Characterization of the mechanical properties of bovine cortical bone treated with a novel tissue sterilization process

Over the past decade chemical processing and engineering of musculoskeletal tissue (tendon and bone) has improved dramatically. The use of bone allograft and xenograft in reconstructive orthopedic and maxillofacial surgeries is increasing, yet severe complications can occur if the material is contaminated in any way. A novel tissue sterilization process, BioCleanse^®, has been developed to clean and sterilize musculoskeletal tissue for implantation. The present study was designed to determine the effect of this novel cleaning process on the biomechanical properties of bovine cortical bone prior to implantation. The mechanical properties of treated bovine bone material were compared to human samples with respect to failure under compression, shear and three-point bending. The data demonstrate that bovine bone treated with the novel sterilization procedure has favorable biomechanical properties compared to that of human bone treated in a similar fashion.     

The effect of sterilization on mechanical properties of soft tissue allografts

One major concern regarding soft tissue allograft use in surgical procedures is the risk of disease transmission. Current techniques of tissue sterilization, such as irradiation have been shown to adversely affect the mechanical properties of soft tissues. Grafts processed using Biocleanse processing (a proprietary technique developed by Regeneration Technologies to sterilize human tissues) will have better biomechanical characteristics than tissues that have been irradiated. Fifteen pairs of cadaveric Achilles tendon allografts were obtained and separated into three groups of 10 each. Three treatment groups were: Biocleanse, Irradiated, and Control (untreated). Each specimen was tested to determine the biomechanical properties of the tissue. Specimens were cyclically preloaded and then loaded to failure in tension. During testing, load, displacement, and optical strain data were captured. Following testing, the cross sectional area of the tendons was determined. Tendons in the control group were found to have a higher extrinsic stiffness (slope of the load–deformation curve, p = .005), have a higher ultimate stress (force/cross sectional area, p = .006) and higher ultimate failure load (p = .003) than irradiated grafts. Biocleanse grafts were also found to be stiffer than irradiated grafts (p = .014) yet were not found to be statistically different from either irradiated or non-irradiated grafts in terms of load to failure. Biocleanse processing seems to be a viable alternative to irradiation for Achilles tendon allografts sterilization in terms of their biomechanical properties.     

The biomechanical effects of limb lengthening and botulinum toxin type A on rabbit tendon

Numerous studies have examined the effects of distraction osteogenesis (DO) on bone, but relatively fewer have explored muscle adaptation, and even less have addressed the concomitant alterations that occur in the tendon. The purpose herein was to characterize the biomechanical properties of normal and elongated rabbit (N=20) tendons with and without prophylactic botulinum toxin type A (BTX-A) treatment. Elastic and viscoelastic properties of Achilles and Tibialis anterior (TA) tendons were evaluated through pull to failure and stress relaxation tests.All TA tendons displayed nonlinear viscoelastic responses that were strain dependent. A power law formulation was used to model tendon viscoelastic responses and tendon elastic responses were fit with a microstructural model. Distraction-elongated tendons displayed increases in compliance and stress relaxation rates over undistracted tendons; BTX-A administration offset this result. The elastic moduli of distraction-lengthened TA tendons were diminished (p=0.010) when distraction was combined with gastrocnemius (GA) BTX-A administration, elastic moduli were further decreased (p=0.004) and distraction following TA BTX-A administration resulted in TA tendons with moduli not different from contralateral control (p>0.05). Compared to contralateral control, distraction and GA BTX-A administration displayed shortened toe regions, (p=0.031 and 0.038, respectively), while tendons receiving BTX-A in the TA had no differences in the toe region (p>0.05). Ultimate tensile stress was unaltered by DO, but stress at the transition from the toe to the linear region of the stress–stretch curve was diminished in all distraction-elongated TA tendons (p<0.05). The data suggest that prophylactic BTX-A treatment to the TA protects some tendon biomechanical properties.     

