Serial Bleeding Research Articles

Improved Pharmacokinetic and Bioavailability Support of Drug Discovery Using Serial Blood Sampling in Mice

Pharmacokinetic studies in mice traditionally require one animal per time point, resulting in dosing and euthanizing a large number of animals and producing suboptimal quality of pharmacokinetic data due to inter-animal variability and dosing error. These studies are time-consuming and labor-intensive. To improve the throughput and quality of pharmacokinetic evaluation in mice, we have developed a serial blood sampling methodology using the lateral saphenous vein puncture technique. Two marketed drugs, indinavir and rosuvastatin, were selected for this validation study because of their distinctly different physicochemical and pharmacokinetic properties. Each compound was dosed orally and intravenously in mice using both discrete and serial blood sampling methods. The pharmacokinetic results from serial bleeding are in excellent agreement with those from discrete sampling for both compounds. Compared to the discrete sampling, the serial sampling procedure is a more humane method, allowing for rapid and repeated sampling from the same site without the need for anesthesia. The application of this new method has led to a remarkable reduction in animal and compound usage, a significant increase in throughput and speed, and a drastic improvement in pharmacokinetic data quality. This approach is especially useful for the first-tier in vivo pharmacokinetic screening of discovery compounds.

Pharmacokinetic optimization of four soluble epoxide hydrolase inhibitors for use in a murine model of inflammation

Early soluble epoxide hydrolase inhibitors (sEHIs) such as 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) are effective anti-hypertensive and anti-inflammatory agents in various animal models. However, their poor metabolic stability and limited water solubility make them difficult to use pharmacologically. Here we present the evaluation of four sEHIs for improved pharmacokinetic properties and the anti-inflammatory effects of one sEHI. The pharmacokinetic profiles of inhibitors were determined following p.o. (oral) administration and serial bleeding in mice. Subsequently the pharmacokinetics of trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), the most promising inhibitor, was further studied following s.c. (subcutaneous), i.v. (intravenous) injections and administration in drinking water. Finally, the anti-inflammatory effect of t-AUCB was evaluated by using a lipopolysaccharide (LPS)-treated murine model. Better pharmacokinetic parameters (higher C(max), longer t(1/2) and greater AUC) were obtained from the tested inhibitors, compared with AUDA. Oral bioavailability of t-AUCB (0.1 mg.kg(-1)) was 68 +/- 22% (n = 4), and giving t-AUCB in drinking water is recommended as a feasible, effective and easy route of administration for chronic studies. Finally, t-AUCB (p.o.) reversed the decrease in plasma ratio of lipid epoxides to corresponding diols (a biomarker of soluble epoxide hydrolase inhibition) in lipopolysaccharide-treated mice. The in vivo potency of 1 mg.kg(-1) of t-AUCB (p.o.) was better in this inflammatory model than that of 10 mg.kg(-1) of AUDA-butyl ester (p.o) at 6 h after treatment. t-AUCB is a potent sEHI with improved pharmacokinetic properties. This compound will be a useful tool for pharmacological research and a promising starting point for drug development.

Antibodies to DNA, chromatin core particles and histones in mice with graft-versus-host disease and their involvement in glomerular injury.

Chronic graft-versus-host disease (GVHD) was induced in female (C57 B10S/DBA/2)F1 hybrid mice with two successive injections of lymphoid cells from parental DBA/2 strain. Serial bleedings of 27 GVHD mice were screened with a panel of antigens including the five histones H1, H2A, H2B, H3 and H4, 15 histone peptides, core particles, dsDNA, heat-shock proteins hsp70 and ubiquitin, a branched peptide of ubiquitinated H2A (U-H2A), poly(ADP-ribose) and SSB/La protein. The predominant IgG response to histone peptides was directed against regions 204-218 of H1, 1-25 of H2B and 1-29 of H4. GVHD mice also produced IgG antibodies to dsDNA and chromatin core particles as reported previously. IgG antibodies reacting with dsDNA appeared before antibodies to core particles and histones. Raised levels of antibodies to U-H2A, but not to monomeric ubiquitin, were also found. While the level of antibodies to dsDNA, histones and core particles decreased significantly before the appearance of proteinuria, suggesting their involvement in glomerular injury, the longitudinal pattern of anti-U-H2A peptide response was apparently not linked to the manifestation of lupus nephritis in GVHD mice.

