Single step purification procedure for the rapid separation of equine leucocytes

Percoll gradients have been used to separate relatively pure populations of viable equine polymorphonuclear (PMN) and mononuclear (MN) cells. In preliminary studies, a continuous density gradient of 70% Percoll solution was used to separate two distinct leucocyte-rich bands. After measurement of the density of each band on the continuous gradient, discontinuous Percoll gradients, using 60% and 75% Percoll solutions, were used to provide a rapid means of separating PMN and MN cells. The yield of viable cells per ml of blood was 3.0 X 10(6) and 3.2 X 10(6) for MN and PMN cells, respectively. Corresponding values for recovery were 45% and 72%. The purity was 94% for PMNs and 99% for MNs.     

α‐6 Integrin Expression in Bovine Spermatogonial Cells Purified by Discontinuous Percoll Density Gradient

The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of α‐6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self‐renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two‐step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of α‐6 integrin by flow cytometry and real‐time RT‐PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two‐step enzymatic digestion. An average of 1 × 10⁵ viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding α‐6 integrin expression. Flow cytometry analysis demonstrated no differences in the α‐6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real‐time PCR analysis (p > 0.05). In addition to α‐6 integrin, the expression of GFRa‐1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to α‐6 integrin expression.     

Platelet activating factor as a mediator of equine cell locomotion

Equine polymorphonuclear (PMN) and mononuclear (MN) leucocytes were separated on Percoll gradients and used to study the chemoattractant properties of the polar ether-linked phospholipid, platelet activating factor (PAF). Six concentrations of PAF ranging from 1 ng/ml to 100 g/ml were studied in each of two in vitro assay systems, the agarose microdroplet and a microfilter technique. Very significant (p<0.01) increases in the movement of both PMN and MN cells were obtained with most concentrations of PAF. In two instances there was no apparent concentration-response relationship, although the action of PAF was approximately bell-shaped in two others. The possible significance of these findings for equine inflammatory conditions is discussed.     

Effect of Swim up and Percoll Treatment on Viability and Acrosome Integrity of Frozen–thawed Bull Spermatozoa

The aim of this study was to investigate the effect of the swim up and Percoll methods to select frozen–thawed bull spermatozoa with high quality membrane and acrosomal integrity and final concentration. Semen samples from six Holstein–Friesian bulls were examined. The whole experiment was repeated three times. Before and after both treatments, spermatozoa were subjected to a double‐staining method and evaluated by brightfield light microscope using 40× dry, or 100× oil immersion objectives. The concentration of spermatozoa evaluated by haemocytometer was 8.8 × 10⁷/ml after thawing, and the percentage of live cells with intact acrosome was 45.8%. Both treatments significantly increased the proportion of live spermatozoa compared with no treatment, and the use of Percoll gradient resulted in a significantly higher percentage of living cells with an intact acrosome (88.2%) than the swim up method (69.4%). The concentration of spermatozoa after Percoll separation (9.3 × 10⁶/ml) was higher than that after the swim up method (5.8 × 10⁶/ml). These results indicate that spermatozoa with a higher viability and acrosome integrity can be obtained by Percoll separation than by the swim up method. Therefore the use of Percoll‐treated spermatozoa in IVF systems can be more expedient.     

Simplified isolation and enrichment of spermatogonial stem-like cells from pubertal domestic cats (Felis catus)

The efficiency of spermatogonial stem cell (SSC) isolation and culture from pubertal donors is currently poor primarily, because of contamination with other testicular cells. This study aimed to purify SSC-like cells using different extracellular matrixes and a discontinuous gradient density. In experiment 1, testes (n=6) were analyzed for histology and SSC-related protein expressions (laminin, SSEA-4, DDX-4 and GFRα-1). After enzymatic digestion, the cell suspension was plated onto either a laminin- or gelatin-coated dish. The number of SSC-like cells was determined at 15, 30 and 60 min of culture (experiment 2). Experiment 3 was performed to test whether or not the additional step of Percoll gradient density centrifugation could really improve purification of SSC-like cells. Testicular histology revealed complete spermatogenesis with laminin expression essentially at the basal lamina of the seminiferous tubules. SSEA-4 and GFRα-1 co-localized with DDX-4 in the spermatogonia. The relative percentage of SSC-like cells, as determined by cells expressing SSEA-4 (59.42 ± 2.18%) and GFRα-1 (42.70 ± 1.28%), revealed that the highest SSC-like cell purity was obtained with the 15-min laminin-coated dish compared with other incubation times and gelatin treatment (P<0.05). Percoll treatment prior to laminin selection (15 min) significantly improved SSC-like cell recovery (91.33 ± 0.14%, P<0.001) and purity (83.82 ± 2.05% for SSEA-4 and 64.39 ± 1.51% for GFRα-1, P<0.05). These attached cells demonstrated a typical SSC-like cell morphology and also expressed POU5F1, RET and ZBTB16 mRNA. In conclusion, double enrichment with Percoll gradient density centrifugation and laminin plating highly enriched the SSC-like cells population.     

