Different Concentrations Of Heparin Research Articles

Influence of Heparin on Fibrinogen Assay by Clauss Method

Objective: To investigate the effect of heparin on fibrinogen detection by Clauss method. Methods: A normal plasma pool (NPP) of 20 healthy people was prepared with 3.2% sodium citrate anticoagulant. For experimental group 1, samples containing different concentrations of heparin were prepared. For experimental group 2, samples of experimental group 1 were diluted twice. For experimental group 3, samples of experimental group 1 were diluted 4 times. For the control group, samples of normal saline with the same volume as heparin in experimental group 1 were prepared. The fibrinogen contents of experimental group 1, experimental group 2, experimental group 3, and control group were detected by Clauss method and prothrombin time (PT)-derived method, and the fibrinogen detection results of different groups were analyzed. Results: The trend of fibrinogen detected by Clauss method and PT-derived method in experimental group 1 was different; there was significant difference between the results of experimental group 1 and the control group (P < 0.05); there was no significant difference between the results of experimental group 2 and the control group (P > 0.05); there was no significant difference between the results of experimental group 3 and the control group (P > 0.05); there was no significant difference between the results of experimental group 2 and experimental group 3 (P > 0.05); the relative deviation between experimental group 1 and the control group was higher in high-concentration heparin sample. Conclusion: Heparin affects fibrinogen detection by Clauss method, and the effect can be reduced by sample dilution.

Effect of heparin as an anticoagulant on measurements of the erythrocyte genome size using flow cytometry in bony fishes of different ploidy

The aim of the study was to verify whether the use of heparin as an anticoagulant can affect the results of genome size measurements using a flow cytometer. The values obtained by measuring blood samples with different concentrations of heparin were compared. The differences observed were highly significant, suggesting that the results of previous comparative studies, or those determining individual polyploidy, may need reinterpretation. We anticipate that our findings will increase the accuracy of flow cytometric analysis and will be useful for further surveys of genome size undertaken using blood samples.

Anticoagulation and antibacterial properties of heparinized nanosilver with different morphologies

Synthesis and characterization of nanoparticles with different morphologies coupled to minimal chemical interventions for sustainable applications is one of the contemporary topics in the field of nanotechnology. In the current study, heparinized silver nanoparticles were synthesized using a chemical reduction method. Different concentrations of heparin were used to investigate its role in the stability and morphological properties of silver nanoparticles. Interestingly, it has been observed that the concentration of the stabilizing agent heparin plays a pivotal role in dictating the size and shape of the nanosilver. As visualized under a transmission electron microscope, nanosilver with different morphological states such as triangles, truncated triangles, hexagon, and spheres has been experimentally trapped. Such modular property of heparin coated nanosilver has also exhibited substantial differences in their anticoagulation and antimicrobial activities.

Heparinized chitosan/hydroxyapatite scaffolds stimulate angiogenesis

Formation of blood vessels during bone regeneration represents a major challenge for tissue engineered constructs. Poor revascularization can lead to scaffold failure and consequently, leads to non-healing fracture. Heparin is known to bind with angiogenic growth factors influencing the process of new blood vessels formation. There are several problems associated with the use of growth factors in clinic such as low stability, uncontrolled delivery to the site, and high price. The aim of the present study was to explore the potential of heparin to produce pro-angiogenic bone regeneration materials. Chitosan/hydroxyapatite freeze-gelled scaffolds were prepared and loaded with heparin. Different concentrations of heparin were successfully loaded onto the scaffolds, its release from the scaffold was analysed by toluidine blue assay and their angiogenic effect was evaluated by chorioallantoic membrane (CAM) assay to determine the optimal concentration of heparin to induce a proangiogenic effect. It was noted that low heparin concentrations exhibited a positive effect, with approximately 28 μg per scaffold indicating a significant increment in blood vessels. The synthesized materials showed no cytotoxic effects when evaluated by using U2OS cell line.