Effects of supercritical fluid CO<Subscript>2</Subscript> and 25 kGy gamma irradiation on the initial mechanical properties and histological appearance of tendon allograft

Tendon allografts, when autograft options are limited or when obtaining an autograft is not aligned with the patients’ best interest, play an important role in tendon and ligament reconstruction. To minimize the risk of infectious disease transmission tissue banks perform screening tests and the allografts cleaned are sterilized. The current study examines and compares the initial mechanical properties and histological appearance of supercritical CO₂ (SCCO₂)-treated and gamma-irradiated porcine extensor tendons. Thirty intact porcine forelimb extensor tendons randomized equally into three groups: control group, gamma-irradiation group, and SCCO₂-treated group. Once treated, histological assessment and histomorphologic measurements were made on the histological sections obtained from each tendon while stiffness and ultimate failure loads were evaluated from tensile testing. Histological evaluation of gamma-irradiated tendons showed significant disruption to the hierarchical morphology of the fascicle bundles, which was not evident in SCCO₂-treated specimens. Histomorphologic measurements showed a significant increase for measured dead space (void) between tendon fibrils of the gamma-irradiated group comparing to both control and SCCO₂ treated groups (p?<?0.01). There was a significant reduction in the ultimate failure load for tendons treated by gamma-irradiation compared to the control group (p?<?0.05). No statistically significant difference was detected between control and SCCO₂-treated tendons in the ultimate failure load. Stiffness values were not significantly different between three-study groups. This study suggests that while gamma-irradiation has a deleterious effect on mechanical properties of tendon tissue, SCCO₂ does not alter the biomechanical properties and the histological structure of porcine extensor tendons.     

Comparison and evaluation of biomechanical,electrical, and biological methods for assessment of damage to tissue collagen

In regard to evaluating tissue banking methods used to preserve or otherwise treat (process) soft allograft tissue, current tests may not be sufficiently sensitive to detect potential damage inflicted before, during, and after processing. Using controlled parameters, we aim to examine the sensitivity of specific biomechanical, electrical, and biological tests in detecting mild damage to collagen. Fresh porcine pulmonary heart valves were treated with an enzyme, collagenase, and incubated using various times. Controls received no incubation. All valves were cryopreserved and stored at ?135 °C until being rewarmed for evaluation using biomechanical, permeability, and cell viability tests. Statistically significant time dependent changes in leaflet ultimate stress, (p = 0.006), permeability (p = 0.01), and viability (p ≤ 0.02, four different days of culture) were found between heart valves subjected to 0–15 min of collagenase treatment (ANOVA). However, no statistical significance was found between the tensile modulus of treated and untreated valves (p = 0.07). Furthermore, the trends of decreasing and increasing ultimate stress and viability, respectively, were somewhat inconsistent across treatment times. These results suggest that permeability tests may offer a sensitive, quantitative assay to complement traditional biomechanical and viability tests in evaluating processing methods used for soft tissue allografts, or when making changes to current validated methods. Multiple test evaluation may also offer insight into the mechanism of potential tissue damage such as, as is the case here, reduced collagen content and increased tissue porosity.     

The effects of lyophilization on flexural stiffness of extrasynovial and intrasynovial tendon