Isolation of Fasciola hepatica metacercariae by density gradients

Fasciola hepatica metacercariae were purified in high yield, removing contaminating cyst walls and plant material by step gradients consisting of 10 ml of 60% Percoll (density = 1.08 g ml-1) and 10 ml of 50% Metrizamide (density = 1.25 g ml-1). Greater than 90% of the metacercariae applied to the density gradients were recovered. These isolated metacercariae had an in vitro excystment rate of greater than 80%, which was the same excystment rate as metacercariae not subjected to density gradient centrifugation.     

Isolation of equine bone marrow‐derived mesenchymal stem cells: a comparison between three protocols

Reason for performing the study: There is a need to assess and standardise equine bone marrow (BM) mesenchymal stem cell (MSC) isolation protocols in order to permit valid comparisons between therapeutic trials at different sites. Objective: To compare 3 protocols of equine BM MSC isolation: adherence to a plastic culture dish (Classic) and 2 gradient density separation protocols (Percoll and Ficoll). Materials and methods: BM aspirates were harvested from the sternum of 6 mares and MSCs isolated by all 3 protocols. The cell viability after isolation, MSC yield, number of MSCs attained after 14 days of culture and the functional characteristics (self‐renewal (CFU) and multilineage differentiation capacity) were determined for all 3 protocols. Results: The mean ± s.d. MSC yield from the Percoll protocol was significantly higher (6.8 ± 3.8%) than the Classic protocol (1.3 ± 0.7%). The numbers of MSCs recovered after 14 days culture per 10 ml BM sample were 24.0 ± 12.1, 14.6 ± 9.5 and 4.1 ± 2.5 × 10 ⁶ for the Percoll, Ficoll and Classic protocols, respectively, significantly higher for the Percoll compared with the Classic protocol. Importantly, no significant difference in cell viability or in osteogenic or chondrogenic differentiation was identified between the protocols. At Passage 0, cells retrieved with the Ficoll protocol had lower self‐renewal capacity when compared with the Classic protocol but there was no significant difference between protocols at Passage 1. There were no significant differences between the 3 protocols for the global frequencies of CFUs at Passage 0 or 1. Conclusions and clinical relevance: These data suggest that the Percoll gradient density separation protocol was the best in terms of MSC yield and self‐renewal potential of the MSCs retrieved and that MSCs retrieved with the Ficoll protocol had the lowest self‐renewal but only at passage 0. Then, the 3 protocols were equivalent. However, the Percoll protocol should be considered for equine MSC isolation to minimise culture time.     

Failure of Lipopolysaccharides to Directly Trigger the Chemiluminescence Response of Isolated Equine Polymorphonuclear Leukocytes

Divergent results have been reported on the effects of lipopolysaccharides (LPS) on the activation of equine polymorphonuclear leukocytes (PMN). We therefore attempted to determine whether LPS alone can stimulate equine PMN or whether plasma factors are necessary. PMN were isolated from citrated blood on a discontinuous density gradient of Percoll. The luminol (10-3 mol/L)-enhanced chemiluminescence (CL) of 1.25 x 106 cells was measured after addition of Escherichia coli LPS (0.001-10 µg/ml) alone or after incubation in autologous plasma (1 h, 37°C). After direct stimulation with LPS, there were random variations of CL in 16 horses that were not reproducible from one sample to the next for the same horse. LPS which had been incubated in plasma gave a dose-dependent stimulation of the CL of the PMN, which did not occur if the plasma had been heat inactivated (1 h, 56°C). These results indicated a role for plasma factors, which were unlikely to be cytokines, as there were no monocytes or lymphocytes in the plasma incubated with LPS, but might have been complement fragments or LPS ligands, such as LPS binding protein. Studies using specific antibodies against these factors are needed to clarify this question.     

