Hyperglycemia associated with increased hepatic glycogen phosphorylase and phosphoenolpyruvate carboxykinase in rats following subchronic exposure to malathion

We examined the effects of subchronic exposure to malathion, an organophosphorous (OP) insecticide, on plasma glucose and hepatic enzymes of glycogenolysis and gluconeogenesis in rats in vivo. Malathion was administered orally at doses of 100, 200 and 400 ppm for 4 weeks. At the end of the specified treatment (18 h fasting after the last dose of malathion), the liver was removed. The activities of glycogen phosphorylase (GP) and phosphoenolpyruvate carboxykinase (PEPCK) were analyzed in the homogenate. Four weeks administration of malathion at doses of 100 ppm, 200 ppm, and 400 ppm increased plasma glucose concentrations by 25% (P < 0.01), 17% (P < 0.01), and 14% (P < 0.01) of control, respectively. Malathion also increased hepatic PEPCK activity by 25% (100 ppm, P < 0.01), 16% (200 ppm, P < 0.01), and 21% (400 ppm, P < 0.01) of control, respectively. In addition, malathion increased hepatic GP by 22% (100 ppm, P < 0.01), 41% (200 ppm, P < 0.01), and 32% (400 ppm, P < 0.01) of controls. We conclude that exposure of rats to malathion as a widely used OP in subchronic exposure, which resembles human exposure, may induce diabetes associated with stimulation of hepatic gluconeogenesis and glycogenolysis in favor of glucose release into the blood. The possible mechanisms including increased energy production to detoxification, depressed paraoxonase activity, and increased production of cyclic nucleotides are discussed.     

Some macromolecular abnormalities developed by the interaction of malathion and permethrin and subsequent refeeding in Tribolium castaneum larvae

The effects of sublethal treatments of malathion and malathion + permethrin combinations on activities of acetylcholine esterase (ChE), carbohydrate-metabolizing enzymes (amylase; lactate dehydrogenase, LDH), protein-metabolizing enzymes (alanine and aspartate aminotransferase, ALAT, ASAT), as well as acid and basic phosphatases (AcP and AkP, respectively), and Kreb's cycle enzymes (isocitrate dehydrogenase, ICDH) were studied on sixth instar larvae of the red flour beetle, Tribolium castaneum. In addition, the levels of lipid, cholesterol, glucose, glycogen, proteins, free amino acids (FAA), urea, and nucleic acids (DNA and RNA) were determined. Malathion (400 ppm) increased the activity of LDH (53%), as well as the concentrations of FAA (31%) and urea (39%). Malathion treatments of 400 ppm decreased the glucose (20%) and glycogen (24%) content but did not affect other enzymes and biochemical components. Permethrin (200 ppm) and malathion (20 ppm) mixtures increased the activities of ChE (708%) and LDH (55%), and raised the concentrations of FAA (26%), urea (24%), glucose (23%), lipid (14%), cholesterol (21%), DNA (24%), and RNA (8%). The decrease in AcP activity and in glycogen concentration observed with malathion was sustained by permethrin in the mixture. Permethrin + malathion mixtures also depressed the levels of AkP (30%), ICDH (24%), and glycogen (36%). The intensity of effects commensurated with the dose and duration of insecticide exposure. Refeeding showed tendencies towards normalization of various biochemical parameters.     

Biochemical evaluation of hepatic damage in subchronic exposure to malathion in rats: effect on superoxide dismutase and catalase activities using native PAGE

The aim of this study was the evaluation of the hepatic damages following a subchronic exposure to malathion, an organophosphorus (OP) insecticide. Malathion was administered intragastrically in 1 ml corn oil containing 100 mg/kg Body Weight daily for 32 days. Malondialdehyde (MDA) concentration superoxide dismutase (SOD) and catalase (CAT) activities were analysed using a non-denaturing electrophoresis. The serum activities of Pseudocholinesterase (PchE), aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) were determined. Malathion exposure leads to a significant decrease in AchE activity, an increase in hepatic MDA, and in serum ASAT and ALAT activities. A positive correlation between serum transaminases levels and hepatic MDA was demonstrated. These results indicate that malathion exposure induced lipid peroxidation LPO, a process of degradation of membrane lipids, involving the deterioration of the cellular integrity. We have recorded a slight increase in antioxidant enzymes activities. This leads us to suggest an insufficient elimination of free radicals, causing cytotoxic effects. To cite this article: R. Rezg et al., C. R. Biologies 331 (2008).     