Sensitivity improved with Parylene-C passivized on Lamb wave sensor for aPTT measurement through monitoring whole blood reaction

Blood clotting time monitoring is of great importance in predicting the risk of haemorrhage and cardiovascular diseases. In this study, a new P-Lamb wave sensor was developed to measure activated partial thromboplastic time (aPTT) with enhanced sensitivity. The sensor utilizes a piezoelectric and hydrophobic material, Parylene-C, as a waveguide for the P-Lamb wave sensor in order to monitor the aPTT in whole blood samples. With a smooth surface, the P-Lamb wave sensor reduced energy loss and prevented leaky modes, with a decrease of 33% ± 1.25% in the surface roughness and an increase of 2.75% ± 0.15% in the signal. The experimental results demonstrated that, compared to a quartz crystal microbalance (QCM), a P-Lamb sensor with high sensitivity (the frequency shift of a P-Lamb sensor is approximately 200 times greater than that of a QCM) improved stability and enhanced repeatability. Parylene-C worked as a phonon coupling layer, which improved the stability and sensitivity of the Lamb sensor. The relative standards deviation (RSD) of the aPTT measurement was 2.11%, which was 1.22 times lower than reported values. The P-Lamb wave sensor was also tested for its sensitivity to different concentrations of heparin and was found to exhibit an increase in coagulation time with an increasing heparin concentration. Compared to the SYSMEX CS 5100 haematology analyser, the clinical coefficient index (R2) was 0.99467. Considering its compact size, low cost, and mass production of the chip unit, the developed P-Lamb wave sensor is a promising device for point-of-care diagnosis of haemostasis and personal health monitoring.

Effect of heparin, caffeine and calcium ionophore A23187 on in vitro induction of the acrosome reaction of fresh ram spermatozoa

Abstract Objective To determine the effect of different concentrations of heparin, caffeine or calcium ionophore and incubation time on motility, hyperactivity (HA) and acrosome reaction (AR) of ram sperm in vitro. Methods Semen samples were collected by artificial vagina from three mature ram. Split fractions (0.1 mL) of the pooled semen were layered under 1 mL of S-TALP medium supplemented with either heparin (10, 25, 50, 75 and 100 μg/mL), caffeine (0.5, 0.75, 1, 2 mg/mL), or calcium ionophore A23187 (0.5, 0.75, 1, 1.55 mM/mL). Individual motility, hyperactivity percentage and acrosomal status were recorded at 0, 1, 2, 3 and 4 h post-incubation for all treatments and control. Moreover, they were examined for ability to fertilize sheep oocyte in vitro. Results Heparin, caffeine and calcium ionophore A23187 at a concentration of 75 μg/mL, 1 mg/mL, and 1.55 mM/mL respectively can be used as a protocol to provide the best results for in vitro cap citation and acrosome reaction in ram. The penetration rates of rate of oocytes inseminated with spermatozoa treated with calcium ionophore A23187, and heparin were higher as compared with caffeine. Moreover, heparin achieved higher fertilization rates but without significant difference with others. Conclusion The best concentration of heparin, caffeine and ionophore A23187 are 75 μg/mL, 1 mg/mL, 1.55 mM/mL for 3, 1, 4 h incubation respectively and can be used for in vitro fertilization in sheep.

Heparin versus 0.9% sodium chloride intermittent flushing for the prevention of occlusion in long term central venous catheters in infants and children.