The purification of sporocysts and sporozoites from Eimeria tenella oocysts using Percoll density gradients

A protocol is presented for the purification of sporozoites from sporulated oocysts of Eimeria tenella. Two Percoll density gradients are the basis of the purification. The first gradient is used after glass-bead grinding to purify undamaged sporocysts; 87% of the sporocysts loaded onto the gradient were recovered in the pellet. The second gradient is used after excystation to purify sporozoites; 98% of the sporozoites loaded onto the gradient were recovered in the pellet. The sporozoites are 99% pure with a final recovery of about three sporozoites per oocyst.     

Isolation of bovine polymorphonuclear leukocytes by density gradient centrifugation

A method for the isolation of an enriched population (greater than 95%) of bovine polymorphonuclear leukocytes (PMNs) was developed using density gradient centrifugation. Leukocytes were isolated from peripheral blood by centrifugation in a density gradient medium (Percoll) of specific gravity 1.092. Viability was greater than or equal to 95% and the isolated PMNs were functional in migration inhibition and chemiluminescence assays. This has proved to be a simple effective method for obtaining bovine PMNs and yields cell populations that can be utilized for a variety of measures of PMN function.     

Effect of density gradient composition on in vitro maturation of stallion sperm

The present study was conducted to determine the influence of density gradient composition on in vitro capacitation of stallion spermatozoa. In Experiment I spermatozoa were isolated on either 90% Percoll (P), 90% arabinogalactan (AG), or 12% BSA gradients and then challenged with 1 μM A23187 (15, 150, and 270 min) and heat-solubilized equine zonae pellucidae (270 min, sEZP, 2 ZP/μl). The P gradient enhanced the percentage of progressively motile spermatozoa (PMS) more (P=.0001) than AG or BSA gradients immediately post-processing, but was not sustained throughout the culture period. The viability for P-separated spermatozoa was higher (P=.01) than that of BSA or AG-separated spermatozoa. Gradient composition had no effect (P=.68) on the percentage of live, acrosome reacted spermatozoa (PAR), before and following ionophore and sEZP challenge. In Experiment II, the number of spermatozoa penetrating the ZP of saltstored oocytes was not influenced (P=.35) by gradient composition; however, the number of spermatozoa bound per oocyte was higher (P=.02) for P-separated spermatozoa than for AG or BSA-separated spermatozoa. These data suggest that isolation on a P density gradient may enhance in vitro capacitation of stallion spermatozoa.     

Effects of Cholesterol on Progesterone Production by Goat Luteal Cell Subpopulations at Two Different Stages of the Luteal Phase

The aim of the present study was to evaluate the effects of cholesterol on progesterone production during long‐term culturing of luteal cell subpopulations at early and late luteal stages of the goat corpora lutea. Corpora lutea were collected from Angora goats on days 5 and 15 of the oestrous cycle. Luteal cells were isolated by collagenase digestion. The cells were separated into two distinct subpopulations by Percoll density‐gradient centrifugation. Both subpopulations of luteal cells staining positively for 3β‐HSD activities (5 × 10⁴ cell/well) were cultured with or without 22(R)‐hydroxycholesterol (22R‐HC) in serum‐free culture medium for periods of up to 7 days. Cells were incubated with serum (10%) for the first 18 h of incubation followed by serum‐free medium. Cell treatment (10 and 20 μg/ml) was performed on days 1, 3 and 5. Treatment of cells with both concentrations of 22R‐HC resulted in significant (p < 0.01) and dose‐dependent stimulation (p > 0.05) on progesterone production in both fractions of cells throughout 7 days of incubation. Treatment of the cells with cholesterol resulted in 2.5‐ and 9.0‐fold increases in progesterone accumulation on day 3 of incubation. Steroid production was maintained throughout the incubations when cells are incubated in serum‐free media treated with cholesterol and ITS premix. Cells collected from higher density of percoll layers produced 2.82 and 2.32 times more progesterone, in comparison to the lover density percoll layer, on days 5 and 15 of the oestrous cycle in untreated cell groups, respectively. Progesterone accumulation was decreased as incubation time advanced in all groups of untreated cells. These results demonstrated that goat luteal cell subpopulations secrete substantial amounts of progesterone in response to cholesterol treatment at least for 7 days, and cholesterol is required as progesterone precursor for maintaining a high‐level steroidogenesis during long‐life culturing of both cell subpopulations.     

