its receptors (EP2, EP4 an FP), the steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid-dehydrogenase (3βHSD) and the progesterone receptor (PR). Peripheral plasma P4 concentrations were determined by RIA. CL were collected via ovariohysterectomy from pregnant bitches (n = 3–5) on days 8–12 (Group 1, pre-implantation period), days 18–25 (Group 2, post-implantation period), days 35–40 (Group 3, mid-gestation period) and during the prepartal progesterone decline (Group 4). Additionally, CL were obtained from groups of 5 mid-pregnant dogs (days 40–45) 24 h, respectively 72 h after the second treatment with Aglepristone. Expression of Cox2 and PGES was highest during the pre-implantation period, that of PGFS and FP during the post-implantation period. EP4 and EP2 revealed a constant expression pattern throughout pregnancy with a prepartal upregulation of EP2. 3βHSD and StAR decreased significantly from the pre-implatation period to prepartal luteolysis, it was matched by the course of P4 concentrations. Expression of the PR was higher during mid-gestation and prepartal luteolysis than in the two preceding periods. After application of Aglepristone the overall mRNA-expression resembled the situation during prepartal luteolysis except for EP2, which remained unchanged.These data suggest that – as in the nonpregnant bitch – also in the pregnant bitch luteal production of prostaglandins is associated with luteal support rather than luteolysis. On the other hand induction of luteolysis by the PR blocker Aglepristone points to a role of luteal P4 as an autocrine factor in a positive loop feedback system controlling the availability of P4, StAR and 3βHSD.     

Prostaglandin E1 and F2α specific binding in bovine corpora lutea: Comparison with luteolytic effects

Preliminary characterization indicated the presence of separate prostaglandin (PG)E₁ and (PG)F_2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10⁻⁹M and 2.1 × 10⁻⁸M for PGE₁ and PGF_2α, respectively. Competition of several natural prostaglandins for the PGE₁ and PGF_2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit ³H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF_2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE₁ resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE₁ treatment compared to pretreatment values. In so far as data obtained on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently on PGF_2α induced luteolysis in the bovine, PGF_2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE₁ relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium

The present study was conducted to assess the capacitation status of fresh and frozen-thawed buffalo spermatozoa and its relationship with sperm cholesterol level, membrane fluidity and intracellular calcium. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris–egg yolk extender, equilibrated (4 °C for 4 h) and frozen at −196 °C in LN₂. The fresh and frozen-thawed spermatozoa were assessed for capacitation status using chlortetracycline (CTC) fluorescent assay, membrane fluidity using merocyanine 540/Yo-Pro-1 assay and intracellular calcium using Fluo-3 AM with flowcytometry. Results revealed a significant (P < 0.01) increase in capacitated sperm population in frozen-thawed semen compared to fresh semen (42.21% vs 14.32%). Similarly, a significantly (P < 0.01) higher proportion of frozen-thawed live spermatozoa showed high membrane fluidity (53.62% vs 25.67%) and high intracellular calcium (43.68% vs 11.72%) compared to fresh semen. The sperm cholesterol was significantly (P < 0.01) reduced after freezing–thawing as compared to fresh semen. The proportion of capacitated spermatozoa (CTC pattern B) was positively correlated with the proportion of sperm with high intracellular calcium (r = 0.81) and high membrane fluidity (r = 0.65), and negatively correlated with cholesterol level (r = −0.56) in frozen-thawed semen. The membrane fluidity was also strongly associated with the cholesterol level and intracellular calcium. The study concluded that changes in buffalo spermatozoa and established the relationship among capacitation status, sperm cholesterol level, membrane fluidity and intracellular calcium concentration in frozen-thawed spermatozoa.     