Guidelines and clinical practice for the prevention of complications associated with central venous catheters (CVC) around the world vary greatly. Most institutions recommend the use of heparin to prevent occlusion, however there is debate regarding the need for heparin and evidence to suggest 0.9% sodium chloride (normal saline) may be as effective. The use of heparin is not without risk, may be unnecessary and is also associated with increased cost. To assess the clinical effects (benefits and harms) of intermittent flushing of heparin versus normal saline to prevent occlusion in long term central venous catheters in infants and children. The Cochrane Vascular Trials Search Co-ordinator searched the Specialised Register (last searched April 2015) and the Cochrane Register of Studies (Issue 3, 2015). We also searched the reference lists of retrieved trials. Randomised controlled trials that compared the efficacy of normal saline with heparin to prevent occlusion of long term CVCs in infants and children aged up to 18 years of age were included. We excluded temporary CVCs and peripherally inserted central catheters (PICC). Two review authors independently assessed trial inclusion criteria, trial quality and extracted data. Rate ratios were calculated for two outcome measures - occlusion of the CVC and central line-associated blood stream infection. Other outcome measures included duration of catheter placement, inability to withdraw blood from the catheter, use of urokinase or recombinant tissue plasminogen, incidence of removal or re-insertion of the catheter, or both, and other CVC-related complications such as dislocation of CVCs, other CVC site infections and thrombosis. Three trials with a total of 245 participants were included in this review. The three trials directly compared the use of normal saline and heparin, however, between studies, all used different protocols for the standard and experimental arms with different concentrations of heparin and different frequency of flushes reported. In addition, not all studies reported on all outcomes. The quality of the evidence ranged from low to very low because there was no blinding, heterogeneity and inconsistency between studies was high and the confidence intervals were wide. CVC occlusion was assessed in all three trials (243 participants). We were able to pool the results of two trials for the outcomes of CVC occlusion and CVC-associated blood stream infection. The estimated rate ratio for CVC occlusion per 1000 catheter days between the normal saline and heparin group was 0.75 (95% CI 0.10 to 5.51, two studies, 229 participants, very low quality evidence). The estimated rate ratio for CVC-associated blood stream infection was 1.48 (95% CI 0.24 to 9.37, two studies, 231 participants; low quality evidence). The duration of catheter placement was reported to be similar between the two study arms, in one study (203 participants). The review found that there was not enough evidence to determine the effects of intermittent flushing of heparin versus normal saline to prevent occlusion in long term central venous catheters in infants and children. Ultimately, if this evidence were available, the development of evidenced-based clinical practice guidelines and consistency of practice would be facilitated.

Inhibition of urinary macromolecule heparin on aggregation of nano-COM and nano-COD crystals.

Purpose: This research aims to study the influences of heparin (HP) on the aggregation of nano calcium oxalate monohydrate (COM) and nano calcium oxalate dihydrate (COD) with mean diameter of about 50 nm. Method: The influences of different concentrations of HP on the mean diameter and Zeta potential of nano COM and nano COD were investigated using a nanoparticle size Zeta potential analyzer. Results: HP could be adsorbed on the surface of nano COM and nano COD crystals, leading to an increase in the absolute value of Zeta potential on the crystals and an increase in the electrostatic repulsion force between crystals. Consequently, the aggregation of the crystals is reduced and the stability of the system is improved. The strong adsorption ability of HP was closely related to the -OSO3− and -COO− groups contained in the HP molecules. X-ray photoelectron spectroscopy confirmed the coordination of HP with Ca2+ ions of COM and COD crystals. Conclusion: HP could inhibit the aggregation of nano COM and nano COD crystals and increase their stability in aqueous solution, which is conducive in inhibiting the formation of calcium oxalate stones.

Thrombin Generation Increasing with Age and Decreasing with Use of Heparin Indicated by Calibrated Automated Thrombogram Conducted in Chinese