Malathion-induced oxidative stress in a parasitoid wasp: effect on adult emergence, longevity, fecundity, and oxidative and antioxidative response of Pimpla turionellae (Hymenoptera: Ichneumonidae)

Effects of an organophosphorus insecticide, malathion, on survivorship and lipid peroxidation of the greater wax moth, Galleria mellonella (L.), pupae were investigated by rearing the newly hatched larvae on an artificial diet containing 0.01, 0.1, 1, 10, and 100 ppm of the insecticide. As bioindicators of long-term physiological stress responses, the adult emergence rate, longevity, and fecundity associated with lipid peroxidation level and antioxidant enzyme activity in the endoparasitoid Pimpla turionellae (L.) (Hymenoptera: Ichneumonidae) were determined by rearing the parasitoid on a factitious host, G. mellonella pupae treated with malathion. At 100 ppm, malathion significantly decreased pupation rate of G. mellonella larvae and the rate of adult emergence of the parasitoid from these pupae. This concentration resulted in a significant increase in the lipid peroxidation product malondialdehyde (MDA) in both the host and the parasitoid. Malathion at 1 and 10 ppm significantly increased pupation rate and lipid peroxidation level of G. mellonella pupae. The adult emergence rate of P. turionellae was significantly decreased from 63.7 to 20% by these concentrations, whereas MDA content was increased by two- and three-fold, respectively, compared with the control (45.3 +/- 3.2 nmol/ g protein). The longevity of adults was significantly extended from 52.5 +/- 5.7 to 75.7 +/- 6.3 d when the parasitoids emerged from host pupae exposed with 0.1 ppm malathion. At low concentrations (0.01 and 0.1 ppm), malathion significantly increased the number of eggs laid per female per day. However, the lowest concentration (0.01 ppm) had no significant effect on hatchability, whereas 0.1 ppm of the insecticide resulted in significant decrease in egg hatch compared with the control. A significant increase in total superoxide dismutase (SOD) activity for low concentrations of malathion (0.01-1 ppm) was found compared with the control. There was a significant positive correlation of SOD activities with adult longevity and fecundity. This study suggested that malathion-induced oxidative stress was causative factor in the deterioration of biological fitness and that increased SOD activities may have resulted in decreased oxidative damage, which retarded the rate of deteriorative physiological changes in P. turionellae in response to sublethal doses of malathion.     

Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of oseltamivir and its metabolite oseltamivir carboxylate in plasma, saliva and urine

A bioanalytical method for the analysis of oseltamivir (OP) and its metabolite oseltamivir carboxylate (OC) in human plasma, saliva and urine using off-line solid-phase extraction and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. OP and OC were analysed on a ZIC-HILIC column (50 mm x 2.1 mm) using a mobile phase gradient containing acetonitrile-ammonium acetate buffer (pH 3.5; 10mM) at a flow rate of 500 microL/min. The method was validated according to published FDA guidelines and showed excellent performance. The lower limit of quantification for OP was determined to be 1, 1 and 5 ng/mL for plasma, saliva and urine, respectively and for OC was 10, 10 and 30 ng/mL for plasma, saliva and urine, respectively. The upper limit of quantification for OP was determined to be 600, 300 and 1500 ng/mL for plasma, saliva and urine, respectively and for OC was 10,000, 10,000 and 30,000 ng/mL for plasma, saliva and urine, respectively. The within-day and between-day precisions expressed as R.S.D., were lower than 5% at all tested concentrations for all matrices and below 12% at the lower limit of quantification. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range. Matrix effects were thoroughly evaluated both graphically and quantitatively. No matrix effects were detected for OP or OC in plasma or saliva. Residues from the urine matrix (most likely salts) caused some ion suppression for both OP and its deuterated internal standard but had no effect on OC or its deuterated internal standard. The suppression did not affect the quantification of OP.     

