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Glass wool column filtration (GWCF) selects human, bull, boar, dog and buffalo spermatozoa, but reports in the horse are scarce. Single-layer colloid centrifugation with Androcoll-E™ is currently the standard procedure to select good-quality equine sperm. This study was designed to assess GWCF (50 and 75 mg columns; GWCF-50 and GWCF-75, respectively) efficacy to select good-quality sperm from fresh and frozen-thawed equine semen, and to compare its performance with Androcoll-E™ colloid centrifugation. Percentage total motile (TM), progressively motile (PM), morphologically normal (MN), osmotically competent (HOS+) and acrosome-intact/osmotically competent (AI/HOS+) sperm were determined. In studies done with fresh semen samples (n = 17), suspensions subjected to GWCF-50 showed an improvement (p < .05) in PM and HOS+ sperm after selection. With GWCF-75, an increase (p < .05) in PM, MN and HOS+ sperm was observed. Results with GWCF were comparable or better than with Androcoll-E™ selection. Sperm recovery was similar between procedures for all semen parameters. Total sperm count recovery was lower after GWCF-75 (GWCF-50 = 60.0; GWCF-75 = 51.0; Androcoll-E™ = 76.0 million sperm; median; p = .013), but results on total progressive sperm count were similar (GWCF-50 = 23.0; GWCF-75 = 27.0; Androcoll-E™ = 24.0 million sperm; median; p = .3850). Using frozen-thawed semen samples (n = 16), an improvement (p < .05) in TM, PM, NM, HOS+ and AI/HOS+ sperm was observed in GWCF-75 filtrates. Results were comparable to Androcoll-E™ centrifugation, except HOS+ that increased (p < .05) only after GWCF-75. Recovery was comparable for all parameters in frozen samples. GWCF is a simple and low-cost procedure that selects equine sperm with a quality comparable to colloid centrifugation with Androcoll-E™.

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Platelet-rich plasma (PRP) has been proposed as an agent to accelerate the healing process and stimulate the regenerative capacity of tissues due to its abundance of growth factors. A large variety of kits and protocols are available to obtain PRP by different cell-separation systems. However, the lack of standardization may lead to inconsistent results. The aim of this study was to characterize cellular composition, platelet parameters using the ADVIA 120 flow cytometer, and TGF-ß1 concentration from the PRP product obtained through a closed system, using simple centrifugation. Six clinically healthy horses were used in this study. The protocol in the closed system resulted in approximately 1.6-fold higher platelet and approximately 2.0-fold lower white blood cell concentrations in comparison with whole blood values. The evaluated system was efficient in concentrating platelets and in retrieving a small number of leukocytes, using a protocol of single centrifugation at low speed.

O plasma rico em plaquetas (PRP) tem sido proposto como um agente para acelerar o processo de cicatrização e estimular a capacidade regenerativa dos tecidos, devido à sua abundância de fatores de crescimento. Uma grande variedade de kits e de protocolos está disponível para obtenção de PRP, utilizando-se diferentes sistemas de separação de células. No entanto, a falta de padronização pode levar a resultados inconsistentes. O objetivo deste estudo foi caracterizar a composição celular, os parâmetros plaquetários pelo citômetro de fluxo ADVIA 120 e a concentração de TGF-ß1 do PRP obtido em sistema fechado, por centrifugação simples. Seis cavalos clinicamente saudáveis ​​foram usados ​​neste estudo. O protocolo no sistema fechado resultou em concentrações de plaquetas aproximadamente 1,6 vez maior e concentrações de leucócitos aproximadamente 2,0 vezes menores em comparação com os valores do sangue total. O sistema avaliado foi eficiente na concentração de plaquetas e na recuperação de um pequeno número de leucócitos, utilizando um protocolo de centrifugação única em baixa velocidade.

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Accelerated healing in wild or captive South American sea lions (Otaria flavescens) is a key tool to help minimize infection and complications associated with open wounds, dental disease, and ocular pathology. Platelet-rich plasma (PRP) is an autogenous source for growth factors based on platelet concentration, which can be obtained by centrifuging whole blood collected in sodium citrate anticoagulant. Currently, there are well-defined PRP concentration protocols for humans and most domestic companion animal species. However, there is no clear centrifugation protocol for obtaining PRP in most marine mammal species. This study aimed to optimize the platelet concentration protocol based on whole blood centrifugation using speeds ranging from 500 to 5,000 rpm and times ranging from 3 to 6 min. Blood was drawn from seven adult South American sea lions, placed into 1-ml sodium citrate tubes, and centrifuged following 12 different centrifugation protocols. PRP was designated as the lower third fraction of the centrifuged plasma. Platelet counts were performed using flow cytometry and statistical analysis was carried out to establish a well-defined protocol for efficient PRP production. Transmission electron microscopy (TEM) analysis was performed to evaluate possible platelet degranulation during the different centrifugation protocols and measure platelet areas. Maximum concentration of platelets in PRP was 4.73-fold higher than the number of platelets in equal volume of whole blood, and significant differences in the concentrations obtained were found between the 12 centrifugation protocols evaluated using different speed and time combinations. The best one-step centrifugation protocol resulted from using 900-rpm speed for 3 min. The highest-fold increase was achieved using a two-step centrifugation protocol, which combined the most efficient one-step centrifugation protocol (900 rpm, 3 min) with a second centrifugation using 2,000-rpm speed for 6 min. TEM analysis confirmed that platelets were complete and maintained integrity after the proposed protocol.