Influence of Inseminate Components on Porcine Leucocyte Migration In Vitro and In Vivo after Pre- and Post-Ovulatory Insemination

A post‐breeding migration of leucocytes (PMN) into the uterus is considered to be an important reason for sperm losses. Minimizing such effects may be necessary for successful insemination with low sperm numbers, as required with sex‐sorted spermatozoa. We examined the magnitude of PMN influx 3 h after pre‐ or post‐ovulatory insemination with various combinations of seminal plasma (SP), semen extender Androhep? (AH; Minitüb, Tiefenbach, Germany) and sperm preparations (S). Pre‐ovulatory inseminations with preparations containing 98% AH caused a massive influx of PMN, independent of whether spermatozoa were present (628 ± 189 × 10⁶ leucocytes/uterine horn) or not (580 ± 153 × 10⁶). Post‐ovulatory, 98% AH caused a comparable immigration only in the absence of sperm cells (AH: 569 ± 198 × 10⁶, AH+S: 162 ± 102 × 10⁶). The presence of SP significantly dampened the numbers of recruited uterine leucocytes. The reaction to all inseminates containing 98% SP both with and without spermatozoa, used before ovulation (SP: 14 ± 6 × 10⁶, SP+S: 73 ± 27 × 10⁶) and after ovulation (SP: 60 ± 32 × 10⁶, SP+S: 51 ± 33 × 10⁶) did not differ significantly from controls using phosphate buffered saline (PBS) (pre‐ovulatory: 1 ± 1 × 10⁶, post‐ovulatory: 11 ± 9 × 10⁶). Quantitative in vitro transmigration assays with blood‐derived PMN proved that AH‐induced leucocyte migration into the uterus to be not as a result of direct chemotaxis, because, on account of the chelator citrate, AH significantly inhibited the transmigration towards recombinant human Interleukin‐8 (rhCXCL8) (AH: 14 ± 5% migration rate vs controls: 37 ± 6%, p < 0.05). Supernatants of spermatozoa incubated in PBS for 1, 12 or 24 h showed neither chemoattractive nor chemotaxis‐inhibiting properties. SP at ≥0.1% [v/v] significantly inhibited the in vitro transmigration of PMN. With respect to in vivo migration of neutrophils, the striking difference in the results between semen extender and seminal plasma suggests that adaptation of extender composition is needed to reflect more closely the in vivo regulatory potential of natural seminal plasma.     

Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid

The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- × 0.5-mL straws (200 × 10⁶/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 × 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 × 10⁶ ± 21, 59 × 10⁶ ± 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination.     

Growth of Chlamydia in pig lung alveolar macrophages; Preparation of macrophages and demonstration of growth

The harvesting, storing and utilisation in tissue culture of lung alveolar macrophages from 2–3 weeks old piglets were investigated. An average yield of 4×10⁸ cells was obtained, which in culture usually remained healthy for at least 8 days. In liquid nitrogen at –173°C about 50% of the cells were still viable after 3 months. Pig cells sustained the growth of ovineChlamydia psittaci and a rising titre was demonstrated over 4 passages.     

Isolation and cryopreservation of functionally competent equine leucocytes

Sufficient numbers of functionally competent polymorphonuclear neutrophil granulocytes (PMN) seem to be of major importance during the course of equine endometritis. In this study, we wanted to establish a method for cryopreservation of functionally competent neutrophils for an intended local endometritis therapy in mares. The separation of leucocytes by hypotonic lysis of whole blood from clinically healthy mares was superior to the separation by dextrose sedimentation. After suspension of the cells in the cryoprotective solution [equine plasma with 5% (v/v) dimethyl sulphoxide (DMSO)], the leucocytes were frozen in liquid nitrogen. A temperature gradient with low cooling velocity (1 degree C/min between 4 and -70 degrees C) resulted in highest numbers of viable cells after thawing. Thawed PMN had a high phagocytic capacity for opsonized streptococci. Their ability to generate reactive oxygen species (ROS) after stimulation with a phorbol ester was even higher than that of freshly isolated PMN and was preserved up to 6 h after thawing. The results of this study indicate that cryopreservation of PMN may provide viable and functionally competent neutrophils for therapeutic use in mares susceptible to endometritis.     