Design and optimization of a multiplex anti-influenza peptide immunoassay

Current flu vaccines are based on killed or attenuated virus vaccines that must be altered each year to include the hemagglutinin and neuraminidase genes from a strain of virus predicted to predominate in the coming year. A vaccine that could protect against multiple strains of influenza A and B would be a major asset in the fight against flu-related mortality and morbidity. To support development of such a vaccine, we have developed a Flu Multiplex Assay based on a Luminex® platform to assess serum antibody levels to two conserved peptides derived from influenza A (M2 protein) and influenza B (hemagglutinin protein). The peptides were synthesized with a biotin label and subsequently coupled to two different LumAvidin™ microspheres. We then tested various sera against both types of peptide in the multiplex assay format. The data show that sera from Rhesus macaques immunized with a single peptide react only with the homologous peptide while Rhesus macaques immunized with both peptides respond well to both peptides. Additionally, we were able to specifically compete reactivity to both peptides. We have tested serial bleeds from 100 pediatric patients at ages ranging from 16 to 56 weeks as well as single bleeds from over 100 healthy adults. No overall trend in titer relative to pediatric age was detected. Both demographics exhibited a minimal response to either the A/M2 or B/HA0 peptides. However, the average titer for the pediatric serum samples was significantly lower than that found in the adult population. The adult population exhibited a higher prevalence of low reactive samples. Assay reagents and parameters have been optimized and the assay is shown to be repeatable and robust. The assay will be used to support clinical vaccine trials of a bivalent peptide vaccine.

Cyclophosphamide Pharmacokinetics in Mice: A Comparison Between Retro Orbital Sampling Versus Serial Tail Vein Bleeding

Preclinical studies on pharmacokinetics in animals usually require at least 3-5 animals per time point. The use of several animals in each time point may increase the result variation due to individual dosing errors and interand intraindividual variation among animals. Moreover, a large number of animals has to be euthanized in these experiments. In the present paper, the pharmacokinetics of the anticancer drug, cyclophosphamide was investigated using serial bleeding from the tail vein and compared with the kinetics obtained from traditional retro-orbital sinus bleeding in mice. Cyclophosphamide (100 mg/kg) was administered intraperitoneally to two groups of mice. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 3, 4, 5 and 6 hr. In the first group (n=6), 20 L blood samples were collected from the tail-vein by serial bleeding. In the second group, 3 animals were killed at each time point and blood was collected from both tail vein and retro-orbital sinus. Cyclophosphamide analysis in whole blood was carried out using LC-MS/MS with on-line sample preparation utilizing microextraction by packed sorbent (MEPS). Pharmacokinetics including AUC, tmax, Cmax and half – life were estimated using WinNonLin. Cyclophosphamide concentrations in blood obtained after sampling through tail vein were close to those obtained after retro-orbital bleeding within the same animal. No significant differences in estimated pharmacokinetic parameters were found when serial tail vein bleeding was compared to retro-orbital bleeding. Our results indicate that serial sampling using tail vein in mice can be a good alternative to retro-orbital sampling method. The method is reliable, easy and can be used for early preclinical kinetic studies. However, more studies are needed to evaluate different drug categories.

A portal vein cannulation technique for drug discovery in mice

One approach to understanding how orally administered drugs are absorbed and metabolized involves measuring compound concentrations in portal vein blood and in systemic circulation at various time points. In mice, blood samples are generally collected through terminal bleeding, a process that requires a large number of mice and is susceptible to variation between individuals. The authors developed a portal vein cannulation procedure for serial bleeding in the mouse, using a modified catheter containing a stainless steel stylet that is implanted directly in the portal vein. To demonstrate the technique, they orally administered two different compounds to mice and obtained blood samples from the tail vein and portal vein at different time points. They analyzed compound concentrations using liquid chromatography-tandem mass spectrometry. The technique refines existing methods for pharmacokinetic studies in the mouse and reduces the number of mice required.

Evaluation of the effects of serial phlebotomy on the transcriptome of major tissues and on the response to toxicants in rats

Preclinical pharmacokinetic (PK) evaluations are typically conducted in rats before in vivo toxicologic evaluations. It is unclear how the serial bleeding procedures in PK studies affect tissue homeostasis or sensitivity to toxicity. In this study, our objective was to evaluate the impact of serial bleeding on the transcriptome of various major tissues (kidney, heart, liver, spleen) and their response to two well-characterized molecules, doxorubicin and cisplatin. Rats received single i.v. injections of saline, doxorubicin (8 mg/kg) or cisplatin (4 mg/kg). In each group, half of the rats were serially bled by tail vein. Serial bleeding was associated with slight decreases of red blood cell parameters, but did not result in histopathological changes or in increased sensitivity to doxorubicin and cisplatin toxicity based on clinical pathology and histopathology evaluation. In addition, serial bleeding did not induce significant gene expression changes in either vehicle- or compound-treated rats when compared to their respective control groups. Overall, these results suggest that the serial bleeding procedure used in PK studies in our institution minimally affects the tissue response to toxicants, and support the use of these studies to generate early toxicology data in drug discovery.