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In semen cryopreservation, egg yolk is still widely used as a non-penetrating cryoprotectant. Much has been developed in the search for alternatives for this biological product. This work aimed to evaluate the processed egg yolk through ultracentrifugation and/or sonication in the cryopreservation of swine semen. Twenty-seven semen doses were purchased from a commercial boar stud and processed for cryopreservation using egg yolk lactose 11% (control) extender, processed using two different methods: high-speed centrifugation and sonication. Then, they were submitted to freeze-thawing protocol and were assessed for kinematic and cell structural parameters. Samples in which extenders underwent centrifugation had better results in velocity parameters, meanwhile those that only sonication was performed had poorest results in this parameter. The preservation of the membrane and mitochondria structure had better results when the diluent was only centrifuged in comparison with the other treatments. Therefore, centrifugation of extender containing egg yolk is important for better cryopreservation of swine semen.

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Separation techniques of seminal plasma [centrifugation (SC) and Sperm Filter® (SF)] and sperm selection [Androcoll-E (SCA) and filtration glass wool (GW)] were used in 24 ejaculates from 6 stallions. In experiment 1, the ejaculates were allocated into control (no spin), centrifugation at 600 g x 10min, SF and GW. In experiment 2, semen was submitted to SC, SGA and filtered through GW. Following the treatments in both experiments, samples were kept chilled at 5°C to 50 x 106 sperm/ml for 48h. The variables measured on fresh and cooling semen were pH, motility, membrane viability function by 6-carboxyfluorescein diacetate and propidium iodide (CFDA / PI), viability or vitality (eosin / nigrosine) and mitochondrial activity. In experiment 1, centrifugation to remove seminal plasma resulted in greater damage to sperm than separation by sperm filter, and selection by glass wool was more efficient in separating viable cells and maintaining viability during cooling. In experiment 2 Androcoll-E and glass wool treatments resulted in higher (P <0.0001) motility, membrane function, mitochondrial activity, and viability than centrifuged semen. Both selection by Androcoll- E and glass wool improved the quality of semen pony stallions for preservation for up to 48h to 5ºC.(AU)

As técnicas de separação do plasma seminal (centrifugação, SpermFilter) e de seleção espermática (Androcoll-E e filtração por lã de vidro) foram aplicadas em 24 ejaculados de seis garanhões da raça Pônei Brasileiro. Após coleta e separação da fração gel, os ejaculados foram diluídos 1:1 com diluente à base de leite em pó. No experimento 1, os ejaculados foram distribuídos em controle (sem centrifugação), centrifugação a 600g x 10min, SpermFilter e filtração por lã de vidro. No experimento 2, o sêmen foi submetido aos procedimentos: centrifugado (SC), centrifugado com Androcoll-E e filtrado por lã de vidro. Após os procedimentos de ambos os experimentos, as amostras foram mantidas refrigeradas a 5ºC, com 50 x 106 espermatozoides/mL, por 48h. As variáveis mensuradas a fresco, 24h e 48h foram: pH, motilidade, funcionalidade de membrana, viabilidade por diacetato de carboxifluoresceína e iodeto de propídio (CFDA/PI, vitalidade (eosina/nigrosina) e atividade mitocondrial. Já osmolaridade e morfologia espermática foram avaliadas somente imediatamente após a coleta. No experimento 1, a centrifugação para retirada do plasma seminal resultou em maiores danos aos espermatozoides do que a separação por SpermFilter. A filtração por lã de vidro mostrou-se mais eficiente em separar células viáveis e manter a viabilidade durante o resfriamento. No experimento 2, os tratamentos com Androcoll-E e filtrado por lã de vidro foram superiores (P<0,0001) ao sêmen centrifugado quanto à motilidade, à funcionalidade de membrana, à atividade mitocondrial e à viabilidade, tanto nas amostras de sêmen fresco como de sêmen refrigerado. O Androcoll-E e a lã de vidro permitiram manter por 48h, a 5ºC, o sêmen de garanhões pôneis utilizando-se diluente à base de leite.(AU)

