Unstable Stifles without Clinical or Radiographic Osteoarthritis in Young Goats: An Experimental Study

Thirteen young, castrated male goats had instability of one stifle (knee joint) created by surgical transection of the cranial cruciate ligament, but did not develop any signs of osteoarthritis (OA) in treated joints when confined in limited space for 8 months. At the end of the experiment, the instability in the stifles had not improved, the joints were normal at radiographic examination, there were no signs of inflammation in the synovial membrane or joint capsule, and fibrosis in these tissues was not evident. The articular cartilage was normal both visually and histologically. This may indicate that the young age of the goats and the restricted physical activity on soft floor had prevented the expected development of OA in the experimantally operated joints. Synovial fluid volumes and proteoglycan concentration were measured in the treated and control joints in 6 of the goats. There seemed to be increased quantity of the proteoglycan aggrecan in the synovial fluid from the treated joints compared to the contralateral joints throughout the course of this study. It was concluded that the turnover of aggrecan in the articular cartilage of the treated joints may have been increased.     

Fluid movement and joint capsule strains due to flexion in rabbit knees

Diarthrodial joints are freely moveable joints containing synovial fluid (SF) within a connective tissue joint capsule that allows for low-friction and low-wear articulation of the cartilaginous ends of long bones. Biomechanical cues from joint articulation regulate synoviocyte and cartilage biology via joint capsule strain, in turn altering the composition of SF. Joint flexion is clinically associated with pain in knees with arthritis and effusion, with the nociception possibly originating from joint capsule strain. The hypothesis of this study was that knee fluid volume distribution and joint capsule strain are altered with passive flexion in the rabbit model. The aims were to (a) determine the volume distribution of fluid in the joint at different total volumes and with flexion of rabbit knees ex vivo, (b) correlate the volume distribution for the ex vivo model to in vivo data, and (c) determine the strains at different locations in the joint capsule with flexion. During knee flexion, ～20% of anteriorly located joint fluid moved posteriorly, correlating well with the fluid motion observed in in vivo joints. Planar joint capsule principal strains were ～100% (tension) in the proximal-distal direction and ～-40% (shortening) in the circumferential direction, relative to the femur axis and 30° strain state. The joint capsule strains with flexion are consistent with the mechanics of the tendons and ligaments from which the capsule tissue is derived. The movement and mixing of SF volume with flexion determine the mechanical and biological fluid environment within the knee joint. Joint fluid movement and capsular strains affect synovial cell biology and likely modulate trans-synovial transport.     

Hyaluronic Acid in Synovial Fluid

Twelve horses with traumatic arthritis were treated with intraarticular injection of hyaluronic acid mixed with cortisone and the results compared with 6 horses treated only with cortisone. There was a significantly better improvement in the group injected with a mixture of hyaluronic acid and cortisone. Further studies have given the same results in traumatic arthritis in horses if hyaluronic acid alone is injected. After injection of hyaluronic acid a large number of granulated monocytes appeared in the synovial fluid, but no inflammatory signs were observed. It is possible that this macrophage invasion is instrumental in producing improvement in the condition of the joint. The injected hyaluronic acid may also adhere to the surface of articular cartilage producing an “clastic cushion” protecting the cartilage surface. Experimental mechanical damage was also inflicted on the surface of articular cartilage in dogs and monkeys, and smoother healing was achieved if hyaluronic acid was injected into the joints after the damage. Injections of hyaluronic acid seem to be of value in treating traumatic arthritis or similar conditions.     

LDH and LDH Isoenzymes of Synovial Fluid in the Horse

LDH is an intracellular enzyme, which when cells degenerate is released to the extracellular spaces and body fluids. Cells and organs in the mammalian body differ from each other with respect to their LDH isoenzyme patterns. These circumstances have led to the use of LDH isoenzyme determinations in laboratory diagnostic work. In the present investigation total LDH activity and LDH isoenzyme distribution in equine synovial fluid from healthy joints, joints with serous arthritis, osteochondrosis dissecans and arthrosis, were determined. The fluids from the diseased joints differed from normal synovial fluid with respect to total LDH activity, and the different joint diseases each seemed to give rise to a characteristic isoenzyme pattern. In order to examine possible sources of the increased LDH activity and altered isoenzyme patterns, blood plasma, red and white blood cells, synovial membrane and articular cartilage were also studied. It was found that LDH4 and LDH5 were present in high amounts in articular cartilage, and an increase in these isoenzymes was the most characteristic feature in synovial fluid from joints with arthrosis. The results were discussed in view of possible diagnostic value of isoenzyme determinations on synovial fluid.     