Relationships of changes in ultrasonographic image attributes to ovulatory and steroidogenic capacity of large antral follicles in sheep

Ovarian steroidogenesis and antral follicular development in ewes, following the treatment with medroxyprogesterone acetate (MAP) and equine chorionic gonadotrophin (eCG), are affected by the reproductive season. The objective of this study was to compare the ultrasonographic attributes of large antral follicles between cyclic (December) and seasonally anovular (June–July) ewes, after a 12-day treatment with MAP-soaked intravaginal sponges, with or without the administration of 500 IU of eCG at sponge removal, and to determine whether there is a correlation between the ultrasonographic attributes of the follicular wall and serum concentrations of oestradiol. Digital images of ovulatory follicles from cyclic ewes and eCG-treated anoestrous ewes (n = 34 follicles), and of anovulatory follicles attaining ≥5 mm in control anoestrous ewes (n = 8 follicles), were analysed using the spot and line techniques designed to determine the echotextural characteristics of the follicular antrum (central and peripheral), follicular wall and perifollicular ovarian stroma. The mean diameter of ovulatory follicles was greater (P < 0.001) in cyclic than anoestrous ewes, with or without the eCG treatment. The mean pixel heterogeneity (SD of numerical pixel values) of the follicular antrum (P < 0.05), as well as mean pixel intensity and heterogeneity of the peripheral antrum, follicular wall proper and perifollicular ovarian stroma (P < 0.05), were consistently greater in anoestrous than cyclic ewes at the time of sponge removal and 24 h after the treatment with MAP sponges or MAP/eCG. Mean oestradiol concentrations were greater (P < 0.05) in cyclic compared to anoestrous ewes in both MAP- and MAP/eCG-treated animals, from 1 to 2 days after sponge withdrawal. There was a moderate negative correlation (r² = 0.12, P < 0.05; Pearson's Product Moment and r² = 0.23, P < 0.05; ANCOVA) between mean pixel heterogeneity (standard deviation of mean pixel values) of the follicular wall proper (all follicles ≥5 mm in diameter) and serum concentrations of oestradiol after sponge withdrawal. Our results indicate that large antral follicles from cyclic and seasonally anovular ewes exhibit distinctive ultrasonographic characteristics. The differences in follicular echotexture appear to be related mainly to seasonal variations in ovarian follicular morphology and oestradiol production.     

Use of gonadotropin releasing hormone to improve reproductive responses of ewes introduced to rams during seasonal anestrus

Three experiments were conducted on anestrous ewes of Suffolk, Dorset, and Katahdin breeding to examine the potential value of GnRH to improve ovulation and pregnancy in response to introduction of rams. In Experiment 1, treatment with GnRH 2 d after treatment with progesterone (P₄; 25 mg i.m.) at introduction of rams was compared to treatment with P₄ alone at the time of introduction of rams. Treatment with GnRH did not increase percentages of ewes with a corpus luteum (CL) 14 d after introduction of rams, pregnant 32 d after treatment with PGF₂α 14 d after introduction of rams, or percent of treated ewes lambing to all services. In Experiment 2, treatments with GnRH on day 2, 7, or both after introduction of rams were compared. Treatments did not differ in mean estrous response, percentages of ewes with a detectable CL or number of CL present on day 11, or mean pregnancy and lambing rates. Therefore, neither one nor two injections of GnRH at these times appeared to be effective to induce anestrous ewes to breed. In Experiment 3, treatments compared included GnRH 4 d before introduction of rams, GnRH 4 d before and 1 d after introduction of rams, ram introduction alone, and treatment with P₄ (25 mg i.m.) at the time of introduction of rams. Percentages of ewes with concentrations of P₄ greater than 1 ng/mL (indicating formation of CL had occurred) 7 d after ram introduction tended to be greater (P < 0.07) in ewes treated with GnRH or P₄ than in control ewes treated with ram introduction alone. However, there was no difference in P₄ concentrations between groups by day 11 or 12 after introduction of rams. Estrous response rates and percentages of ewes pregnant 95 d after PGF₂α was administered (on day 12 after introduction of rams) tended to be greater (P = 0.08 and 0.06, respectively) in ewes treated with GnRH or P₄ than in ewes exposed to rams only. There was no difference in response variables between ewes treated with GnRH 4 d before introduction of rams and ewes treated with GnRH 4 d before and 1 d after introduction of rams. In conclusion, treatment with GnRH 4 d before ram introduction showed promise as an alternative to treatment with P₄ to improve the ovulatory response and reproductive performance of ewes introduced to rams during seasonal anestrus.     