Immunological differences between susceptible and resistant sheep during the preclinical phase of scrapie infection

In order to investigate the relationship between the immune response to scrapie infection and genetic susceptibility to the disease in sheep, immune cell subsets and prion protein (PrP) expression were determined in susceptible and resistant Suffolk sheep in the preclinical phase of infection. At 6 months of age, 12 ARQ/ARQ (susceptible) and nine ARR/ARR (resistant) scrapie-free Suffolk lambs were challenged subcutaneously with scrapie inoculum. Prefemoral lymphadenectomies were carried out at 14 and 180 days post-inoculation (p.i.) and serial bleeds were collected at monthly intervals for up to 1 year p.i. An indirect double-labelling procedure was carried out on peripheral blood mononuclear cells (PBMCs) and lymph node cell preparations and analysed using flow cytometry. Prior to scrapie challenge, significantly more PrP(+) cells were detected in PBMCs from the susceptible sheep. Furthermore, following challenge, significantly more CD8(+) and gammadelta(+) T cells were detected in the PBMCs of the resistant sheep. However, at both 14 and 180 days p.i, CD21(+) cell expression was significantly higher in the lymph node preparations of the susceptible sheep. In contrast, more CD4(+) cells were detected in the lymph nodes of the resistant sheep at both time points. It was concluded that significant differences in immune cell subsets and PrP expression occur between ARQ/ARQ and ARR/ARR Suffolk sheep in the preclinical phase of infection.

Effect of pentobarbital on pH and electrolyte levels after induced seizure in rats

We studied the effects of high doses of pentobarbital (PB) and carbamazepine (CBZ) on electrolyte levels and pH in an epileptic animal model. Pentobarbital decreased Ca2+ and Na+ levels without pentylenetetrazole (PTZ). After this, Ca2+ and Na+ levels continued to decrease except when CBZ was used, which preserved the Ca2+ levels PTZ may have opposed effects on PB. Our results suggest that PB causes changes in electrolyte levels and pH, but these changes are diminished by CBZ.

Development of an epitope-blocking-enzyme-linked immunosorbent assay to differentiate between animals infected with and vaccinated against foot-and-mouth disease virus

An epitope-blocking ELISA (EB-ELISA) was developed to distinguish animals infected with foot-and-mouth-disease (FMDV) from those immunized with commercial vaccines. The assay used monoclonal antibodies to target the 3B core repeat motif (QKPLK) and purified recombinant 3AB proteins from the major B cell line epitopes of FMDV. Sera from uninfected and regularly vaccinated cattle, pigs, goats, and sheep (raised in FMDV free areas) were screened to evaluate the specificity of the EB-ELISA. The specificity scores of the assays were 99.8–100% and 100%, respectively. Reference sera from cattle, pigs, goats, and sheep experimentally infected with FMDV tested positive, with only a single exception. Antibodies formed in response to FMDV 3B appeared 1 week after infection and persisted at high levels for more than 8 weeks within the sera collected from serial bleeding of animals infected with FMDV O/SKR/2000. The EB-ELISA was used to differentiate between farms vaccinated against and those infected with FMDV (FMDV Asia serotype) during the 2005 epidemic in Mongolia by detecting antibodies against the FMDV Asia serotype in outbreak farms. This EB-ELISA method shows promise as an effective tool for FMDV control and eradication.