Tendon or ligament reconstructions often use autologous or allogenic tendons from either extrasynovial or intrasynovial sources. Allograft tendons must be lyophilized for preservation before transplantation, a process which can impact mechanical properties of the graft. Reconstituted graft properties that are similar to native tendon are desirable. Although tensile and compressive properties of tendons have been investigated, there is a paucity of information describing flexural properties of tendon, which can impact the gliding resistance. This study aims to design a testing method to quantify tendon flexural modulus, and investigate the effects of lyophilization/rehydration procedures on tendon flexibility. A total of 20 peroneus longus tendons (extrasynovial) and 20 flexor digitorum profundus tendons (intrasynovial) were collected. Ten of each tendon were processed with 5 freeze–thaw cycles followed by lyophilization and rehydration with saline solution (0.9%). Bend testing was conducted on tendons to quantify the flexural modulus with and without processing. As canine FDP tendons contain fibrous and fibrocartilaginous tissue regions, the flexural moduli were measured in both regions. Flexural modulus of rehydrated, lyophilized extrasynovial PL tendon was significantly lower than that of similarly processed intrasynovial FDP tendon (p < 0.001). Flexural moduli of both the fibrocartilaginous and non-fibrocartilaginous regions of intrasynovial tendon significantly increased after lyophilization (p < 0.001). The flexural modulus of the fibrocartilaginous region was significantly higher than that of the non-fibrocartilaginous region in intrasynovial tendon (p < 0.001). Lyophilization significantly increases the flexural modulus of extrasynovial and intrasynovial tendons, and flexural modulus differs significantly between these two tendon types. Increases in stiffness caused by lyophilization may impact the mechanical performance of the allograft in vivo.     

In vivo biostability of four types of arterial grafts with impervious walls; their haemodynamic and pathological characteristics

We describe the haemodynamic and pathological characteristics of four types of impervious arterial prostheses, two alloplastic (Milrathane^® and Gore-Tex^®), and two chemically processed bovine heterografts (Solcograft^® and Solco P^®). They were implanted in the thoracic aortae of dogs for durations of 24 hours, 48 hours, one weeks, two weeks, one month, three months and six months. Haemodynamic analyses showed no relation between the shear rate index, I·_Y, and compliance, C_D. The observed shear rates are 6.5 times lower than those likely to damage the endothelial cell layer. Macroscopic and microscopic observations of explanted grafts showed the presence of obstructive thrombi at the anastomoses of Mitrathane^® grafts as early as one week. Gore-Tex^® grafts develop in the area of anastomoses parietal-thrombi which reorganize and become covered with pseudo-endothelial cells. The bovine heterografts show a similar behaviour. However, whereas Solcograft^® has an irregular thin wall, Solco P^® had improved characteristics except in the graft implanted for three months which demonstrated some manufacturing weaknesses. Both types showed the development of anastomotic pannus covered with endothelial-like cells. All grafts, whether alloplastic or chemically processed, suffered from an absence of healing of the middle part of the prosthesis. The cause of this problem will be found in the analysis of the biochemical and enzymatic reactations between the material used and its physiological surrounding.     

Effect of commercial herbal toothpastes and mouth rinses on the prevention of enamel demineralization using a microcosm biofilm model

Abstract

This work evaluated the effects of commercial toothpastes and mouth rinses containing natural/herbal agents on biofilm viability, extracellular polysaccharide (EPS) production and on enamel demineralization in vitro. Microcosm biofilm was produced on bovine enamel for 5?days and treated daily with: Orgânico natural^® (toothpaste/mouth rinse), Boni Natural Menta &; Malaleuca^® (toothpaste/mouth rinse), Propolis &; Myrrh^® (toothpaste), Colgate Total 12 Clean Mint^® (toothpaste, positive control), Malvatricin^® Plus (mouth rinse), PerioGard^® (mouth rinse, positive control) or PBS (negative control). Tom’s Propolis &; Myrrh^® and Colgate Total 12^® toothpastes and Malvatricin^® Plus and PerioGard^® mouth rinses significantly reduced biofilm viability (p?® had significant effects on biofilm thickness and EPS. Despite the indication that Tom’s Propolis &; Myrrh^® significantly reduced lesion depth, only Colgate Total 12^® significantly reduced mineral loss. Malvatricin^® Plus significantly reduced mineral loss and lesion depth, as did PerioGard^®. Some herbal products, Malvatricin^® Plus and Tom’s Propolis &; Myrrh^®, showed anticaries effects.     