Viscosity of Equine Synovial Fluid

Synovial fluid samples from 51 light horses were examined with respect to their rhéologie properties. The analyses were made with a Rotovisco RV3 rotational viscosimeter. Samples from carpal, stifle and hock joints and from healthy joints, joints with synovitis and joints with infectious arthritis were studied. The analyses showed that synovial fluids from both healthy and diseased joints have complex rhéologie properties. In most samples the viscosity varied with the shear rates, the main exceptions being synovial fluids from joints with infectious arthritis. Flow curves (flow behaviour), rather than single observations on viscosity, were considered to be representative for the synovial fluids examined.     

Validation of equilibration and chromium reduction methods for deuterium measurements of fluid volumes.

Determinations of fluid volumes are of importance for correct treatment of patients subjected to shock and trauma. Gas isotope ratio mass spectrometry (GIRMS) is an advanced method for analysis of stable isotopes. These can be used as tracers for measurement of various fluid volumes. In the current in vitro study, deuterium was used to determine different volumes of water simulating a range of body fluid volumes from neonates to adults. A high-precision scale gave control weights (i.e., volumes), and two methods, equilibration (EQ) and chromium reduction (CR), were compared by use of a GIRMS. The coefficient of variation was <1% when using both EQ (0.45%) and CR (0.79%). The variability was greater at small volumes, and, when regression equations for the relation between measured and calculated volumes were used as formulas, the deviation was 0.4% using EQ and 2.8% using CR at the volume of 1,000 ml. At larger volumes, the deviation when using CR approached 1%. These variations are better than previously published data using other methods. It was concluded that GIRMS is a suitable technique for fluid volume determinations in neonates as well as in adult patients, using deuterium as a tracer. EQ and CR methods were both regarded to give acceptable variabilities in this in vitro study. GIRMS may in the future increasingly be used clinically for accurate measurements of body fluid volumes.     

Synovium Fragment-Derived Cells Exhibit Characteristics Similar to Those of Dissociated Multipotent Cells in Synovial Fluid of the Temporomandibular Joint

Multipotent mesenchymal stem cells (MSCs) found in the synovial fluid (SFMSCs) of the tempromandibular joint (TMJ) remain poorly understood. During TMJ arthrocentesis, we discovered that synovial fluid collected from some patients with TMJ disorders contained not only SFMSCs but also synovium fragments (SFs). In this study, we attempted to characterize both the SFMSCs and SF-derived cells (SFCs) in order to further understand the role of MSCs in the synovial fluid of the TMJ. The SFs were membranous and translucent and consisted of several cell layers, indicating that their origin was only from the intima. SFCs were obtained by digestion of the SFs and subsequently expanded in vitro. SFMSCs were enriched by centrifugation of the synovial fluid and expanded in vitro. SFCs and SFMSCs displayed a similar fibroblast-like, spindle-shaped morphology, and we observed that some SFMSCs grew out of small tissue masses in culture. Flow cytometric analysis showed that both groups of cells expressed similar surface markers, including CD90, CD44, CD105, and CD73. However, both were negative for Stro-1, CD146, CD45, CD34, CD11b, CD19, and HLA-DR. Immunofluorescent staining showed that both SFs and SFMSCs expressed vascular cell adhesion molecule 1. Both SFCs and SFMSCs could be induced to differentiate down osteogenic, chondrogenic, adipogenic, and neurogenic lineages in vitro. Together, our results indicate that the intima is the most likely tissue origin of SFMSCs in the TMJ. Moreover, the SFs are composed of only intima and thus offer an improved source of synovium-derived MSCs compared to synovium specimens obtained by surgery, which contain both intima and subintima.     

