Centrifugal force assessment in ram sperm: identifying species-specific impact.

BACKGROUND: Centrifugation is routinely employed in handling the ejaculates of some species, but it is not part of the commonly used protocols in ram. However, the development and implementation of new assisted reproductive technologies, alternative preservation models based on washing sperm from a cellular ageing-accelerating substance such as the seminal plasma, and basic studies in spermatology is associated with the use of centrifugation. This requires a specific evaluation of the centrifugation protocols considering the species-specific relationship with the potential damage produced by this procedure. No previous studies have determined the effect of different centrifugation forces on ram sperm. Therefore, we aimed to assess the performance of three centrifugal forces (600×g, 3000×g, and 6000×g for 10 min at room temperature) and their effects on ram sperm motility and functionality. RESULTS: Sperm motility and functionality parameters were assessed at 0 h and after 2 h of incubation at 37 °C. As expected, a higher cell packaging degree was obtained at high centrifugation forces (P ≤ 0.0001). Cell packaging was unstable at all centrifugal forces. Thus, there was a high cell resuspension rate after less than 2 min. Regarding sperm quality, there was a change in movement pattern of 3000×g and 6000×g centrifuged sperm after 2 h of incubation at 37 °C, characterized by an increase in rapid progressive motility, linearity, straightness, and beat frequency, and a decrease in medium progressive motility, curvilinear velocity, path velocity, and head lateral amplitude. Non-significant differences were obtained among the different treatments concerning the total viability. However, we observed a significant increase (P ≤ 0.05) in the percentage of viable apoptotic sperm in the samples centrifuged at 6000×g at 0 h. CONCLUSIONS: Centrifugal forces equal to or greater than 3000×g induced some deleterious effects in ram sperm quality, and lower forces did not provide a successful cell packaging degree.

Cytocentrifuge preparation in veterinary cytology: a quick, simple, and affordable manual method to concentrate low cellularity fluids.

BACKGROUND: Smears prepared by cytocentrifugation, the so-called cytospins, are widely used in human and veterinary cytology. However, the high cost has hampered the availability of commercial cytospin centrifuges in some veterinary clinics and laboratories. Nevertheless, cytospins are important for evaluating fluids with very low cellularity such as cerebrospinal fluid (CSF) or bronchoalveolar lavage fluid (BALF). OBJECTIVES: The aim of this study was to devise and test the use of a low-cost, in-house manual cytocentrifuge to obtain cytospin preparations. METHODS: Twenty-two fluid samples (including CSF and BALF) were collected from dogs and cats. These were processed in a conventional cytocentrifuge and in an in-house, manual centrifuge (salad spinner). The cytospins obtained by the 2 methods were compared by scoring cellularity, number of cells per field, hemodilution, cell preservation, and proportion of ruptured cells. Additionally, cell number and size were compared by morphometry. Differences between the automated and manual method were statistically assessed. RESULTS: The morphology and cellular detail of cytospin preparations produced by both methods were identical. There was an almost perfect agreement for cellularity, number of cells per HPF, hemodilution and cell preservation (kappa ≥ 0.85), and a moderate agreement for the amount of ruptured cells. Cell recovery was comparable (including in CSF and BALF). CONCLUSIONS: The manual cytocentrifuge produced cytospins with similar cell yield as the automated cytocentrifuge. Considering the low cost and portability, this new method should be particularly useful for cytologic diagnosis in small clinics, developing countries, and in field studies.

A simple centrifugation method for improving the detection of Ostreid herpesvirus-1 (OsHV-1) in natural seawater samples with an assessment of the potential for particulate attachment.

Ostreid herpesvirus-1 (OsHV-1) is responsible for massive mortality events in commercially farmed Pacific oysters (Crassostrea gigas) in Australia, New Zealand, Europe and the USA. Economic losses have been severe in many countries since 2008, associated with a strain known as OsHV-1µ-var. Despite intensive studies of the virus itself, there is almost no information on its detection in natural seawater, how it is spread over wide geographic distance in water or on how it is transmitted from oyster to oyster via seawater. The aim of the current work was to (1) assess and compare several centrifugation methods in order to detect OsHV-1 in natural seawater samples using real-time quantitative PCR, in such a way that large numbers of samples could be processed efficiently and (2) assess the potential for particulate attachment of OsHV-1 using filtration. Compared to testing unprocessed seawater samples, centrifugation of seawater at 1000×g for 20 min with testing of the pellet improved OsHV-1 detection rates by two fold. Results suggest that OsHV-1 may be attached to particles large enough to be pelleted at low g-force, as well as in the form of small particles, free virus or free viral DNA. Filtration of seawater using low protein binding filters could not be used to assess OsHV-1 particle attachment, due to interactions between particles, free virus or free viral DNA and the membranes.