ObjectiveCalibrated Automated Thrombogram(CAT) is a test to monitor the generation of thrombin. It can be described by four parameters: lag time, peak thrombin, endogenous thrombin potential (ETP) and time to peak (ttPeak). This study aims to determine the normal ranges of CAT parameters in Chinese, and evaluate whether thrombin generation is correlated with the concentration of heparin/low molecular weight heparin. MethodsPlasma from 120 healthy subjects were collected to determine the normal rangea of CAT parameters in Chinese. Normal plasma pool (NPP, n=25) spiked with different concentrations of heparin or enoxaparin were used to detecte CAT parameters. The overall and age specific normal ranges of CAT parameters were calculated using descriptive statistics method with mean±2SD. The correlation between CAT parameters and age or concentrations of heparin, enoxaparin were analyzed with linear regression model. ResultsThe normal ranges for lag time, peak thrombin, ETP, ttPeak in the subjects were 3.648±2.465 min, 367.39±151.93 nmol/L, 2277±1030 nmol/L·min and 6.372±4.280 min respectively. Age was linearly correlated with lag time (r=-0.6583, P<0.0001), peak thrombin (r=0.4863, P<0.0001), ETP (r=0.3608, P<0.0014) and ttPeak (r=-0.6313, P<0.0001). The values of ETP/peak ratio were linearly correlated with concentrations of heparin. ConclusionThe normal ranges of four CAT parameters for Chinese were determined. CAT parameters are associated with age. ETP/peak ratio could be used to monitor the process of anticoagulation therapy.

Real-time measurement of free thrombin: Evaluation of the usability of a new thrombin assay for coagulation monitoring during extracorporeal circulation

In patients undergoing cardiac surgery with heart-lung machine support, adequate anticoagulation to mitigate blood clotting caused by the artificial surfaces of the extracorporeal circulation (ECC) system is essential. These patients routinely receive heparin, whose effectiveness is monitored by measurements of the activated clotting time (ACT). However, ACT values only poorly correlate with the actual hemostatic status. The aim of our study was to evaluate the detection of free thrombin in heparinized human blood as a monitor of anticoagulation during ECC. Human whole blood was anticoagulated with different concentrations of heparin (0.75, 1, 2 or 3 IU/ml) and circulated in the Chandler-loop model for up to 240 min at 37 °C. Next to ACT, ECC-mediated changes in free active thrombin, prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin-III (TAT) levels were measured before and during circulation. Platelet activation and cell count parameters were further investigated. Our study shows that detection of ECC-mediated changes in free thrombin is possible in blood anticoagulated with 0.75 or 1 IU/ml heparin, whereas no thrombin was detectable at higher heparin concentrations. Thrombin generation during 240 min of ECC is comparable to F 1+2 and TAT plasma levels during ECC. Thrombin is the key enzyme in the coagulation cascade and hence represents a promising marker for monitoring the coagulation status of patients. Although detection of free thrombin was not feasible at high heparin concentrations, the employed test represents an additional test to current laboratory methods investigating blood coagulation at low heparin concentrations.

Hollow core photonic crystal fiber as a reusable Raman biosensor

We report that a single hollow core photonic crystal fiber (HC-PCF) can be used for repetitive characterization of multiple samples by Raman spectroscopy. This was achieved by integrating the HC-PCF to a differential pressure system that allowed effective filling, draining and re-filling of samples into a HC-PCF under identical optical conditions. Consequently, high-quality and reliable spectral data could be obtained which were suitable for multivariate analysis (partial least squares). With the present scheme, we were able to accurately predict different concentrations of heparin and adenosine in serum. Thus the detection scheme as presented here paves a path for the inclusion of HC-PCFs in point-of-care technologies and environmental monitoring where rapid sample characterization is of utmost importance.

Raman spectroscopy for clinical-level detection of heparin in serum by partial least-squares analysis

Heparin is the most widely used anti-coagulant for the prevention of blood clots in patients undergoing certain types of surgeries including open heart surgeries and dialysis. The precise monitoring of heparin amount in patients' blood is crucial for reducing the morbidity and mortality in surgical environments. Based upon these considerations, we have used Raman spectroscopy in conjunction with partial least squares (PLS) analysis to measure heparin concentration at clinical level which is less than 10 United States Pharmacopeia (USP) in serum. The PLS calibration model was constructed from the Raman spectra of different concentrations of heparin in serum. It showed a high coefficient of determination (R2>0.91) between the spectral data and heparin level in serum along with a low root mean square error of prediction ~4 USP/ml. It enabled the detection of extremely low concentrations of heparin in serum (~8 USP/ml) as desirable in clinical environment. The proposed optical method has the potential of being implemented as the point-of-care testing procedure during surgeries, where the interest is to rapidly monitor low concentrations of heparin in patient's blood.