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The objective of this study was to design a protocol to separate spermatozoa from seminal plasma of raw llama semen without prior enzymatic treatment using a single-layer centrifugation with Androcoll-E™ (AE). Two experiments were performed: (a) samples were divided into three aliquots (1 ml) that were deposited on the top of 4, 5 or 6 ml of AE and were centrifuged at 800g for 20 min and (b) samples were divided into two aliquots (1 ml) that were deposited on the top of 4 ml of AE and were centrifuged at 600g or 1,000g for 20 min. Columns of 5 and 6 ml of AE showed a total sperm motility (TM) significantly lower, while in the 4 ml column, this parameter was not different from the TM of samples before the AE treatment. The percentage of spermatozoa with intact and functional membranes, normal morphology and intact acrosomes, as well as the percentages of sperm with highly condensed chromatin, was conserved (p Ëƒ .05) in the three column heights and in the two centrifugation speeds evaluated. In conclusion, the different column heights of AE (4, 5 and 6 ml) and the different centrifugation speeds used (600, 800 and 1,000g) allow separating spermatozoa of raw llama semen without enzymatic treatment, preserving the evaluated sperm characteristics. However, of all the studied treatments, centrifugation in the 4 ml column of AE at 800g would be the method of choice to process raw llama semen samples destined for reproductive biotechnologies.

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Background: Canine distemper has been classified as highly contagious for most of domestic and wild carnivores, and theinfection can be fatal. Canine distemper inclusion bodies, also denominated Lenz inclusion bodies, are large aggregatesof viral nucleocapsid particles that can be form in red blood cells (RBCs), white blood cells (WBCs) and epithelial cellsin many tissues during the acute phase of infection. Their presence in blood is transient and rarely encountered in lightmicroscopy but are pathognomonic when identified in blood smears. The objective of this study was to investigate thefrequency of distemper inclusions in erythrocytes according to the fraction of the sample used for blood smears.Materials, Methods & Results: The study was conducted with routine blood sample provided by the Veterinary Laboratory of Clinical Analysis from the Veterinary Teaching Hospital of Universidade Federal do Rio Grande do Sul. TheEDTA-K2 blood sample of a 40 days old male dog, mixed breed, no immunization records, presenting diarrhea, hyporexia,myoclonus and pustules in the abdomen, was selected. In a routine peripheral blood smear examination, several distemperinclusions were observed in the erythrocytes. From this sample, ten smears were performed using a whole blood (WB)and top erythrocyte fraction combined with buffy coat, denominated of expanded buffy coat (EBC). The EBC fraction wasobtained after centrifugation of EDTA whole blood in microhematocrit tubes at 9600 g for 5 min to obtained the packedcell volume (PCV) and buffy coat. After centrifugation, the blood cells are separated into three layers based on density:platelets (adjacent to supernatant), WBCs, and RBCs in the bottom. The PCV was measured and the microhematocrit tubewas ruptured 2% below the interface between leukocytes and plasma, deposited into a plastic microtubes, homogenized andused for blood smear preparation. All smears were stained with Diff-Quick Stain...(AU)

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Background: Canine distemper has been classified as highly contagious for most of domestic and wild carnivores, and theinfection can be fatal. Canine distemper inclusion bodies, also denominated Lenz inclusion bodies, are large aggregatesof viral nucleocapsid particles that can be form in red blood cells (RBCs), white blood cells (WBCs) and epithelial cellsin many tissues during the acute phase of infection. Their presence in blood is transient and rarely encountered in lightmicroscopy but are pathognomonic when identified in blood smears. The objective of this study was to investigate thefrequency of distemper inclusions in erythrocytes according to the fraction of the sample used for blood smears.Materials, Methods & Results: The study was conducted with routine blood sample provided by the Veterinary Laboratory of Clinical Analysis from the Veterinary Teaching Hospital of Universidade Federal do Rio Grande do Sul. TheEDTA-K2 blood sample of a 40 days old male dog, mixed breed, no immunization records, presenting diarrhea, hyporexia,myoclonus and pustules in the abdomen, was selected. In a routine peripheral blood smear examination, several distemperinclusions were observed in the erythrocytes. From this sample, ten smears were performed using a whole blood (WB)and top erythrocyte fraction combined with buffy coat, denominated of expanded buffy coat (EBC). The EBC fraction wasobtained after centrifugation of EDTA whole blood in microhematocrit tubes at 9600 g for 5 min to obtained the packedcell volume (PCV) and buffy coat. After centrifugation, the blood cells are separated into three layers based on density:platelets (adjacent to supernatant), WBCs, and RBCs in the bottom. The PCV was measured and the microhematocrit tubewas ruptured 2% below the interface between leukocytes and plasma, deposited into a plastic microtubes, homogenized andused for blood smear preparation. All smears were stained with Diff-Quick Stain...