Malathion-induced Oxidative Stress in Rat Brain Regions

Malathion is a pesticide with high potential for human exposure. However, it is possible that during the malathion metabolism, there is generation of reactive oxygen species (ROS) and malathion may produce oxidative stress in intoxicated rats. The present study was therefore undertaken to determine malathion-induced lipid peroxidation (LPO), protein carbonylation and to determine whether malathion intoxication alters the antioxidant system in brain rats. Malathion was administered intraperitoneally in the acute and chronic protocols in the doses of 25, 50, 100 and 150 mg malathion/kg. The results showed that LPO in brain increased in both protocols. The increased oxidative stress resulted in an increased in the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), observed in cortex, striatum in the acute malathion protocol and hippocampus in the chronic malathion protocol. Our results demonstrated that malathion induced oxidative stress and modulated SOD and CAT activity in selective brain regions.     

Permethrin- and malathion-induced macromolecular abnormalities in adult Tribolium castaneum (herbst)

In Tribolium castaneum adults, sublethal doses of 1 and 2 ppm permethrin and 300 ppm malathion led to significant changes in amylase, trehalase, acid phosphatase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and isocitrate dehydrogenase activities. Malathion at 150 ppm did not affect phosphatases and lactate dehydrogenase. Both malathion and permethrin significantly increased cholinesterase activity. Mixing of the two insecticides resulted in antagonistic action with reference to various enzymatic activities. Glucose and glycogen contents were at first mobilized for energy supply under insecticidal stress conditions followed by lipid and cholesterol. Soluble protein, total protein, free amino acids, and urea contents remained unaltered under all experimental conditions.     

Effects of Subchronic Administration of Metrifonate on Cholinergic Neurotransmission in Rats

The effects of subchronic oral administration of metrifonate, a long-acting cholinesterase (ChE) inhibitor, on cholinergic neurotransmission were assessed in young adult male Wistar rats. Animals were treated twice daily with metrifonate. In a pilot study testing a 100 mg/kg dose of metrifonate for up to 14 days, ChE activity was found to steadily decrease to reach maximum inhibition levels of about 55%, 80% and 35% in brain, erythrocytes and plasma. Steady-state inhibition levels were attained by the 10th day of treatment. When metrifonate-treatment was discontinued, ChE activity in plasma returned to control levels within another day, while erythrocyte and brain ChE activity took more than 2 weeks to recover. In subsequent dose-response studies, metrifonate treatment was given for 3 and 4.5 weeks at doses of 0, 12.5, 25, 50, and 100 mg/kg, to different groups of animals, respectively. Correlation analysis indicted that brain ChE inhibition was more accurately reflected by erythrocyte than by plasma ChE inhibition, although all effects were highly correlated. The changes in ChE activity were not paralleled by changes in other parameters of the cholinergic neurotransmission, such as acetylcholine synthesis rate or acetylcholine receptor binding. It is therefore concluded that repeated administration of metrifonate to rats induces a long-lasting inhibition of ChE activity in a dose-related and predictable manner, which is neither subject to desensitization nor paralleled by counterregulatory downregulation of muscarinic or nicotinic receptor binding sites in brain.     

Mitochondrial Respiratory Dysfunction and Oxidative Stress after Chronic Malathion Exposure

Malathion is a pesticide used on a large scale and with high potential risk for human exposure. However, it is reasonable to hypothesize that while the malathion is metabolizing reactive oxygen species (ROS) can be generated and subsequently there is onset of an oxidative stress in central nervous system (CNS) structures: hippocampus, cortex, striatum and cerebellum of intoxicated rats due to mitochondrial respiratory chain disfunctions. The present study was therefore undertaken to evaluate malathion-induced lipid peroxidation (LPO), superoxide production from sub-mitochondrial particles and the activity of complexes II and IV of the mitochondrial respiratory chain. Malathion was administered in doses of 25, 50, 100 and 150 mg malathion/kg. After malathion administration LPO increased in hippocampus and striatum. This was accompanied by an increase in the formation of superoxide in submitochondrial particles in the hippocampus. Complex IV suffered significant inhibition of its activity. We could demonstrate in this study that malathion induces oxidative stress and it could be due to inactivation of mitochondrial respiratory complexes.     