Effect of estradiol-17β on peripheral plasma concentration of 15-keto,14-dihydro PGF2α and luteolysis in cyclic cattle

Friesian heifers (n = 10) were assigned randomly to receive an intravenous injection of estradiol-17^β (E₂; 3 mg) or saline: ethanol vehicle solution (6 ml; 1:1) on day 13 of the estrous cycle. Blood was collected collected from the jugular vein by venipuncture into heparinized vacutainer tubes at 30 minute intervals for 2 hours (h) preinjection, 10.5 h postinjection and then at 3 h intervals until estrus. Repeated hormone measurements of 15-keto-13,14-dihydro-PGF_2α (PGFM) and progesterone (P₄) were evaluated by split-plot analysis of variance. Mean concentration of PGFM for the 12.5 h acute sampling phase was 164.1 ± .14 pg/ml. A treatment by time interaction was detected (P < .01). After treatment with E₂, PGFM concentrations began to increase at approximately 3.5 h, reached a mean peak of 330.4 ± 44.5 pg/ml (n = 5) at 5.5 ± .3 h, and returned to basal concentration by 9.0 ± .6 h. Vehicle treatment did not alter concentrations of PGFM. Injections of E₂ on day 13 of the estrous cycle caused luteolysis (P₄ concentration < 1 ng/ml) to occur earlier following injection (96.9 ± 10.6 h < 153.6 ±17.7 h; P, 0.05) than did the vehicle control treatment. During the chronic sampling phase of 3 h intervals, 39 of 606 samples (6.4%) were classified as PGFM spikes (323.0 ± 50.0 pg/ml); 21 (53%) of the spikes occurred at a mean interval of 18.9 ± 3.86 h before the time of completed luteolysis. Exogenous E₂ induced an acute increase in PGFM that may be indicative of uterine PGF_2α production. Peaks of PGFM in plasma were temporally associated with luteolysis on a within cow basis.     

Effects of dose and route of administration of cloprostenol on luteolysis,estrus and ovulation in beef heifers

Four experiments were conducted (with crossbred beef heifers) to determine the effects of dose and route of administration of cloprostenol on luteolysis, estrus and ovulation. In Experiment 1, 19 heifers with a CL > or = 17 mm in diameter were randomly allocated to receive cloprostenol as follows: 100 microg s.c., 250 microg s.c., or 500 microg i.m. Heifers given 100 microg s.c. had a longer (P<0.03) interval (120.0 h+/-10.7 h; mean+/-S.E.M.) from treatment to ovulation than those given either 250 microg s.c. or 500 microg i.m. (92.0 h+/-7.4 h and 84.0 h+/-8.2 h, respectively). In Experiment 2, 28 heifers were given porcine LH (pLH), followed in 7 days by cloprostenol (same doses and routes as in Experiment 1), and a second dose of pLH 48 h after cloprostenol. Luteolysis occurred in all heifers, and no difference was detected among treatment groups in the interval from cloprostenol treatment to ovulation (mean, 101 h; P<0.9). In Experiment 3, 38 heifers at random stages of the estrous cycle (but with plasma progesterone concentrations > or =1.0 ng/ml) received 500 or 125 microg cloprostenol by either i.m. or s.c. injection (2/2 factorial design). There was no difference (P<0.4) among groups in the proportions of heifers that were detected in estrus or that ovulated. However, the interval from cloprostenol treatment to estrus was shorter (P<0.02) in the group that received 500 microg i.m. (58.5h) than in the other three groups (500 microg s.c., 75.0 h; 125 microg i.m., 78.0 h; and 125 microg s.c., 82.3h). In Experiment 4, 36 heifers were treated (as in Experiment 3) on Day 7 after ovulation. The proportions of heifers detected in estrus and ovulating after 125 microg s.c. (33 and 44%, respectively) or 125 microg i.m. (55 and 55%) were lower (P<0.05) than in those that received 500 microg s.c. (100 and 100%), but not different from those receiving 500 microg i.m. (78 and 89%, respectively). Overall, ovulation was detected in 9/18 heifers given 125 microg and 17/18 heifers given 500 microg of cloprostenol, on Day 7 (P<0.01) and was detected in 17/20 heifers given 125 microg and 18/18 heifers given 500 microg of cloprostenol, at random stages of the estrous cycle (P>0.05). Although there was no significant difference in luteolytic efficacy between i.m. and s.c. injections of the recommended dose (500 microg) of cloprostenol, variability in responsiveness to a reduced dose depended upon CL sensitivity, therefore, reduced doses cannot be recommended for routine use.     