Effects of <Superscript>60</Superscript>Co gamma radiation dose on initial structural biomechanical properties of ovine bone—patellar tendon—bone allografts

Gamma radiation is established as a procedure for inactivating bacteria, fungal spores and viruses. Sterilization of soft tissue allografts with high dose ⁶⁰Co gamma radiation has been shown to have adverse effects on allograft biomechanical properties. In the current study, bone-patellar tendon-bone (BPTB) allografts from 32 mature sheep were divided into two treatment groups: low-dose radiation at 15 kGy (n = 16) and high-dose radiation at 25 kGy (n = 16) with the contralateral limb serving as a 0 kGy (n = 32) non-irradiated control. Half of the tendons from all treatment groups were biomechanically tested to determine bulk BPTB mechanical properties, cancellous bone compressive properties, and interference screw pull-out strength. The remaining tissues were prepared, implanted, and mechanically tested in an acute in vitro anterior crucial ligament (ACL) reconstruction. Low-dose radiation did not adversely affect mechanical properties of the tendon allograft, bone, or ACL reconstruction compared to internal non-irradiated control. However, high-dose radiation compromised bulk tendon load at failure and ultimate strength by 26.9 and 28.9%, respectively (P < 0.05), but demonstrated no negative effect on the cancellous bone compressive properties or interference screw pull-out strength. Our findings suggest that low dose radiation (15 kGy) does not compromise the mechanical integrity of the allograft tissue, yet high dose radiation (25 kGy) significantly alters the biomechanical integrity of the soft tissue constituent.     

A quantitative method to evaluate the donor corneal tissue quality used in a comparative study between two hypothermic preservation media

To standardize a new evaluation technique for calculating the overall quality (OQ) of the donor cornea and validate it using a comparative study of corneas preserved in Optisol-GS and Cornea Cold^®. Thirty pairs of donor corneas were selected for a 4 week in vitro comparative study using masked observers. Physiological parameters like thickness, transparency, viable endothelial cell density (VECD) and morphology were transformed to numerical range (0–4) to obtain the OQ. Microbiological examination was performed using Bactec instrument. Students t test showed statistically better results (p < 0.05) from week 3 for thickness, week 2 for transparency and week 1 for morphology and VECD; statistical significance (p < 0.05) was found for OQ from week 2 for the corneas preserved in Cornea Cold^® compared to Optisol-GS. Epithelial quality was similar regardless of the medium. Microbiological examination showed absence of aerobic and anaerobic microorganisms in both media. OQ method is efficient, consistent and easy, now validated for comparative studies. Further refinement is necessary for its use at eye-banks, bio-banks and research or transplantation purposes. Cornea Cold^® is a promising hypothermic corneal storage medium with preservation time ≤21 days. This permits higher flexibility, evaluation accuracy, longer duration for surgical preparation and ease of transportation.     

The effect of tendon loading on in-vitro carpal kinematics of the wrist joint

Measurements of in-vitro carpal kinematics of the wrist provide valuable biomechanical data. Tendon loading is often applied during cadaver experiments to simulate natural stabilizing joint compression in the wrist joint. The purpose of this study was to investigate the effect of tendon loading on carpal kinematics in-vitro.A cyclic movement was imposed on 7 cadaveric forearms while the carpal kinematics were acquired by a 4-dimensional rotational X-ray imaging system. The extensor- and flexor tendons were loaded with constant force springs of 50 N, respectively. The measurements were repeated without a load on the tendons. The effect of loading on the kinematics was tested statistically by using a linear mixed model.During flexion and extension, the proximal carpal bones were more extended with tendon loading. The lunate was on the average 2.0° (p=0.012) more extended. With tendon loading the distal carpal bones were more ulnary deviated at each angle of wrist motion. The capitate was on the average 2.4° (p=0.004) more ulnary deviated.During radioulnar deviation, the proximal carpal bones were more radially deviated with the lunate 0.7° more into radial deviation with tendon loading (p<0.001). Conversely, the bones of distal row were more flexed and supinated with the capitate 1.5° more into flexion (p=0.025) and 1.0° more into supination (p=0.011).In conclusion, the application of a constant load onto the flexor and extensor tendons in cadaver experiments has a small but statistically significant effect on the carpal kinematics during flexion–extension and radioulnar deviation.     