Effect of malathion on antioxidant defence system in human fetus--an in vitro study.

Malathion under in vitro condition even at lower concentration (250 ppm) altered the level of enzymes associated with glutathione cycle and antioxidant defence system in human fetal brain and liver. Such changes involved alterations in glutathione status and extent of lipid peroxidation. The inhibitory effect of malathion was dose dependent in case of human fetal brain and was more vulnerable than fetal liver. This alteration (inhibition or activation) was maximum in case of tissues from fetuses of early period of development, suggesting greater susceptibility of human fetus towards this organophosphorus insecticide.     

In vivo dose response relationship between physostigmine and cholinesterase activity in RBC and tissues of rats

Dose response of physostigmine (Phy) was studied in rat using various doses (25-500 micrograms/kg i.m.). Rats were sacrificed 15 min after Phy administration. Blood and tissues were analyzed for ChE activity by radiometric method and Phy concentration by HPLC method. A comparison of ChE values in different tissues of rats indicated that ChE activity was highest in brain (7.11 mumol/min/g) and least in diaphragm (0.67 mumol/min/g). The enzyme activity was eleven times more in brain as compared to diaphragm. Phy produced a dose-dependent inhibition of ChE in RBC (18-42%), brain (23-35%) and diaphragm (25-35%) from 50 to 200 micrograms/kg, then ChE inhibition was plateaued from 200 to 500 micrograms/kg in these tissues. A dose related ChE inhibition was seen in heart (16-50%) and thigh muscle (8-53%) from 50 to 500 micrograms/kg. Phy concentration increased linearly from 50 to 400 micrograms/kg in plasma, brain, heart and thigh muscle. These results indicate that ChE inhibition is linear up to 200 micrograms/kg in RBC, 150 micrograms/kg in brain and 300 micrograms/kg in heart. This linearity is not consistent in other tissues.     

Protective effect of vitamin E in dimethoate and malathion induced oxidative stress in rat erythrocytes

Organophosphate (OP) pesticides such as dimethoate and malathion intoxication has been shown to produce oxidative stress due to the generation of free radicals and alter the antioxidant defense system in erythrocytes. It is possible that vitamin E being present at the cell membrane site may prevent OP-induced oxidative damage. In the present study, rats were pretreated orally with vitamin E (250 mg/kg body wt, twice a week for 6 weeks) prior to oral administration of a single low dose of dimethoate and/or malathion (0.01% LD(50)). The result showed that treatment with OP increased lipid peroxidation (LPO) in erythrocytes, however, vitamin E pretreated rats administered OP's showed decreased LPO in erythrocytes. The increase in the activities of superoxide dismutase (SOD) and catalase (CAT) and total-SH content in erythrocytes from dimethoate and/or malathion treated rats as compared to control appears to be a response towards increased oxidative stress. Vitamin E pretreated animals administered OP's showed a lowering in these parameters as compared to OP treated rats which indicates that vitamin E provide protection against OP-induced oxidative stress. The glutathione-S-transferase (GST) activity in erythrocytes was inhibited in OP intoxicated rats which partially recovered in vitamin E pretreated animals administered OP's. Inhibition in erythrocyte and serum acetylcholinesterase (AChE) activity was not relieved in vitamin E pretreated rats administered OP's probably due to the competitive nature of enzyme inhibition by OP's. The results show that vitamin E may amelierate OP-induced oxidative stress by decreasing LPO and altering antioxidant defense system in erthrocytes.     

Advanced glycation end products and antioxidant status in nondiabetic and streptozotocin induced diabetic rats: effects of copper treatment