Preparation and evaluation of a recombinant Rift Valley fever virus N protein for the detection of IgG and IgM antibodies in humans and animals by indirect ELISA

This paper describes the cloning, sequencing and bacterial expression of the N protein of the Rift Valley fever virus (RVFV) ZIM688/78 isolate and its evaluation in indirect ELISAs (I-ELISA) for the detection of IgM and IgG antibodies in human and sheep sera. Sera used for the evaluation were from 106 laboratory workers immunised with an inactivated RVF vaccine, 16 RVF patients, 168 serial bleeds from 8 sheep experimentally infected with wild type RVFV and 210 serial bleeds from 10 sheep vaccinated with the live attenuated Smithburn RVFV strain. All human and animal sera that tested positive in the virus neutralisation test were also positive in the IgG I-ELISA. There was a high correlation ( R 2 = 0.8571) between virus neutralising titres and IgG I-ELISA readings in human vaccinees. In experimentally infected sheep IgG antibodies were detected from day 4 to 5 post-infection onwards and IgM antibodies from day 3 to 4. The IgG I-ELISA was more sensitive than virus neutralisation and haemagglutination-inhibition tests in detecting the early immune response in experimentally infected sheep. The I-ELISAs demonstrated that the IgG and IgM response to the Smithburn vaccine strain was slower and the levels of antibodies induced markedly lower than to wild type RVFV infection.

Multiplexed Detection of Antibodies to Nonstructural Proteins of Foot-and-Mouth Disease Virus

Liquid array technology was used to develop a multiplexed assay for the detection of antibodies to viral nonstructural proteins (NSPs), raised in cattle in response to infection with foot-and-mouth disease (FMD) virus. Two assays, one based on recombinant NSPs and the other on synthetically produced peptides, were developed and compared side-by-side. Serum samples from serial bleeds of cattle, each experimentally infected with one of the seven serotypes (C, A, O, Asia, SAT1, SAT2, SAT3) of FMD virus were analyzed. A distinct pattern in the detection of NSP antibodies and a close correlation of the recombinant protein and peptide-based assays were observed. The detection of antibodies to NSPs is a method to differentiate FMD-infected and FMD-vaccinated animals, and a high-throughput assay would be an invaluable tool in the case of an outbreak of FMD in North America, when emergency vaccination may be utilized to spare vaccinated, noninfected animals from slaughter and subsequent disposal.

CHANGES IN ECHOCARDIOGRAPHIC VARIABLES OF LEFT VENTRICULAR SIZE AND FUNCTION IN A MODEL OF CANINE NORMOVOLEMIC ANEMIA

The objective of this study was to document changes in echocardiographic variables of left ventricular size and function noninvasively during acute normovolemic anemia. This model was developed as a pilot study with the purpose of providing baseline information to investigate the pathophysiology, and more specifically the effect on the heart, of canine babesiosis-induced anemia. The study group comprised of 11 mature healthy Beagle dogs that weighed between 9 and 15 kg. Severe normovolemic anemia was induced over a 3-4-day period by serial bleeding while maintaining normovolemia by autotransfusing plasma and infusing crystalloids. The dogs were then allowed to recover. Preanemic (mean Hct 46.7%, standard deviation [SD] 2.4%) echocardiographic variables of left ventricular performance (Fractional shortening, ejection fraction, end-systolic and end-diastolic ventricular volumes, cardiac index, and heart rate) were compared with those in the severely (mean Hct 15.3%, SD 1.1%), moderately (Hct mean 24.7%, SD 1.5%), and mildly (mean Hct 33.5%, SD 2.5%) anemic states, and between the anemic states. With the exception of end diastolic volume, there was a statistically significant (P < 0.05) increase in all variables in the severely anemic state vs. the preanemic and the mild and moderate anemic states. In concordance with previous invasive models, a hyperdynamic state of the left ventricle develops in response to experimentally induced acute canine normovolemic anemia in the conscious dog. Echocardiography has promise as a noninvasive technique of evaluating the cardiac changes in dogs having canine babesiosis.

Supercritical Fluid Chromatography−Tandem Mass Spectrometry for the Enantioselective Determination of Propranolol and Pindolol in Mouse Blood by Serial Sampling

Packed-column supercritical fluid chromatography (pSFC) coupled to an atmospheric pressure chemical ionization (APCI) source and a tandem mass spectrometer (MS/MS) with minimum sample pretreatment was explored for the rapid and enantioselective determination of (R,S)-propranolol in mouse blood. Serial bleeding of mice is advantageous for the reduction of animal usage, dosing errors, and animal-to-animal variation. The effects of the eluent flow rate and composition as well as the nebulizer temperatures on the ionization efficiency of racemic propranolol and pindolol as model compounds in the positive ion mode under pSFC conditions were studied. The fundamental parameters on the proposed hyphenated system such as matrix ionization suppression and chromatographic performances were investigated in improving sensitivity and enantiomeric separation for the detection of the analytes. The proposed chiral pSFC-APCI/MS/MS approach requiring approximately 3 min/sample for the determination of (R,S)-propranolol at a low nanogram per milliliter region was partially validated with respect to specificity, linearity, reproducibility, and accuracy and was applied to support a pharmacokinetic study.