Assessment of different carbohydrates as exogenous carbon source in cultivation of cyanobacteria

Glucose is the substrate most widely used as exogenous carbon source for heterotrophic cultivation of cyanobacteria. Due to limited information about the use of different carbohydrates as carbon sources to support cyanobacterial heterotrophic metabolism, the objective of this work was to evaluate different monosaccharides (arabinose, fructose, galactose, glucose, mannose and xylose), disaccharides (lactose, maltose, sucrose and trehalose) and polysaccharides (carboxymethylcellulose, cassava starch, Hi-maize^®, maltodextrin Corn Globe 1805^® and xylan) as exogenous carbon source for heterotrophic culture of cyanobacterium Phormidium sp. The batch cultivation using fructose as organic carbon source resulted in the highest (p < 0.05) cell biomass (5,540 mg/L) in parallel with the highest (p < 0.05) substrate yield coefficient (0.67 mg_biomass/mg_fructose). Mannose was the carbon source with the highest (p < 0.05) substrate consumption rate (3,185.7 mg/L/day) and maltodextrin was the carbohydrate with major potential to produce biomass (1,072.8 mg_biomass/L/day) and lipids (160.8 mg_lipids/L/day). Qualitatively, the fatty acid profiles of the lipid extract from Phormidium sp. showed predominance of saturated chains for the cultures grown with most of the carbon sources, with the exception of the ones grown with xylose and maltodextrin.     

Effect of addition of buserelin acetate to the extender on motility and viability of bovine spermatozoa

The present study was aimed to determine the effect of GnRH analog (buserelin acetate) on the quality of bovine spermatozoa stored at 16°?C for 24?h. Semen collected in the summer season from June to September from healthy Polish Holstein–Friesian bulls. Ejaculates were centrifuged, divided and diluted to the final concentration of 240?×?10⁶ spermatozoa/mL using animal protein–free commercial BIOXcell^® extender (IMV Technologies, L’aigle, France) (Control) or with BIOXcell^® extender supplemented with buserelin acetate and stored 0, 8 and 24?h. Sperm motility parameters analysis was performed using a computer-assisted sperm analysis (CASA) system. The viability of spermatozoa was performed using flow cytometer. The addition of buserelin acetate to BIOXcell^® extender did positively affect the total motility (was higher in the observed samples with the addition of 2?µg/mL and 4?µg/mL than in the control group), progressive motile (forward progressing sperm was significantly increased (p?<?0.05) over the control group at the 0?h and 8?h of incubation following the supplementation of 2, 4 and 8?μg/mL buserelin acetate) and viability of spermatozoa (the number of live spermatozoa was significantly higher (p?<?0.05) in 2?µg/mL and 4?µg/mL samples with buserelin acetate at 8th hour of incubation and in sample with 4?µg/mL at 24th hour of incubation compared to the control group). We recommend adding 4?µg/mL to the extender to improve the quality of bovine semen.     

Resistance training minimizes the biomechanical effects of aging in three different rat tendons

Aging process is characterized by a decline in the organism functionality, especially in the decrease of muscle function, which also affects tendons. On the other hand, the resistance training (RT) has been used as an important tool to increase muscle and tendineous function during aging. Thus, this study aim has been to verify the effects of RT on the biomechanical properties of three different aged rat tendons. For this purpose, 20 wistar rats have been divided into four groups (5 rats per group): young sedentary (YS), trained (YT), old sedentary (OS) and old trained (OT). The RT has been performed through climb protocol for 12 weeks. After RT, the calcaneal tendon (CT), superficial flexor tendon (SFT) and deep flexor tendon (DFT) have been used for analysis. The results indicate that the RT in aged rats can prevent tendon function decrease (p<0.05). Although RT has prompted significant biomechanical changes in trained aged rats, there has been no increase in cross-section area or tendon stiffness reduction. Thus, the OT group showed better biomechanical responses when compared with OS (p<0.05). Therefore, RT can be used as an excellent strategy for increasing in tendon capacity during aging.     

