Influence of haematological parameters on size of the advanced platelet-rich fibrin+ (A-PRF+) in the horse.

The advanced-PRF+ (A-PRF+) is a platelet concentrate, showing a higher concentration of growth factors, an increased number of cells and looser structure of the fibrin clot than leukocyte-PRF. A high variability in the size of PRF associated with patients, haematological features and centrifugation protocols was reported. The aims of this study were to evaluate the feasibility of A-PRF+ production in the field and the correlation between haematological parameters, macroscopic and microscopic features in equine A-PRF+. Samples from twenty Standardbred horses (3-7 years) were harvested with glass tubes without anticoagulants, previously heated at 37 °C. Blood samples were centrifugated at 1300 rpm for 8 min with a fixed-angle centrifuge and a horizontal centrifuge in the field, at a temperature of 15-17 °C. Clots were measured and placed on the Wound Box® for a 2-min compression. Membranes were measured and fixed in 10% formalin for histological examination. Clot and membrane surface did not differ between sex and centrifuge. Haematological parameters did not show a significant correlation to clot and membrane size. Membranes obtained from both centrifugation protocols showed a loose fibrin structure and cells evenly distributed throughout the clot. Tubes' warming was effective to obtain A-PRF+ clots from all samples, regardless the environmental temperature. Further studies are needed to evaluate the influence of other blood molecules on the A-PRF+ structure and size.

Sow reproductive performance following artificial insemination with semen doses processed using Single Layer Centrifugation without antibiotics in the tropics.

Single Layer Centrifugation (SLC) through a low density colloid offers an alternative solution to antibiotic use in boar semen extenders, with lower costs compared to high density colloids. The aim of this study was to explore the reproductive performance of sows when using SLC-prepared semen doses without antibiotics, employing low density Porcicoll to prepare semen doses for artificial insemination in a commercial swine herd in Thailand. Ejaculates were divided into two equal parts to create insemination doses, with each dose containing 3000 × 106 sperm/80 ml for intra-uterine insemination in individual sows. The sows were inseminated twice, with the interval between the two inseminations ranging from 8 to 16 h. The CONTROL group consisted of 206 semen doses treated with antibiotics, prepared for insemination in 103 sows, while the SLC group comprised 194 SLC-prepared semen doses without antibiotics for inseminating 97 sows. Fertility and fecundity traits, including non-return rate, conception rate, farrowing rate, and litter traits (i.e., the total number of piglets born per litter, number of piglets born alive per litter, number of stillborn piglets, and number of mummified fetuses), were compared between groups. Furthermore, data on piglet characteristics, including live-born and stillborn piglets (i.e., the prevalence of stillbirth (yes, no), birth weight, crown-rump length, body mass index (BMI), and ponderal index (PI)), were determined. No significant differences in non-return rate (75.7 % vs. 77.3 %), conception rate (73.8 % vs. 73.2 %), and farrowing rate (71.8 % vs. 73.2 %) were observed between the CONTROL and SLC groups, respectively (P > 0.05). Nevertheless, the total number of piglets born per litter in the SLC group was higher than in the CONTROL group (14.6 ± 0.9 vs. 12.3 ± 0.6, respectively, P = 0.049). Interestingly, the prevalence of stillbirth in the SLC group was lower than in the CONTROL group (6.2 % vs. 11.6 %, respectively, P < 0.001). Moreover, the newborn piglets in the SLC group exhibited higher birth weight and BMI compared to those in the CONTROL group (1.36 ± 0.03 vs. 1.26 ± 0.02 kg, P = 0.005, and 18.3 ± 0.3 vs. 17.3 ± 0.2 kg/m2, P = 0.003). In conclusion, employing sperm doses after SLC through a low density colloid in artificial insemination within a commercial breeding operation did not have a detrimental impact on either fertility or fecundity traits but showed potential benefits in increasing the total number of piglets born per litter. Moreover, improvements were observed in the birth weight and body indexes of piglets, and the percentage of stillbirths was reduced. Our findings introduce new possibilities for antibiotic alternatives in semen extenders to reduce the risk of antimicrobial resistance in the swine industry. Additionally, they provide compelling reproductive outcomes supporting the integration of SLC-prepared semen doses into artificial insemination practices.

Effect of storage and single layer centrifugation before cryopreservation on stallion sperm cryosurvival.