Colloid centrifugation of boar semen.

Colloid centrifugation of boar semen has been reported sporadically for at least the last two decades, beginning with density gradient centrifugation (DGC) and progressing more recently to single layer centrifugation (SLC). Single layer centrifugation through a species-specific colloid has been shown to be effective in selecting the best spermatozoa (spermatozoa with good motility and normal morphology) from boar sperm samples. The method is easier to use and less time-consuming than DGC and has been scaled-up to allow whole ejaculates from other species, e.g. stallions, to be processed in a practical manner. The SLC technique is described, and various scale-up versions are presented. The potential applications for SLC in boar semen preservation are as follows: to improve sperm quality in artificial insemination (AI) doses for 'problem' boars; to increase the shelf-life of normal stored sperm samples, either by processing the fresh semen before preparing AI doses or by processing the stored semen dose to extract the best spermatozoa; to remove pathogens (viruses, bacteria), thus improving biosecurity of semen doses and potentially reducing the use of antibiotics; to improve cryosurvival by removing dead and dying spermatozoa prior to cryopreservation; to select spermatozoa for in vitro fertilization. These applications are discussed and practical examples are provided. Finally, a few thoughts about the economic value of the technique to the boar semen industry are presented.

Simple and economic colloidal centrifugation protocols may be incorporated into the clinical equine sperm processing procedure.

For many years in human assisted-reproduction procedures there have been special protocols to prepare and improve sperm quality. Colloidal centrifugation (CC) is a useful technique that has been proved to enhance semen quality by selection of the best spermatozoa for different species. Its use is recommended to improve fertility of subfertile stallions but current CC protocols are clinically complicated in the equine sperm processing technique due to economic and technical difficulties. The aim of this study was to determine the optimal processing procedures to adapt the use of a CC product (EquiPure™) in the equine reproduction industry. A total of nineteen ejaculates were collected from 10 Purebred Spanish Horses (P.R.E horses) using a Missouri artificial vagina. Gel-free semen aliquots were analyzed prior to treatment (control). Semen was subjected to one of six CC protocols with EquiPure™ and centrifuged samples were statistically evaluated by ANOVA and Duncan tests (p<0.05) for sperm quality and recovery rate. We obtained higher values by colloidal centrifugation in LIN, STR and BCF variables and DNA fragmentation index trended to be lower in most of the CC protocols. The studied protocols were shown to be as efficient in improving equine sperm quality as the current commercial EquiPure™, with the added advantage of being much more economical and simple to use. According to these results it seems to be possible to incorporate single layer and or high colloidal centrifugation volume protocols what would make them simple, economic and clinically viable for the equine sperm processing procedure.

Assessment of platelet growth factors in supernatants from rehydrated freeze-dried equine platelets and their effects on fibroblasts in vitro.

OBJECTIVE: To determine whether platelet growth factors are preserved in supernatants obtained from rehydrated trehalose-stabilized, freeze-dried (lyophilized) equine platelets and whether those growth factors stimulate fibroblast proliferation and migration and enhance fibroblast-associated contraction in a collagen gel assay. ANIMALS: 6 clinically normal adult horses. PROCEDURES: Blood samples were obtained from 6 horses, and washed platelets were prepared via differential centrifugation. Washed platelets were freeze-dried in a physiologic buffer with a mixture of trehalose and polyethylene glycol 4000. Rehydrated platelet supernatants and releasates prepared from fresh washed platelets stimulated with thrombin or platelet-activating factor were evaluated for transforming growth factor beta1 and platelet-derived growth factor-BB by use of ELISAs. Effects of rehydrated freeze-dried platelet supernatants on fibroblast proliferation, migration, and collagen gel contraction were compared with effects of 1%, 2.5%, or 10% fetal bovine serum (FBS). RESULTS: Supernatants from freeze-dried platelets contained similar amounts of growth factors as thrombin- and platelet-activating factor-stimulated platelet releasates. The supernatants significantly enhanced fibroblast proliferation and migration in a scratch assay, compared with FBS-free control or low (1%) FBS conditions. Additionally, supernatants from freeze-dried platelets enhanced contraction of fibroblast-seeded collagen gels, compared with the effect of 1% FBS. CONCLUSIONS AND CLINICAL RELEVANCE: The preparation technique preserved platelet growth factors, enhanced fibroblast proliferation and migration, and improved fibroblastseeded collagen gel contraction under conditions of low FBS concentration; these platelet supernatant preparations may prove useful as an aid to conventional wound management.