Hypobaric hypoxia (380 Torr) decreases intracellular and total body water in goats

The effects of prolonged hypoxia on body water distribution was studied in four unanesthetized adult goats (Capra lircus) at sea level and after 16 days in a hypobaric chamber [(380 Torr, 5,500 m, 24 +/- 1 degrees C); arterial PO2 = 27 +/- 2 (SE) Torr]. Total body water (TBW), extracellular fluid volume (ECF), and plasma volume (PV) were determined with 3H2O, [14C]inulin, and indocyanine green dye, respectively. Blood volume (BV) [BV = 100PV/(100 - hematocrit)], erythrocyte volume (RCV) (RCV = BV - PV), and intracellular fluid (ICF) (ICF = TBW - ECF) and interstitial fluid (ISF) (ISF = ECF - PV) volumes were calculated. Hypoxia resulted in increased pulmonary ventilation and arterial pH and decreased arterial PCO2 and PO2 (P less than 0.05). In addition, body mass (-7.1%), TBW (-9.1%), and ICF volume (-14.4%) all decreased, whereas ECF (+11.7%) and ISF (+27.7%) volumes increased (P less than 0.05). The decrease in TBW accounted for 89% of the loss of body mass. Although PV decreased significantly (-15.3%), BV was unchanged because of an offsetting increase in RCV (+39.5%; P less than 0.05). We conclude that, in adult goats, prolonged hypobaric hypoxia results in decreases in TBW volume, ICF volume, and PV, with concomitant increases in ECF and ISF volumes.     

Targeted Synovial Fluid Proteomics for Biomarker Discovery in Rheumatoid Arthritis

Objective  Rheumatoid arthritis (RA) is an autoimmune disease that targets the synovium. The autoantigens involved in the autoantibody responses in RA are unknown. A targeted proteomics approach was used to identify proteins in RA synovial fluid (SF) that are recognized by autoantibodies in RA sera. Methods  RA SF, depleted of abundant proteins, was fractionated by two-dimensional liquid chromatography (chromatofocusing followed by reverse phase HPLC). Protein arrays constructed from these fractions were probed with RA and normal control sera, and proteins within reactive fractions were identified by mass spectrometry. The reactivity of RA sera to an identified peptide was confirmed by ELISA. Results  RA sera specifically reacted to a SF fraction containing fibrin. Mass spectrometry analyses established the presence of a citrullinated arginine at position 271 of the fibrin fragment present in RA SF. A synthetic peptide corresponding to fibrin residues 259–287, containing the citrulline substitution at Arg 271, was recognized by 10 of 12 RA sera, but by two of 18 normal control sera and one of 10 systemic lupus erythematosus sera. Conclusion  Proteomics methodology can be used to directly characterize post-translational modifications in candidate autoantigens isolated from sites of disease activity. The finding that RA sera contain antibodies to the citrullinated fibrin 259–287 peptide may ultimately lead to improved diagnostic tests for RA and/or biomarkers for disease activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Relationship between Spinal Cord Volume and Spinal Cord Injury due to Spinal Shortening

Vertebral column resection is associated with a risk of spinal cord injury. In the present study, using a goat model, we aimed to investigate the relationship between changes in spinal cord volume and spinal cord injury due to spinal shortening, and to quantify the spinal cord volume per 1-mm height in order to clarify a safe limit for shortening. Vertebral column resection was performed at T10 in 10 goats. The spinal cord was shortened until the somatosensory-evoked potential was decreased by 50% from the baseline amplitude or delayed by 10% relative to the baseline peak latency. A wake-up test was performed, and the goats were observed for two days postoperatively. Magnetic resonance imaging was used to measure the spinal cord volume, T10 height, disc height, osteotomy segment height, and spinal segment height pre- and postoperatively. Two of the 10 goats were excluded, and hence, only data from eight goats were analyzed. The somatosensory-evoked potential of these eight goats demonstrated meaningful changes. With regard to neurologic function, five and three goats were classified as Tarlov grades 5 and 4 at two days postoperatively. The mean shortening distance was 23.6 ± 1.51 mm, which correlated with the d-value (post-pre) of the spinal cord volume per 1-mm height of the osteotomy segment (r = 0.95, p < 0.001) and with the height of the T10 body (r = 0.79, p = 0.02). The mean d-value (post-pre) of the spinal cord volume per 1-mm height of the osteotomy segment was 142.87 ± 0.59 mm³ (range, 142.19–143.67 mm³). The limit for shortening was approximately 106% of the vertebral height. The mean volumes of the osteotomy and spinal segments did not significantly change after surgery (t = 0.310, p = 0.765 and t = 1.241, p = 0.255, respectively). Thus, our results indicate that the safe limit for shortening can be calculated using the change in spinal cord volume per 1-mm height.     

Degeneration of normal articular cartilage induced by late phase osteoarthritic synovial fluid in beagle dogs

Objective

To investigate the pathogenesis of late phase osteoarthritic (OA) synovial fluid (SF) on normal articular cartilage in vivo and provide an understanding of degenerative cartilage extending in OA joint.