The development of hospital manufactured ready to use heparin solution to flush catheters

BackgroundHeparin flush solution is a sterile preparation of heparin sodium with sufficient sodium chloride to make it isotonic with blood. So far heparin was mainly prepared on wards from concentrated...

Microfluidic device and system for point-of-care blood coagulation measurement based on electrical impedance sensing

Blood coagulation tests are an integral part of diagnosis and treatment of patients with cardiac disorders. The activated partial thromboplastin time test (aPTT or APTT) is performed on a large scale, in laboratories, to monitor the functioning of the intrinsic and common pathways of the blood coagulation cascade and the concentration of anticoagulants such as heparin among patients. A polydimethylsiloxane (PDMS) based microfluidic device has been fabricated and tested to perform APTT as a point-of-care device using whole blood samples by detecting the change in electrical impedance of blood during coagulation. A portable setup consisting of the device and a lock-in amplifier circuit was built and tested for repeatability and stand alone usage. A frequency sweep test was performed on the device and the optimal frequency of operation for maximum output sensitivity was determined. The devices were also tested for their sensitivity to different concentrations of heparin and found to exhibit an expected increase in coagulation time for increasing heparin concentration.

Heparin changes the conformation of high-mobility group protein 1 and decreases its affinity toward receptor for advanced glycation endproducts in vitro

High-mobility group protein 1 (HMGB1) has been identified as a late-acting mediator of inflammation. The receptor for advanced glycation end products (RAGE) is the main receptor and mediates the cytokine activity of HMGB1. Since HMGB1 also exhibits heparin-binding activity, we investigated whether heparin interferes with HMGB1/RAGE interaction and prevents the cytokine activity. We used fluorescence spectrometry, circular dichroism spectrometry and SPR biosensor technique to evaluate the effect. After treatment of HMGB1 with different concentrations of heparin (0, 50, 100 and 1000U/L), the fluorescence peak values of HMGB1 increased and the emission wavelength showed red shifts; further, the secondary structure of HMGB1 showed a marked change in that the content of β-pleated sheet reduced while that of α-helix increased. The equilibrium dissociation constants (KD) were determined by SPR technique; KD=4.5×10−9mol/L for heparin and HMGB1 and KD=9.77×10−8mol/L for HMGB1 and RAGE, respectively. Heparin and RAGE had no interaction. The amount of HMGB1 and RAGE bound forms reduced after treatment with heparin. ELISA revealed that addition of heparin inhibited the TNF-α and IL-6 released by macrophages RAW264.7 and HUVEC; 10U/L and 50U/L of heparin showed the most marked inhibitory effect in RAW264.7 cells and in HUVEC, respectively. In conclusion, heparin can combine with HMGB1 and affect the affinity of HMGB1/RAGE by changing the conformation of HMGB1. And this effect was independent of heparin concentration, so that a low dose of heparin was sufficient to achieve the best anti-inflammatory effect in our test.

In Vitro Modeling of the Influence of FVIII Activity and Heparin Induced Prolongation of APTT

Anticoagulant therapy is most commonly assessed by measuring the effect of the drug on global clotting assay, such as APTT. It is known that response of the APTT to heparin may be decreased in patients with high levels of factor VIII. In this work, we have attempted to determine in vitro conditions of experiment for obtaining relationship between different concentrations of heparin and values of APTT, and to investigate influence of factor VIII on correlation between concentrations of heparin and APTT. Measurement of the effect of heparin, added in vitro in normal coagulation control plasma (NCCP) showed that heparin in concentrations from 0.1 to 1.0 IU/mL prolonged APTT from 0.73 s to 99.26 s. Linearity of the relation of natural logarithm of APTT and concentration of added heparin in plasma for concentrations from 0.5 to 1.0 IU/mL (r = 0.995), and other characteristics of the validated method (RSD = 1.17%), made possible investigation of the influence of factor VIII addition in the solution. The addition of the Factor VIII concentrate, markedly influenced these APTT results. Increased factor VIII activity shortened the APTT, having more pronounced effect in the presence of the large amounts of heparin. Increased factor VIII was associated with downward shift in the concentration -- logAPTT response curve (y = 24644 x + 30.17 vs. y = 10.864 x + 27.256). This finding suggests the possibility for modeling of ex vivo establishment of correlation between plasma activity of FVIII and needed doses of heparin for appropriate management of heparin therapy.