The effects of Cu(II) supplementation on glycemic parameters, advanced glycation end products (AGEs), antioxidant status (glutathione; GSH and total antioxidant capacity; TAOC) and lipid peroxidative damage (thiobarbituric acid-reactive substances, TBARS) were investigated in streptozotocin (STZ) induced diabetic rats. The study was carried out on Wistar albino rats grouped as control (n = 10), CuCl₂ treated (n = 9), STZ (n = 10) and STZ,CuCl₂ treated (n = 9). STZ was administered intraperitoneally at a single dose of 65 mg/kg and CuCl₂, 4 mg copper/kg, subcutaneously, every 2 days for 60 days. At the end of this period, glucose(mg/dl), Cu(μg/dl), TBARS(μmol/l), TAOC(mmol/l) were measured in plasma, GSH(mg/gHb) in erythrocytes and glycated hemoglobin (GHb)(%) in blood. Plasma AGE-peptides(%) were measured by HPLC flow system with spectrofluorimetric and spectrophotometric detectors connected on-line. Data were analyzed by the non-parametric Kruskal–Wallis and Mann–Whitney U test. In the STZ group glucose, GHb and AGE-peptide levels were all significantly higher than the control group (P < 0.01, P < 0.05, and P < 0.01, respectively). CuCl₂ treated group had significantly lower glucose but significantly higher GHb, TAOC and TBARS levels than the control group (P < 0.05, P < 0.001, P < 0.05 and P < 0.001, respectively). STZ,CuCl₂ treated group had significantly higher GHb, TAOC and TBARS levels compared with the control group (P < 0.001, P < 0.05 and P < 0.05, respectively); but only TAOC level was significantly higher than the STZ group (P < 0.01). This experimental study provides evidence that copper intake increases total antioxidant capacity in both nondiabetic and diabetic states. However despite the potentiated antioxidant defence, lipid peroxidation and glycation enhancing effects of CuCl₂ are evident under nondiabetic conditions.     

Cadmium-induced alterations in the antioxidant defense system of the rat eye in relation to dietary selenium intake

Cytosolic activity of glutathione peroxidase (GSH-Px), selenium-independent GSH-Px, and catalase, thiobarbituric acid reactive substances (TBARS), and glutathione and selenium (Se) concentration were measured in ocular tissues of rats maintained on a low (0.05 ppm) or adequate (0.10 ppm) Se diet and treated with 0 or 25 ppm cadmium (Cd) in their drinking water for fourteen weeks. Feeding rats a low Se diet resulted in a significant decrease in GSH-Px activity when compared to rats maintained on adequate dietary Se, irrespective of Cd treatment. Se-independent GSH-Px activity of rats maintained at 0.05 ppm Se decreased 27% when compared to Se-adequate controls, whereas activity increased 38% in the Cd-treated low-Se group. When comparisons were made between ocular TBARS in rats maintained at either level of dietary Se and treated with 0 or 25 ppm Cd, a trend toward decreased amounts of TBARS in Cd-treated groups was observed. A significant decrease in ocular Se concentration occurred in rats fed 0.05 ppm Se when compared to the Se-adequate group. Administering Cd to the low-Se group increased ocular Se levels 100%. A negative correlation between ocular Se concentration and TBARS was observed, suggesting a possible alternate role for Se as an antioxidant in the eye.     

Evaluation of biomarkers in oyster larvae in natural and polluted conditions

Crassostrea gigas D-shaped larvae were subjected to different conditions of temperature and salinity for 24 h and four biomarkers (acetylcholinesterase (AChE) activity, thiobarbituric acid reactive substances (TBARS) levels, glutathione S-transferase (GST) and catalase (CAT) activities) were measured. AChE activity decreased when salinity increased from 25 to 30 and 35 psu at 20 and 25 degrees C. Temperature did not seem to have an influence on AChE activity. TBARS levels increased as a function of salinity when the temperature was maintained at 20 degrees C, whereas at 25 degrees C no effect of salinity could be observed. Variations in GST and CAT activities were not significant with salinity and temperature except that catalase activity was higher at 25 degrees C than at 20 degrees C. Exposure experiments were conducted at 23 degrees C and 30 psu with carbofuran (100 and 1000 microg/l) and malathion (100 and 300 microg/l). There was an inhibition of AChE activity with carbofuran, and a toxic effect shown by an increase in TBARS levels counteracted by increases in GST and CAT activities which protected the larvae. When two pairs of adults producing larvae were taken into consideration, significant differences in biomarker levels were noted between the larval offspring of each pair. Malathion induced a decrease in AChE activity and an increase in CAT activity.     