Methods

A random knee, each of 8 beagle dogs, received anterior cruciate ligament transection (ACLT) and was confirmed to have late phase OA degenerative changes at 24 weeks after operation. Thereafter, one random elbow of each canine was injected with autologous late phase OA knee SF. The contralateral elbow was injected with normal saline (NS) of the same volume as SF aspirated from ACLT knee. These two groups of elbows were labeled “SF” and “NS”. 8 other beagle dogs were left intact and placed in Group Control. After aseptic arthrocentesis was performed weekly on both elbows for 24 weeks, morphological changes were observed in the cartilage of the elbows, and expressions of 7 biological etiological factors of chondrocytes of the elbows were determined in Group SF, Group NS and Group Control, respectively.

Results

Morphological changes were observed in articular cartilage of the elbows in Group SF. Levels of unit area of collagen type I in the noncalcified, calcified and full zones of articular cartilage of the elbows in Group SF increased significantly. Level of unit area of collagen type III in the calcified zone of articular cartilage of the elbows in Group SF remained unchanged. Meanwhile, expressions of MMP-1 and MMP-3 of chondrocytes of the elbows in Group SF increased significantly. There was almost no difference between articular cartilage in Group NS and Group Control.

Conclusion

Based on these results, we conclude that OA degeneration of normal articular cartilage can be independently induced by late phase OA SF. Endogenous OA biological etiological factor may be one of the reasons causing degenerative cartilage extending in OA joint.     

Synergistic Effects of Ethanol and Isopentenyl Pyrophosphate on Expansion of γδ T Cells in Synovial Fluid from Patients with Arthritis

Low to moderate ethanol consumption has been associated with protective effects in autoimmune diseases such as rheumatoid arthritis, RA. An expansion of γδ T cells induced by isopentenyl pyrophosphate, IPP, likewise seems to have a protective role in arthritis. The aim of this project was to test the hypothesis that low doses of ethanol can enhance IPP-induced expansion of synovial fluid γδ T cells from patients with arthritis and may thereby potentially account for the beneficial effects of ethanol on symptoms of the arthritic process. Thus, mononuclear cells from synovial fluid (SF) from 15 patients with arthritis and from peripheral blood (PB) from 15 healthy donors were stimulated with low concentrations of ethanol and IPP for 7 days in vitro. IPP in combination with ethanol 0.015%, 2.5 mM, equivalent to the decrease per hour in blood ethanol concentration due to metabolism, gave a significantly higher fractional expansion of SF γδ T cells compared with IPP alone after 7 days (ratio 10.1+/−4.0, p<0.0008, n = 12) in patients with arthritis. Similar results were obtained for PB γδ T cells from healthy controls (ratio 2.0+/−0.4, p<0.011, n = 15). The augmented expansion of γδ T cells in SF is explained by a higher proliferation (p = 0.0034, n = 11) and an increased survival (p<0.005, n = 11) in SF cultures stimulated with IPP plus ethanol compared to IPP alone. The synergistic effects of IPP and ethanol indicate a possible allosteric effect of ethanol. Similar effects could be seen when stimulating PB with ethanol in presence of risedronate, which has the ability to increase endogenous levels of IPP. We conclude that expansion of γδ T cells by combinatorial drug effects, possibly in fixed-dose combination, FDC, of ethanol in the presence of IPP might give a protective role in diseases such as arthritis.     

A Comparative Study between Use of Arthroscopic Lavage and Arthrocentesis of Temporomandibular Joint Based on Computational Fluid Dynamics Analysis

Arthroscopic lavage and arthrocentesis, performed with different inner-diameter lavage needles, are the current minimally invasive techniques used in temporomandibular joint disc displacement (TMJ-DD) for pain reduction and functional improvement. In the current study, we aimed to explore the biomechanical influence and explain the diverse clinical outcomes of these two approaches with computational fluid dynamics. Data was retrospectively analyzed from 78 cases that had undergone arthroscopic lavage or arthrocentesis for TMJ-DD from 2002 to 2010. Four types of finite volume models, featuring irrigation needles of different diameters, were constructed based on computed tomography images. We investigated the flow pattern and pressure distribution of lavage fluid secondary to caliber-varying needles. Our results demonstrated that the size of outflow portal was the critical factor in determining irrigated flow rate, with a larger inflow portal and a smaller outflow portal leading to higher intra-articular pressure. This was consistent with clinical data suggesting that increasing the mouth opening and maximal contra-lateral movement led to better outcomes following arthroscopic lavage. The findings of this study could be useful for choosing the lavage apparatus according to the main complaint of pain, or limited mouth opening, and examination of joint movements.     