Decidual Cells Produce a Heparin-binding Prolactin Family Cytokine with Putative Intrauterine Regulatory Actions

Pregnancy in mice and rats is associated with the production of a large family of hormones/cytokines related to prolactin (PRL). The hormones/cytokines are hypothesized to coordinate maternal and fetal adaptations to pregnancy. In this study, PRL-like protein-J (PLP-J, also known as PRL family 3, subfamily c, member 1 (Prl3c1)) is shown to be a product of the uterine decidua and a regulator of postimplantation intrauterine events. PLP-J-specific antibodies and a series of recombinant PLP-J proteins were generated and used to investigate PLP-J expression and as ligands for investigating biological targets. Decidual PLP-J migrates as a 29-kDa protein and localizes to a band of decidual cells surrounding the trophoblast cell layer on gestation day 8.5. PLP-J ligands specifically bound in situ to the surrounding uterine stromal cells and vasculature within the decidua of gestation day 8.5 implantation sites. We then investigated the in vitro actions of PLP-J on uterine stromal cells and endothelial cells. PLP-J specifically interacted with both cell populations. PLP-J promoted uterine stromal cell proliferation and inhibited endothelial cell proliferation. We determined that PLP-J does not interact with PRL receptors. Instead, PLP-J interacts with heparin-containing molecules, including syndecan-1, which is expressed in gestation day 8.5 pregnant uteri, as well as in uterine stromal cells and endothelial cells. The restricted expression of PLP-J and its specific interactions with uterine stromal cells and endothelial cells suggests that it acts locally and regulates decidual cell development and the endometrial vasculature.

Heparin effect onin vitronuclear maturation of bovine oocytes

Oocytes undergo numerous biochemical and morphological changes during their development from preantral to preovulatory phases. In vitro studies have suggested several compounds that might induce oocyte maturation. Heparin is a natural component of ooplasm, follicular fluid and uterine fluid and previous studies indicated that it might act as a chromatin maturation factor in bovine oocytes. We tested this hypothesis in vitro by timing germinal vesicle breakdown (GVBD) and first polar body (PB) formation without any other natural or introduced factors that might influence the rate of oocyte maturation. We also determined if these oocytes could be fertilized. Bovine oocytes were incubated in a salt medium and TCM 199 supplemented with different concentrations of heparin for 24 h at 37.5 degrees C in a humidified atmosphere of 5% CO2. With 1.0 and 6.5 mg/ml heparin, the time of GVBD was reduced from 4.7+/-1.1 h to about 1.5 h and the time of first PB formation was reduced from 22.0+/-1.1 h to 9.0-11.0 h in salt medium. In TCM 199, only 6.5 mg/ml heparin significantly reduced the time of PB formation. In both incubation media, 1.0 and 6.5 mg/ml heparin induced GVBD, extrusion of the first PB and formation of the metaphase II nucleus. Moreover, heparin did not interfere with the fertilization of oocytes matured in TCM 199. Based on the results, we propose that heparin plays an important role in the rearrangement of the oocyte chromatin and acts as an oocyte maturation factor.