Cross-Linking in Collagen by Nonenzymatic Glycation Increases the Matrix Stiffness in Rabbit Achilles Tendon

Nonenzymatic glycation of connective tissue matrix proteins is a major contributor to the pathology of diabetes and aging. Previously the author and colleagues have shown that nonenzymatic glycation significantly enhances the matrix stability in the Achilles tendon (Reddy et al., 2002, Arch. Biochem. Biophys., 399, 174–180). The present study was designed to gain further insight into glycation-induced collagen cross-linking and its relationship to matrix stiffness in the rabbit Achilles tendon. The glycation process was initiated by incubating the Achilles tendons (n = 6) in phosphate-buffered saline containing ribose, whereas control tendons (n = 6) were incubated in phosphate-buffered saline without ribose. Eight weeks following glycation, the biomechanical attributes as well as the degree of collagen cross-linking were determined to examine the potential associations between matrix stiffness and molecular properties of collagen. Compared to nonglycated tendons, the glycated tendons showed increased maximum load, stress, strain, Young''s modulus of elasticity, and toughness indicating that glycation increases the matrix stiffness in the tendons. Glycation of tendons resulted in a considerable decrease in soluble collagen content and a significant increase in insoluble collagen and pentosidine. Analysis of potential associations between the matrix stiffness and degree of collagen cross-linking showed that both insoluble collagen and pentosidine exhibited a significant positive correlation with the maximum load, stress, and strain, Young''s modulus of elasticity, and toughness (r values ranging from .61 to .94) in the Achilles tendons. However, the soluble collagen content present in neutral salt buffer, acetate buffer, and acetate buffer containing pepsin showed an inverse relation with the various biomechanical attributes tested (r values ranging from .22 to .84) in the Achilles tendons. The results of the study demonstrate that glycation-induced collagen cross-linking is directly associated with the increased matrix stiffness and other mechanical attributes of the tendon.     

<Emphasis Type="Italic">Apis mellifera</Emphasis> and <Emphasis Type="Italic">Osmia cornuta</Emphasis> as carriers for the secondary spread of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> on apple flowers

The efficiency of two pollinators, Apis mellifera L. (Hymenoptera: Apidae) and the mason bee Osmia cornuta (Latreille) (Hymenoptera: Megachilidae), as carriers of biocontrol agents (BCA) from flower to flower (secondary colonisation) was investigated on apple cv ‘Golden Delicious’. The BCA tested was Bacillus subtilis, strain BD170 (Biopro®) developed for the control of the ‘fire blight’ caused by Erwinia amylovora (Burril) Winslow et al. The two insect species were studied as secondary BCA carriers on apple plants in pots under net screened tunnels. Their behaviour and capacity to deposit the BCA in the most receptive flower parts were compared both by washing, diluting and plating the flower organs on a recovery medium and by means of PCR analyses based on a molecular marker. O. cornuta showed better performances with respect to A. mellifera. For the field trials, pollinators were introduced in four apple orchards. During apple’s flowering, the BD170 (100 g hl^?l) was sprayed once in two fields, and twice in the others. The pollinators’ efficacy in carrying the BCA from sprayed flowers to the stigmas of newly opened ones at different times after the spray treatment was evaluated. The detection of the BCA was performed by PCR analysis. The percentages of positive PCR flower samples were higher in the internal treated areas of the fields with respect to the external untreated ones, but the high colonisation level found in the latter and in the flowers opened in both areas several days after the treatment(s) demonstrated that pollinators can play an important role as secondary carriers.     