High-performance liquid chromatographic determination of the antifungal drug fluconazole in plasma and saliva of human immunodeficiency virus-infected patients

A high-performance liquid chromatographic (HPLC) assay has been developed for the determination of the antifungal drug fluconazole in saliva and plasma of patients infected with the human immunodeficiency virus (HIV). Samples can be heated at 60°C for 30 min to inactivate the virus without loss of the analyte. The sample pretreatment involves a liquid-liquid extraction with chloroform-1-propanol (4:1, v/v). The chromatographic analysis is performed on a Lichrosorb RP-18 (5 μm) column by isocratic elution with a mobile phase of 0.01 M acetate buffer (pH 5.0)-methanol (70:30, v/v) and ultraviolet (UV) detection at 261 nm. The lower limit of is 100 ng/ml in plasma (using 500-μl samples) and 1 μg/ml in saliva (using 250-μl samples) and the method is linear up to 100 μg/ml in plasma and saliva. At a concentration of 5 μg/ml the within-day and between-day precision in plasma are 7.1 and 5.7%, respectively. In saliva the within-day and between-day precision is 10.8% (at 5 μg/ml). The methodology is now being used in pharmacokinetic studies in HIV-infected patients in our hospital.     

Human Exposure to Malathion during a Possible Vector-Control Intervention against West Nile Virus. II: Evaluation of the Toxicological Risks Using a Probabilistic Approach

In order to prevent the propagation of West Nile Virus (WNV), insecticide sprayings have been carried out in several locations in North America since 1999 with the objective of controlling the mosquito populations that transmit this pathogen. An attempt to quantitatively compare the risk of developing a health response to WNV infection with the toxicological risk of insecticides is presented here. First, the acute and subchronic environmental concentrations resulting from repeated spraying events were modeled according to a reasonable worst-case spraying sequence established in an intervention program proposed by the Government of Quebec (Canada). Second, probability density functions (PDF) were established for some exposure parameters according to the data for the concerned population, when feasible. Monte Carlo analyses were performed by incorporating these PDF into the equations used to calculate the daily absorbed doses resulting from the exposure scenarios presented in the companion article (this issue). The results showed that for a significant proportion of the population, aerial and, to a lesser extent, ground sprayings of malathion can generate acute and subchronic exposure that may exceed some levels of toxicological concern based on the USEPA's reference values. Indeed, in the case of acute exposure following aerial spraying for infants, toddlers, and children, these proportions were respectively 37.1%, 59.5%, and 32.8% of the individuals, and 27.3%, 41.3%, and 24.9% following subchronic exposure. For ground spraying, these values were 12.5%, 24.2%, 8.8%; and 9.8%, 16.5%, and 7.4%. These results allowed the comparison of the probability of exceeding a level of toxicological concern for malathion exposure with the probability of developing WNV symptoms. This comparison shows that in some circumstances, the toxicological risk of malathion may exceed the infectious risk of WNV.     

Evaluation of malathion-induced cytogenetical effects and oxidative stress in plants using Allium test

The present study emphasized to explore the toxicity effect of malathion on plants using Allium test. The experiments explored the mitotic inhibition, growth and activity of antioxidant enzymes in roots of Allium cepa at different concentrations (50, 125, 250 and 375 ppm) of malathion under different exposure periods (3, 9 and 18 h). The results revealed that all concentrations of malathion were capable to decline the root growth. Malathion-induced mitotic alterations varying from reduction in mitotic index (MI), relative division rate (RDR) and phase distribution along with large number of chromosomal aberrations. These changes were of varying degree depending on the concentration and treatment period. The roots treated with malathion (375 ppm) showed significant (p ≤ 0.05) higher levels of superoxide dismutase and catalase than the control, while the activity of peroxidase was significantly (p ≤ 0.05) low. At 375 ppm malathion, malondialdehyde content was significantly high (p ≤ 0.05) that was increased with the treatment period. Findings concluded that variations in mitotic index, chromosomal aberrations, alterations in malondialdehyde content and activities of antioxidant enzyme could serve as the useful indicators for monitoring the effects of malathion exposures in the real scenario. Superoxide dismutase and catalase enzyme play vital roles in the antioxidant defence mechanisms under malathion toxicity. According to the data on the malondialdehyde content show malathion to be capable of producing superoxide radicals indirectly, and to result in membrane damage and oxidative stress.     

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.