Influence of lung volume and left atrial pressure on reverse pulmonary venous blood flow

Infarction of the lung is uncommon even when both the pulmonary and the bronchial blood supplies are interrupted. We studied the possibility that a tidal reverse pulmonary venous flow is driven by the alternating distension and compression of alveolar and extra-alveolar vessels with the lung volume changes of breathing and also that a pulsatile reverse flow is caused by left atrial pressure transients. We infused SF6, a relatively insoluble inert gas, into the left atrium of anesthetized goats in which we had interrupted the left pulmonary artery and the bronchial circulation. SF6 was measured in the left lung exhalate as a reflection of the reverse pulmonary venous flow. No SF6 was exhaled when the pulmonary veins were occluded. SF6 was exhaled in increasing amounts as left atrial pressure, tidal volume, and ventilatory rates rose during mechanical ventilation. SF6 was not excreted when we increased left atrial pressure transients by causing mitral insufficiency in the absence of lung volume changes (continuous flow ventilation). Markers injected into the left atrial blood reached the alveolar capillaries. We conclude that reverse pulmonary venous flow is driven by tidal ventilation but not by left atrial pressure transients. It reaches the alveoli and could nourish the alveolar tissues when there is no inflow of arterial blood.     

Fluid and sodium loss in whole-body-irradiated rats

Whole-body and organ fluid compartment sizes and plasma sodium concentrations were measured in conventional, GI decontaminated, bile duct ligated, and choledochostomized rats at different times after various doses of gamma radiation. In addition, sodium excretion was measured in rats receiving lethal intestinal radiation injury. After doses which were sublethal for 3-5 day intestinal death, transient decreases occurred in all the fluid compartments measured (i.e., total body water, extracellular fluid space, plasma volume). No recovery of these fluid compartments was observed in rats destined to die from intestinal radiation injury. The magnitude of the decreases in fluid compartment sizes was dose dependent and correlated temporally with the breakdown and recovery of the intestinal mucosa but was independent of the presence or absence of enteric bacteria or bile acids. Associated with the loss of fluid was an excess excretion of 0.83 meq of sodium between 48 and 84 h postirradiation. This represents approximately 60% of the sodium lost from the extracellular fluid space in these animals during this time. The remaining extracellular sodium loss was due to redistribution of sodium to other spaces. It is concluded that radiation-induced breakdown of the intestinal mucosa results in lethal losses of fluid and sodium as evidenced by significant decreases in total body water, extracellular fluid space, plasma volume, and plasma sodium concentration, with hemoconcentration. These changes are sufficient to reduce tissue perfusion leading to irreversible hypovolemic shock and death.     

Th1-Induced CD106 Expression Mediates Leukocytes Adhesion on Synovial Fibroblasts from Juvenile Idiopathic Arthritis Patients

This study tested the hypothesis that subsets of human T helper cells can orchestrate leukocyte adhesion to synovial fibroblasts (SFbs), thus regulating the retention of leukocytes in the joints of juvenile idiopathic arthritis (JIA) patients. Several cell types, such as monocytes/macrophages, granulocytes, T and B lymphocytes, SFbs and osteoclasts participate in joint tissue damage JIA. Among T cells, an enrichment of classic and non-classic Th1 subsets, has been found in JIA synovial fluid (SF), compared to peripheral blood (PB). Moreover, it has been shown that IL-12 in the SF of inflamed joints mediates the shift of Th17 lymphocytes towards the non-classic Th1 subset. Culture supernatants of Th17, classic and non-classic Th1 clones, have been tested for their ability to stimulate proliferation, and to induce expression of adhesion molecules on SFbs, obtained from healthy donors. Culture supernatants of both classic and non-classic Th1, but not of Th17, clones, were able to induce CD106 (VCAM-1) up-regulation on SFbs. This effect, mediated by tumor necrosis factor (TNF)-α, was crucial for the adhesion of circulating leukocytes on SFbs. Finally, we found that SFbs derived from SF of JIA patients expressed higher levels of CD106 than those from healthy donors, resembling the phenotype of SFbs activated in vitro with Th1-clones supernatants. On the basis of these findings, we conclude that classic and non-classic Th1 cells induce CD106 expression on SFbs through TNF-α, an effect that could play a role in leukocytes retention in inflamed joints.     