High-throughput pharmacokinetic method: Cassette dosing in mice associated with minuscule serial bleedings and LC/MS/MS analysis

A method for pharmacokinetic studies using cassette dosing associated with serial bleeding in mice is described. PK profiles of four soluble epoxide hydrolase inhibitors were determined following oral, subcutaneous or intraperitoneal administration individually or in cassette dosing. Parent analyses were performed on only 5 μL of whole blood from serial bleeds (up to 10 per animal), by LC/MS/MS. An accuracy (88–100%) and precision (<10% RSD) were observed, leading to reliable datum points for PK calculation. PK profiles, T max, C max and half-life values after cassette dosing were similar to the individual PK results. This method dramatically increases speed of data collection while dramatically reducing cost and animal usage. The results presented here clearly indicate that this proposed method could be applicable to high-throughput PK studies.

Duration of the reproductive period decreases the frequency of preovulatory luteinizing hormone surges in heavy weight-sire line turkey hens

The frequency of preovulatory luteinizing hormone (LH) surges is an important determinant of ovulation and oviposition rates in turkeys. Egg production rate is relatively poor in heavy weight-sire line type turkey hens and declines with advancing duration of the reproductive period. The purpose of this study was to measure frequency and characteristics of preovulatory LH surges in turkey hens of a heavy weight-sire line type early, at peak of egg production (Early), and late, after egg production rate had declined (Late), in a reproductive period. The Early hens were photostimulated with a continuous photoperiod [24 h light (L):0 h dark (D)] at 40 weeks of age and sampled during peak egg production at about 47 weeks of age. The Late hens were photostimulated at 40 weeks of age with a long day photoperiod (14L:10D). After a 27-week egg production period, the Late hens were switched to the 24L:0D photoperiod and sampled at 74 weeks of age. Continuous lighting was used during blood sampling to allow the rhythm of preovulatory LH surges to free run. All hens were cannulated 3–5 days before starting sampling and hourly blood samples were collected for 200 h. All hens were necropsied and ovarian and oviductal morphologies were measured after serial bleeding. The Late hens had a longer interval between intra-clutch preovulatory LH surges than the Early hens, and a higher incidence of atretic ovarian follicles. The Early hens had higher baseline and surge amplitude LH concentrations but lower progesterone (P 4) surge amplitude concentrations than the Late hens. The duration of preovulatory LH surges, incidence of “blind” preovulatory LH surges, baseline P 4 concentrations, and overall estradiol-17β (E 2) concentrations were not different between Early and Late hens. In conclusion, a longer interval between preovulatory LH surges, lower LH baseline and surge amplitude concentrations, a higher incidence of atretic follicles, and higher P 4 surge amplitude concentration were associated with the decline in egg production late in the reproductive period in a heavy weight-sire line of turkey hens.

An inhibition enzyme-linked immunosorbent assay for the detection of antibody to Rift Valley fever virus in humans, domestic and wild ruminants

This paper describes the development and validation of an inhibition ELISA based on gamma-irradiated tissue culture-derived antigen for the detection of antibody to Rift Valley fever virus (RVFV) in humans, domestic and wild ruminants. Validation data sets derived from field-collected sera in Africa (humans = 1367, cattle = 649, goats = 806, sheep = 493, buffalo = 258, camels = 156) were categorized according to the results of a virus neutralisation test. In addition, individual sera from 93 laboratory workers immunized with inactivated RVF vaccine, 136 serial bleeds from eight sheep experimentally infected with wild-type of RVFV, and 200 serial bleeds from 10 sheep vaccinated with the live-attenuated strain of the virus, were used to study the kinetics of RVFV antibody production under controlled conditions. At cut-off values selected at 95% accuracy level by the two-graph receiver operating characteristic analysis the ELISA sensitivity ranged from 99.47% (humans) to 100% (sheep, buffalo, camels). The specificity ranged from 99.29% (sheep) to 100% (camels). Compared to virus neutralisation and haemagglutination-inhibition tests, the ELISA was more sensitive in detection of the earliest immunological responses in experimentally infected and vaccinated sheep. Our results demonstrate that the ELISA format reported here can be used as a safe, robust and highly accurate diagnostic tool in disease-surveillance and control programmes, import/export veterinary certification, and for monitoring of the immune response in vaccinees.