Heparan sulfate on activated glomerular endothelial cells and exogenous heparinoids influence the rolling and adhesion of leucocytes

Proliferative glomerulonephritides are characterized by the influx of leucocytes. Heparan sulfate (HS) plays an important role in the recruitment, rolling and firm adhesion of leucocytes to activated endothelium. Recently, we have shown the importance of HS on activated mouse glomerular endothelial cells (mGEnC-1) for the firm adhesion of leucocytes in a static adhesion assay. In the present study, we evaluated the role of HS on glomerular endothelial cells and the effect of adding heparinoids on the leucocyte-glomerular endothelium interaction under dynamic flow conditions. The number of rolling and firmly adhering leucocytes, and the rolling velocity of leucocytes was determined on a monolayer of unactivated or TNF-alpha-activated mGEnC-1 under dynamic flow conditions using physiological relevant shear stress rates in a flow chamber system. Furthermore, the effects of removal of HS on TNF-alpha-activated mGEnC-1 by heparinase III treatment, and of different concentrations of heparin, tinzaparin and HS, on the rolling and adhesion of leucocytes were evaluated. At the calculated physiological shear stress rate of 0.8 dynes/cm2 the number of rolling and firmly adhering leucocytes to mGEnC-1 increased 2-fold after activation with TNF-alpha, whereas the rolling velocity of the leucocytes decreased 2-fold. Addition of heparin, tinzaparin or HS, and the removal of HS on mGEnC-1 reduced the number of leucocytes rolling and adhering to activated mGEnC-1 about 2-3-fold, while the rolling velocity increased more than 2-fold. HS on activated glomerular endothelial cells is important for the interaction with leucocytes under flow conditions, while exogenous heparinoids interfere with this interaction. These results suggest that supplementary treatment of proliferative glomerulonephritides with heparinoids is an interesting option to pursue.

307 ADDITION OF HIGH CONCENTRATIONS OF HEPARIN DURING IN VITRO FERTILIZATION IMPROVES PERCENTAGES OF CLEAVAGE AND BLASTOCYST FORMATION WHEN BOVINE OOCYTES ARE INSEMINATED IN VITRO WITH SEX-SORTED SPERM

The developmental potential of oocytes fertilized in vitro by sexed–sorted frozen–thawed sperm remains low. The aim of this study was to find an optimal concentration of heparin during IVF using frozen–thawed sex-sorted sperm to improve the percentages of fertilization and blastocyst formation. A total of 1708 matured bovine oocytes were randomly allocated among different concentrations of heparin (0, 2.5, 5, 10, 20, and 40 µg mL−1) in Bracket-Olifant medium and co-incubated with non-sexed or sex-sorted sperm for 6 h (Day 0). The sperm (sorted or not) used for IVF was from one bull of proven fertility. Presumptive zygotes then were cultured in CR1aa + 6 mg mL−1 of BSA in 5% O2, 5% CO2, and 90% N2 at 39°C until Day 8. Percentages of cleavage were recorded on Day 2. Data (3 replicates) were analyzed by ANOVA. The results (Table 1) demonstrated that addition of different concentrations of heparin in IVF medium for non-sexed sperm groups did not alter cleavage and blastocyst formation. However, the lowest percentages of cleavage were observed with 0, 2.5, and 5 µg mL−1 of heparin in sexed sperm groups, demonstrating that high concentrations of heparin (20 and 40 µg mL−1) positively affected the percentages of cleavage and blastocyst formation. A greater proportion of zygotes in 20 and 40 µg mL−1 sexed sperm groups developed into grade 1 blastocysts on Day 7, and subsequently formed more blastocysts on Day 8 compared with 0, 2.5, and 5 µg mL−1 of heparin in sexed sperm groups. In conclusion, addition of heparin was not necessary when frozen–thawed non-sexed sperm were used for IVF with this particlar bull. However, addition of higher concentrations of heparin during IVF improved fertilization and blastocyst formation in vitro (40 µg mL−1 of heparin could be used as an alternative concentration for frozen–thawed sex-sorted sperm with this particular bull). The incidence of polyspermy as well as polyploidy in blastocysts is currently being studied. Table 1.Effect of heparin concentrations during IVF on cleavage and blastocyst formation