Effects of Pinus brutia bark extract and Pycnogenol® in a rat model of carrageenan induced inflammation

The present study was conducted to explore the anti-inflammatory activities of Pinus brutia bark extract and Pycnogenol^® in a rat model of carrageenan-induced inflammation. Firstly, the compositions of both samples were determined using HPLC. Then, carrageenan-induced paw edema was used to assess anti-inflammatory activity in mice. Paw volume was measured before and 1, 2, 3, 4, 5 and 6 h after the injection of carrageenan. Intraperitoneal administration of both the extract and Pycnogenol^® inhibited paw swelling dose-dependently at 2, 3, 4, 5 and 6 h after carrageenan injection. Both samples exhibited significant anti-inflammatory activities at doses of 75 and 100 mg/kg body wt. between 2 and 4 hours after administration (p<0.05), respectively. Additionally, P. brutia bark extract showed significantly better activity at doses of 75 and 100 mg/kg body wt. than indomethacine at the dose of 10 mg/kg body wt. (p<0.05). No acute toxicity was identified in intraplantar injection of the extract at a dose of 2000 mg/kg body wt.. Therefore, P. brutia bark extract possessing 3.3-fold more total catechins and 9.8-fold more taxifolin than Pycnogenol^® can be utilized as an anti-inflammatory agent.     

Non-suture repair of tendons

A set of metal clamps cooled by liquid nitrogen were developed to hold the ends of tendons during in vitro tension tests. These were then used to determine the strength of whole human tendons, and human tendons repaired in vitro by sutures or by cyanoacrylate glues with or without suturing. The mean ultimate load of the intact human tendons was 1065 N, and the ultimate stress was 99.02 MN m⁻². The repairs gave values of 0.3–2.3% of those of intact human tendon. Tendons were then repaired in vivo by a Kessler suture technique using braided polyester sutures with or without augmentation by a butyl cyanoacrylate glue (Histoacryl). At six months post-repair all tendons were as strong as the unoperated contralateral tendons. The glue augmented sutures were stronger than those not so augmented. A scanning electron micrograph of a tension tested glued tendon showed that it had failed by cohesive failure, i.e. failure of the inter-glue bonds rather than failure at the glue/tendon interface.     

Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM

The brilliant cresyl blue (BCB) test determines the activity of glucose-6-phosphate dehydrogenase (G6PDH); the activity of this enzyme is greatest in growing oocytes, but it declines as oocytes mature. The objective was to develop and evaluate this test for assessing development of buffalo oocytes (to select developmentally competent oocytes for increased in vitro embryo production). Oocytes were exposed to BCB stain diluted in mDPBS (DPBS with 0.4% BSA) for 90 min at 38.5 °C in a humidified air atmosphere; those with or without blue coloration of the cytoplasm were designated as BCB+ and BCB−, respectively. In Experiment 1, oocytes were exposed to 13, 26, or 39 μM BCB. There were fewer BCB+ oocytes after exposure to 13 μM BCB (10%) than after exposure to 26 or 39 μM BCB (57.2 and 61.8%; P < 0.05), but there was no significant difference among treatments for blastocyst production rate. In Experiment 2, the diameter of BCB+ oocytes (144.4 ± 4.2 μm; mean ± S.E.M.) was higher (P < 0.05) than that of BCB− oocytes (136.8 ± 4.6 μm). In Experiment 3, oocytes were allocated into three groups: control (immediately cultured); holding-control (kept in mDPBS for 90 min before cultured); and treatment-incubation (incubated with 26 μM BCB). After IVM, oocytes were fertilized in vitro and cultured on an oviductal monolayer. The nuclear maturation rate was higher (P < 0.05) in BCB+ (86.2%), control (83.4%) and holding-control (82.6%) oocytes than BCB− (59.2%) oocytes. The BCB+ oocytes yielded more blastocysts than control or holding-control oocytes (33.4, 20.2, and 21.0%, P < 0.05); blastocyst development was lowest in BCB− oocytes (5.2%). In conclusion, staining of buffalo oocytes with BCB before IVM may be used to select developmentally competent oocytes for increased in vitro embryo production.