Synovial autoreactive T cells in rheumatoid arthritis resist IDO-mediated inhibition

A hallmark of T cell-mediated autoimmunity is the persistence of autoreactive T cells. However, it remains to elucidate the manner in which synovial T cells are sustained in patients with rheumatoid arthritis (RA). We found that dendritic cells (DC) and tissues from the synovial joints of RA patients expressed higher levels of IDO than DC from healthy donors. Interestingly, T cells derived from the joint synovial fluid (SF) of RA patients proliferated in response to either autologous or allogeneic IDO-positive DC, an outcome that was not affected by the addition of IDO inhibitor 1-methyl-D-tryptophan (1-MT). In contrast, addition of 1-MT to the culture stimulated with allogeneic or autologous IDO-positive DC significantly enhanced the proliferation of T cells derived from peripheral blood of healthy donors or from peripheral blood of RA patients. Furthermore, we found that functionally active tryptophanyl-tRNA-synthetase (TTS) was significantly elevated in T cells derived from the SF of RA patients, leading to enhanced storage of tryptophan in T cells and to subsequent resistance to IDO-mediated deprivation of tryptophan. The RA SF enhancement of TTS expression in T cells was blocked by mAb to IFN-gamma and TNF-alpha. These results suggest that the resistance of T cells to IDO-mediated deprivation of tryptophan represents a mechanism by which autoreactive T cells are sustained in vivo in RA patients. Specifically, blocking of the up-regulation of TTS expression in T cells presents an avenue for development of a novel therapeutic approach to treatment of RA.     

Production of alloantibodies against caprine lymphocyte antigens

In all, 217 primiparous goats were each injected with blood cells from their own newborn kid. Eighty-five goats were given mononuclear cells, 61 were given leucocytes and 71 received whole blood. The goats were injected one, two or three times before collection of sera. Sera were also collected from 42 non-injected, primiparous goats. The sera were compared with regard to their potential value in class I histocompatibility typing. The percentage of potentially valuable sera was highest in the group of animals injected twice with whole blood (66 X 7%). However, this percentage was not significantly higher than the percentage in the group of animals injected once with whole blood (54 X 7%). It is concluded that injecting primiparous goats once with whole blood from their own newborn kid, is a rapid and easy method, which gives a high yield of alloantisera with potential value in class I histocompatibility typing.     

Assessment of a monoclonal antibody-based competitive enzyme linked immunosorbent assay (cELISA) for diagnosis of brucellosis in infected and Rev. 1 vaccinated sheep and goats

In this study a cELISA for the diagnosis of brucellosis due to B. melitensis in sheep and goats was evaluated and its capability of discriminating vaccinated from infected animals was assessed. Information is provided indicating that the cELISA has a diagnostic sensitivity (99.4%) and specificity (98.9%) in sheep and goats comparable to that of many standard indirect ELISA methods. In addition, the test proved able to distinguish between vaccinated and infected animals with an accuracy of up to 90% and results reproducibility of 93%. It was concluded that the cELISA could be useful for differentiation of Rev.1 vaccinated and naturally infected sheep and goats.     

Immunohistochemical localization of phospholipase A2 in the bovine seminal vesicle and on the surface of the ejaculated spermatozoa.

1. Approximately 150-fold purified phospholipase A2 (PLA2) from bovine seminal vesicle fluid was injected into rabbit to prepare antibodies. 2. Produced antisera blocked PLA2 activity in bovine seminal plasma, seminal vesicles and its fluid and it gave single precipitation lines with the same samples. No cross-reactivity was detected with other reproductive tissues of bull as well as human seminal plasma. 3. Using indirect peroxidase technique PLA2 was localized in the apical part of epithelia cells of the bull seminal vesicle and also some minor immunohistochemical reactions were observed in the tubular lumen. Indirect peroxidase staining gave weak or no reaction at all to seminal vesicles of immature bulls. This suggests that the enzyme may be under hormonal control. 4. By indirect immunofluorescence method ejaculated spermatozoa of bull revealed immunoreaction which was not uniform and it was restricted to the middle piece, acrosome as well as postacrosomal region, but no specific immunostaining could be found on the surface of the epididymal spermatozoa. 5. Enzyme visualization by immunoelectron microscopic labelling showed a predominant localization in membrane particles inside the lumen of bovine seminal vesicle but some gold particles were also seen in granules, larger vacuoles and in cytoplasm of epithelia cells.