Prostaglandin E1 and F2α specific binding in bovine corpora lutea: Comparison with luteolytic effects

Preliminary characterization indicated the presence of separate prostaglandin (PG)E₁ and (PG)F_2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10⁻⁹M and 2.1 × 10⁻⁸M for PGE₁ and PGF_2α, respectively. Competition of several natural prostaglandins for the PGE₁ and PGF_2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit ³H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF_2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE₁ resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE₁ treatment compared to pretreatment values. In so far as data obtained in vitro on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently in vitro on PGF_2α induced luteolysis in the bovine, PGF_2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE₁ relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

The chlorophenoxy herbicide dicamba and its commercial formulation banvel induce genotoxicity and cytotoxicity in Chinese hamster ovary (CHO) cells

The sister chromatid exchange (SCE) frequency, the cell-cycle progression analysis, and the single cell gel electrophoresis technique (SCGE, comet assay) were employed as genetic end-points to investigate the geno- and citotoxicity exerted by dicamba and one of its commercial formulation banvel^® (dicamba 57.71%) on Chinese hamster ovary (CHO) cells. Log-phase cells were treated with 1.0–500.0 μg/ml of the herbicides and harvested 24 h later for SCE and cell-cycle progression analyses. All concentrations assessed of both test compounds induced higher SCE frequencies over control values. SCEs increased in a non-dose-dependent manner neither for the pure compound (r = 0.48; P > 0.05) nor for the commercial formulation (r = 0.58, P > 0.05). For the 200.0 μg/ml and 500.0 μg/ml dicamba doses and the 500.0 μg/ml banvel^® dose, a significant delay in the cell-cycle progression was found. A regression test showed that the proliferation rate index decreased as a function of either the concentration of dicamba (r = −0.98, P < 0.05) or banvel^® (r = −0.88, P < 0.01) titrated into cultures in the 1.0–500.0 μg/ml dose-range. SCGE performed on CHO cells after a 90 min pulse-treatment of dicamba and banvel^® within a 50.0–500.0 μg/ml dose-range revealed a clear increase in dicamba-induced DNA damage as an enhancement of the proportion of slightly damaged and damaged cells for all concentrations used (P < 0.01); concomitantly, a decrease of undamaged cells was found over control values (P < 0.01). In banvel^®-treated cells, a similar overall result was registered. Dicamba induced a significant increase both in comet length and width over control values (P < 0.01) regardless of its concentration whereas banvel^® induced the same effect only within 100.0–500.0 μg/ml dose range (P < 0.01). As detected by three highly sensitive bioassays, the present results clearly showed the capability of dicamba and banvel^® to induce DNA and cellular damage on CHO cells.     