Changes in spermatogenesis and endocrine function in the ram testis due to irradiation and active immunization against luteinizing hormone-releasing hormone1,2

Spermatogonial stem cell transplantation is a technique that has potential in livestock to enhance genetic gain and generate transgenic offspring through the male germ line. A means for depletion of endogenous germ cells in a recipient's seminiferous tubules is necessary for this technology to be applied. The objectives of this study were to evaluate several methods for depletion of endogenous germ cells in the testes of adult rams and to evaluate ultrasound-guided injections into the rete testes as a means for infusing a suspension into the seminiferous tubules. Sixteen adult rams were randomly divided into 4 treatment groups (n = 4 per group). Treatments consisted of active immunization against LHRH (IMM), localized testicular irradiation (IR), LHRH immunization + irradiation (IMM+IR), and untreated control. Serial bleedings were conducted pretreatment and monthly after treatment for 4 mo, at which time all rams were castrated. Both IMM and IMM+IR rams received exogenous gonadotropin in the form of Perganol weekly for 8 wk before castration to bypass the immunization. All rams also received an ultrasound-guided injection of PBS containing 0.4% trypan blue into the rete testis of one testicle before castration. Rams receiving IMM and IMM+IR treatments had higher (P < 0.05) average percentages of seminiferous tubule cross sections with depleted germ cells compared with controls. Serum testosterone was decreased (P < 0.05) in IMM and IMM+IR rams 1 mo after treatment and throughout the remainder of the study compared with controls and IR rams, which were not different from each other. Serum inhibin concentration was unchanged in all rams following treatment indicating that Sertoli cell function was unaltered. A greater (P < 0.05) average percentage of the total testicular area could be filled with the trypan blue solution by rete testis injection in IMM and IMM+IR rams. These data demonstrate the depletion of endogenous germ cells in adult ram testes without alteration of Sertoli cell viability and function that have potential as methods for preparing recipient animals for germ cell transplantation.

Development of synthetic peptide ELISA based on nonstructural protein 2C of foot and mouth disease virus

It was reported that the sera of convalescent animals contain antibodies to foot and mouth disease (FMD) virus (FMDV) 2C, highly conserved nonstructural protein (NSP), whereas the sera of vaccinated animals do not. But ELISA methods using this protein were not reported and developed until recently. In this study, NSP 2C peptides were synthesized within the amino acid sequence of the conserved 2C nonstructural region of FMDV according to the sequences from Genbank database and used for identifying antigenic determinants. One of the synthesized thirteen peptides gave strong positive reactivity with most of the sera from 13 FMD infected farms, but not with sera from vaccinated and non-infected animals. Moreover, with the sera collected through serial bleedings from four cattle and five goats infected with FMDV O/SKR/2000 experimentally, positive results were obtained in two species after 10 days post infection (DPI). Therefore, we tried to develop and evaluate this ELISA based on 2C peptides. In comparison with the commercial NSP ELISA, the 2C peptide based ELISA method showed good specificity and sensitivity. These results demonstrate that the synthetic 2C peptide ELISA can be a complementary marker to differentiate FMDV-infected from vaccinated on a herd basis.

Development of a cannulation procedure for broiler breeder hens

A cannulation and serial bleeding procedure has been developed to monitor the peripheral patterns of hormones associated with reproduction for up to 10 d in broiler breeder hens. Hens were cannulated via the jugular vein and returned to individual cages. The unrestrained cannulated hens were connected to a tether and swivel system that permitted constant infusion for maintenance of the cannula prior to serial bleeding and unrestrained long-term serial bleeding for up to 10 d. In a short-term experiment hens were bled every 12 min for 36 h, and in a long-term experiment hens were bled hourly for 10 d. In these experiments, 1.5-mL blood samples were collected at each time point with sodium citrate as the anticoagulant. To avoid hemodilution, after removal of plasma the red blood cells were reconstituted with saline to the original volume and returned to the hen of origin. Collection of serial blood samples was successful from 94% of hens in the short-term experiment and 79% of hens in the long-term experiment. Egg production was not affected (P > 0.05) during the 6 wk following serial bleeding in the short-term experiment. For hens that continued laying, egg production for 10 d prior to cannulation was not different (P > 0.05) from egg production for the 10 d during serial bleeding in the long-term experiment. However, late in the reproductive cycle many hens (25%) stopped laying when serially bled. It is concluded that this cannulation procedure can be used to study short-term or long-term peripheral patterns of hormones associated with oviposition and ovulation in laying broiler breeder hens.