Evaluation In Vitro of Canine Neutrophil Function

Polymorphonuclear granulocyte (PMN) phagocytosis may be affected by many pathological changes. A panel of tests requiring relatively small volumes of blood was applied to 16 healthy dogs in order to obtain normal values and to standardize techniques. PMNs were isolated by discontinuous Percoll gradients; chemotaxis was tested in a modified Boyden chamber using the leading front method; fluorescinated yeast uptake was evaluated on a slide and superoxide (SO) production and adherence was carried out on a microtitre plate. The different aspects of phagocytosis showed no correlation with one another. Better results were obtained using a 60 min incubation period using interleukin-8 (25 ng/mL) as an activator for chemotaxis, and incubating plates for 30 min with phorbol myristate acetate (10(-6)mol/L) to assess SO production.     

Application of a novel sorting system for equine mesenchymal stem cells (MSCs)

The objective of this study was to validate non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs) into subpopulations, for use with MSCs derived from equine muscle tissue, periosteal tissue, bone marrow, and adipose tissue. Cells were collected from 6 young, adult horses, postmortem. Cells were isolated from left semitendinosus muscle tissue, periosteal tissue from the distomedial aspect of the right tibia, bone marrow aspirates from the fourth and fifth sternebrae, and left supragluteal subcutaneous adipose tissue. Aliquots of 800 × 10³ MSCs from each tissue source were separated and injected into a ribbon-like capillary device by continuous flow (GrFFF proprietary system). Cells were sorted into 6 fractions and absorbencies [optical density (OD)] were read. Six fractions from each of the 6 aliquots were then combined to provide pooled fractions that had adequate cell numbers to seed at equal concentrations into assays. Equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells were consistently sorted into 6 fractions that remained viable for use in further assays. Fraction 1 had more cuboidal morphology in culture when compared to the other fractions. Statistical analysis of the fraction absorbencies (OD) revealed a P-value of < 0.05 when fractions 2 and 3 were compared to fractions 1, 4, 5, and 6. It was concluded that non-equilibrium GrFFF is a valid method for sorting equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells into subpopulations that remain viable, thus securing its potential for use in equine stem cell applications and veterinary medicine.     

Purification of Cowdria ruminantium by density gradient centrifugation

The isolation of Cowdria ruminantium by differential and isopycnic density gradient centrifugation is reviewed with special reference to the suitability of Percoll as density gradient medium. Infected sheep brain, Amblyomma hebraeum nymphae and various mouse organs were used as starting material. By these methods, partially purified viable populations of the organism with distinctly different densities were obtained. The conclusions are based upon results of analyses of density fractions by inoculation into sheep or mice, protein determination, electron microscopy and enzyme-linked immunosorbent assay. Morphological differences were observed in the density fractions obtained from infected brain tissue and A. hebraeum.     

Separation of equine bronchopulmonary lavage cells by density gradient centrifugation and expression of procoagulant activity in unpurified cells and cell subpopulations

Bronchopulmonary lavage was performed in 10 healthy horses and in 39 horses with chronic pulmonary disease. The predominant cell types were macrophages in healthy horses and neutrophils in severely diseased horses. Procoagulant activity (PCA) was detected in all 32 cell-free supernatants examined and in all 49 unpurified cell suspensions. Cells were separated by centrifugation on discontinuous gradients prepared either with Percoll or with Metrizamide. Macrophages were enriched in subpopulations of low density. Neutrophils could not be purified by density gradient centrifugation using either gradient medium. PCAs of cell subpopulations were plotted against their respective macrophage, neutrophil, and lymphocyte content. PCA was positively correlated with macrophage content (P less than 0.001) and negatively correlated with neutrophil (P less than 0.02) and lymphocyte (P less than 0.001) content. Therefore, PCA of equine lung cells most likely originates from macrophages as shown in other species. The density shift of lung neutrophils requires further investigation.