The efficacy of recombinant equine follicle stimulating hormone (reFSH) to promote follicular growth in mares using a follicular suppression model

The efficacy of a recently engineered single chain recombinant equine follicle stimulating hormone (reFSH) was investigated in estrous cycling mares whose gonadotropins and follicular activity had been suppressed by concurrent treatment with progesterone and estradiol (P&E). Time of estrus was synchronized in 15 estrous cycling mares during the breeding season with prostaglandins F_2α (PGF_2α). The day after ovulation, mares were treated once daily with P&E for 14 days. Mares received a second injection of PGF_2α on day 6 of the synchronized estrous cycle to induce luteolysis. On day 8 post-ovulation mares were randomly assigned to three groups: small dose reFSH-treatment group (0.5 mg reFSH IV, twice daily); large dose reFSH-treatment group (0.85 mg reFSH IV twice daily); control group (saline IV, twice daily). reFSH treatment occurred concurrently with the last week of P&E treatment. After a follicle or cohort of follicles reached 35 mm in diameter, mares were injected with 0.75 mg of recombinant equine luteinizing hormone (reLH) to induce ovulation. Post-treatment ovulation was assessed. Daily blood samples were collected for analysis of FSH, LH, estradiol, progesterone, and inhibin by radioimmunoassay (RIA). On the first day of reFSH/saline treatment, blood samples were collected periodically from 1 h prior to treatment to 6 h post-injection via an indwelling jugular catheter to determine acute changes in FSH concentrations. Monitoring of follicular activity, estrus, and ovulation was performed daily by utilizing a stallion and transrectal ultrasonography.A difference (p ≤ 0.05) between the largest diameter follicle in the reFSH-treatment groups compared to controls occurred on day 14 post-ovulation, the day treatments ended, and the difference continued until day 21 post-ovulation. reFSH-treatment groups had larger (p ≤ 0.05) numbers of 20–29 mm follicles (days 13–18), 30–34 mm follicles (days 15–20) and ≥35 mm follicles (days 16–21) than controls. Mares treated with reFSH, at either dose, took less time (average: 2.95 ± 0.42 days) to develop 2–3 times more pre-ovulatory follicles than control mares (7.8 ± 0.51 days) (p ≤ 0.05). The number of ovulations between treated mares and controls were similar due to a greater incidence of ovulation failure in reFSH-treated mares. During reFSH treatment, concentrations of plasma FSH, inhibin and estradiol were greater (p ≤ 0.05) compared to control concentrations. Plasma LH concentrations in reFSH-treated mares were suppressed and did not exhibit the ovulatory surge of controls (p ≤ 0.05). Plasma progesterone concentrations were not different across groups.These findings demonstrate the specific effects of reFSH to increase number of total follicles including pre-ovulatory follicles in mares with endogenous pituitary gonadotropins and follicular growth suppressed by a regimen of P&E.     

Major human plasma lipid classes determined by quantitative high-performance liquid chromatography, their variation and associations with phospholipid fatty acids

An HPLC method with evaporative light-scattering detection (ELSD) was optimized and validated for the simultaneous quantitation of cholesteryl esters (CEs), triacylglycerols (TGs), free cholesterol (FC) and phosphatidylcholine (PC) in human plasma. The separation of CEs from TGs, the most variable plasma lipid class, was improved by speeding up the gradient steps and by increasing the re-equilibration time between runs. The calibrations were made at levels of 0.14–14 μg lipid/injection. The intra- and inter-day precision values of the method ranged between 1.9 and 4.5 and 2.3–7.2% (RSD, n=6), respectively, including determinations at two concentration levels. In comparison to other lipid classes, quantitation of PC proved to be equally repeatable despite its lowest detector response. The relative recoveries varied from 97.0 to 110.3%, showing good accuracy of the method. The methodological variation of the lipid classes covered 0.6–3.1% of their total variation in the study population (n=48). The CE/FC ratio showed an even closer relationship with phospholipid linoleic acid (18:2n−6; r=0.65, P<0.001) than with serum cholesterol levels, while eicosapentaenoic acid (20:5n−3) was significantly associated with PC (r=0.41, P<0.01). The CE/FC ratio increased (P<0.01) during soyabean oil substitution and the level of PC increased (P<0.01) during cold-pressed rapeseed oil substitution.     