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This study aimed to evaluate the effect of the force and duration of centrifugation and the impact of cushioned centrifugation on sperm selection by Percoll gradient, on sperm quality and development kinetics of in vitro produced bovine embryos. Two experiments were performed. In Experiment I, a pool of semen was selected by Percoll gradients and the pellet was divided into four groups and distributed in a 2 × 2 factorial, with two forces (2200 × g or 9000 × g) and two durations (1 min or 3 min) of centrifugation. In Experiment II, semen was divided into two groups and selected by Percoll gradient with Cushion Fluid (CF) or without CF (Control) in the second centrifugation. The morphofunctionality, biochemical characteristics and fertilizing capacity of the selected sperms were evaluated. In addition, the development of the resulting bovine embryos was monitored for 48 h post-insemination. Duncan and Chi-square tests (P < 0.05) were used to compare the means. In Experiment I, there was a significant increase in sperm vigor (P < 0.05) after sperm selection in all treatments. The force and duration of centrifugation did not have any effect on sperm motility, vigor, and recovery rate among the different treatments (P > 0.05). In Experiment II, the recovery rate and reactive oxygen species (ROS) production in semen were similar among treatments (P > 0.05) although a higher ROS production was observed in the CF fertilization medium. Total fertilization rate was superior in the CF group (65.4 ± 5.3%) compared to that in Control (39.6 ± 4.9%). However, the normal fertilization and cleavage rate did not differ between the Control (94 ± 6.3% and 58.3 ± 8.3%) and CF (89 ± 7.1% and 75.0 ± 7.3%) groups. The reduction in the force and duration of centrifugation did not decrease the sperm recovery during selection by the Percoll gradient and the use of CF in the second centrifugation did not affect the normal fertilization and development of bovine IVF embryos up to 48 h.(AU)

Esse estudo objetivou avaliar o efeito da força e duração da centrifugação, e o impacto da centrifugação amortecida na seleção espermática por gradientes de Percoll, na qualidade espermática e cinética do desenvolvimento de embriões bovinos produzidos in vitro. Dois experimentos foram realizados. No experimento I, um pool de sêmen foi selecionado por gradientes de Percoll e o pellet dividido em quatro grupos e distribuído em um fatorial 2 x 2, com duas forças (2200 e 9000 X g) e dois tempos (1 e 3 min) de centrifugação. No Experimento II, o sêmen foi dividido em dois grupos e selecionado com (CF) ou sem CushionFluid (Controle) na segunda centrifugação. A morfofuncionalidade, características bioquímicas e capacidade fecundante dos espermatozoides selecionados foram avaliadas. Além disso, o desenvolvimento dos embriões bovinos resultantes foi monitorado por 48 horas pós-inseminação. Os testes Duncan e Qui-quadrado (P<0,05) foram usados para comparar as médias. No Experimento I, houve um aumento significativo no vigor após a seleção espermática para todos os tratamentos. A força e a duração da centrifugação não tiveram nenhum efeito na motilidade, vigor e recuperação espermática entre os diferentes tratamentos (P > 0.05). No Experimento II, a taxa de recuperação e a produção de espécies reativas de oxigênio (EROs) no sêmen foram similares entre os tratamentos (P>0,05), embora a maior produção de EROs foi observada no meio de fecundação do grupo CF. A taxa de fecundação total, foi superior no grupo CF (65.4±5.3%) comparada com o Controle (39.6±4.9%). Entretanto a fecundação normal e a taxa de clivagem não diferiram entre os grupos Controle (94±6.3% e 58.3±8.3%) e CF (89±7.1% and 75.0±7.3%). A redução na força e duração da centrifugação não diminuiu a recuperação espermática durante a seleção por gradientes de Percoll e o uso de CF na segunda centrifugação não influenciou a fecundação normal e o desenvolvimento de embriões bovinos PIV até 48 horas.(AU)

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Straws of sex-sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 106  sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex-sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane-coated silica colloid dilutions and layering configurations during centrifugation of sex-sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex-sorted sperm samples were centrifuged using mini-volume single-layer (40%, 60% and 80%) and mini-volume two-layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm® . A single layer of 40% PureSperm® recovered significantly more sex-sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two-layer PureSperm® dilution of 45%/90% (47.57% ± 2.33%). Single-layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini-volume single layer of 80% PureSperm® was determined to be an effective alternative to a two-layer centrifugation configuration for sex-sorted sperm selection. In addition, single-layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.

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