Effect of exogenous administration of buffalo follicular fluid on follicular development, estrus response and luteal function in anoestrous goats (Capra hircus)

The effect of buffalo follicular fluid (buFF) on follicular development, estrus response and luteal function was investigated in anoestrous does. Treatment with buFF (18 ml/doe) had no significant effect on the number of antral follicles of all class categories during the period of administration. However, after cessation of buFF treatment, the number of total antral follicles increased significantly with time (P < 0.003) as well as due to the treatment × time interaction (P < 0.02), without any influence on follicle size. Injection of buFF also caused a marked increase (P < 0.049) with time in the number of medium-sized follicles at cessation. Approximately 60 and 20% of buFF-treated anoestrous does showed behavioural and silent estrus, respectively, compared to none in the control. The mean interval between cessation of buFF treatment to onset of oestrus and oestrus duration was 67.0 ± 18.5 and 17.0 ± 3.6 h, respectively. Corpus lutea size varied between 4.6 and 5.8 mm with an average diameter of 5.2 ± 0.3 mm. Only 33.3% of does showed serum progesterone levels above 1 ng/ml, while the remainder (66.7%) had below 0.5 ng/ml. Our results indicate that exogenous administration of buFF causes enhanced follicular activity following cessation of treatment, which results in behavioural oestrus and corpus luteum (CL) development in anoestrous does. CL development and its function is, however, inadequate in buFF-treated anoestrous does.     

Effect of time interval between prostaglandin F2α 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.     

Rates of luteolysis and pregnancy in dairy cows after treatment with cloprostenol or dinoprost

Our objective was to determine whether rates of luteolysis or pregnancy differed in lactating dairy cows of known progesterone status and either known or unknown luteal status after either cloprostenol or dinoprost was injected as part of a timed-insemination program. In Experiment 1, 2358 lactating dairy cows in six herds were given two injections of PGF_2α 14 d apart (Presynch), with the second injection given 12 to 14 d before the onset of a timed AI protocol (Ovsynch). Cows (n = 1094) were inseminated when detected in estrus after the Presynch PGF_2α injections. Cows not inseminated (n = 1264) were enrolled in the Ovsynch protocol and assigned randomly to be treated with either cloprostenol or dinoprost as part of the timed-AI protocol. In cows having pretreatment concentrations of progesterone ≥ 1 ng/mL and potentially having a functional corpus luteum (CL) responsive to cloprostenol (n = 558) or dinoprost (n = 519), dinoprost increased (P < 0.05) luteal regression from 86.6 to 91.3%. Despite a significant increase in luteolysis, pregnancies per AI did not differ between luteolytic agents (dinoprost = 37.8% and cloprostenol = 36.7%). Fertility was improved in cows of both treatments having reduced concentrations of progesterone at 72 h and in cows showing signs of estrus. In Experiment 2, an ovulation-resynchronization program was initiated with GnRH or saline in 427 previously inseminated lactating dairy cows of unknown pregnancy status in one herd. Seven days later, pregnancy was diagnosed and nonpregnant cows were blocked by number of CL and assigned randomly to be treated with cloprostenol or dinoprost. Compared with cloprostenol, dinoprost increased (P < 0.05) luteal regression from 69.1 to 78.5%, regardless of the number of CL present or the total luteal volume per cow. Pregnancies per AI did not differ between dinoprost (32.8%) and cloprostenol (31.3%). Although dinoprost was more effective than cloprostenol at inducing luteolysis in lactating dairy cows exposed to an Ovsynch or ovulation-resynchronization protocol, resulting fertility did not differ between products.