The objectives of this study were to evaluate the effect of a short, cooled storage before cryopreservation on sperm progressive motility (PM) and compare the effect of different centrifugation methods on post-thaw PM of stored samples. Semen was diluted in chilling extender and aliquoted in 6 protocols: i) Standard centrifugation (SC) followed by freezing; ii) Single Layer Centrifugation (SLC) followed by freezing; iii) Storage for 8 h/5 °C before SC; iv) Storage for 8 h/5 °C before SLC; v) Storage for 8 h/15 °C before SC; and vi) Storage for 8 h/15 °C before SLC. PM was assessed before centrifugation, after centrifugation, and post-thawing. Stallions were classified as "good freezers" (GF) or "bad freezers" (BF). The PM in samples immediately frozen was greater than in the stored ones (71.98 ± 14.2, 52.91 ± 17.8, 53.93 ± 18.9 for no storage, 5 ºC storage and 15 ºC storage, respectively) (P˂ 0.0001). There was an effect of storage condition (p ˂ 0.0001), centrifugation method (p ˂ 0.0001), and freezability (P=0.0016), with an interaction between them (P= 0.0004), on PM after centrifugation. Post-thaw PM was greater in samples treated by SLC than in samples processed by SC, for all storage conditions (p ˂ 0.05). All BF stallions 'showed post-thaw PM ˂ 30 % when samples were previously stored. Storage at 5 ºC or 15º C for 8 h maintains an appropriate quality in GF stallions. Applying a sperm selection technique as SLC is suggested to improve post-thaw motility, allowing GF straws to be frozen after storage, although BF semen should be prepared by SLC immediately after collection.

Reduced bacterial load in stallion semen by modified single layer centrifugation or sperm washing.

The presence of bacteria poses a significant challenge to the quality of stallion semen used in artificial insemination. The bacterial content of insemination doses arises from various sources, such as the healthy stallion, environment, and collection equipment, and is implicated in fertility problems as well as reduced sperm quality during storage. The conventional approach of adding antibiotics to semen extenders raises concerns about antimicrobial resistance and potential negative effects on sperm characteristics, and may not be effective in inhibiting all bacteria. The objective of this study was to determine whether an innovative alternative to antibiotic usage - centrifugation through a single layer of a low density colloid (SLC) - could reduce the bacterial load in stallion semen, and to compare sperm characteristics in samples arising from this procedure, or simple extension of the ejaculate in semen extender, or from sperm washing, i.e. adding extender and then centrifuging the sample to allow the removal of most of the seminal plasma and extender. Eighteen semen samples were collected from six stallions. The semen samples were split and extended prior to washing or SLC, or received no further treatment other than extension. After preparation aliquots from each type of sample were sent for bacteriological examination; the remaining samples were stored for up to 72 h, with daily checks on sperm quality. The low density colloid SLC outperformed sperm washing or extension for bacterial reduction, effectively removing several bacterial species. The bacterial load in the samples was as follows: extended semen, 16 ± 6.7 × 105; washed, 5.8 ± 2.0 × 105; SLC, 2.3 ± 0.88 × 105, p < 0.0001. In addition, SLC completely removed some bacterial species, such as Staphylococcus xylosus. Although there is no selection for robust spermatozoa with the low density colloid, sperm motility, membrane integrity, and DNA fragmentation were not different to washed sperm samples. These findings suggest that SLC with a low density colloid offers a promising method for reducing bacterial contamination in stallion semen without resorting to antibiotics.

Comparison of two processing techniques on the sperm quality of semen contaminated with urine.

Urospermia in stallions can occur intermittently, consistently, or as an isolated event, and may result in reduced sperm quality which is often assumed to reduce fertility. Although sperm quality declines in urospermic ejaculates, fertility has not been assessed in mares bred with urine contaminated semen. The aims of this study were to compare sperm quality after simple dilution (SD), cushioned centrifugation (CC) alone, or cushioned centrifugation combined with a 40 % silane-coated silica solution (SC) in semen contaminated with 0, 20, or 40 % (v/v) urine. Sperm quality values tended to decrease as the percent urine increased within all treatments (SD, CC, SC) after 24 h of cooled storage. However, SC treated groups had higher sperm quality compared to SD and CC when exposed to 20 or 40 % (v/v) urine. Differences in pregnancy rates among treatment groups (SD with 0 or 40 % (v/v) urine, or 40 % (v/v) urine followed by SC) were unable to be detected.

Cushioned and high-speed centrifugation improve sperm recovery rate but affect the quality of fresh and cryopreserved feline spermatozoa.

The development of endoscopic transcervical catheterization (ETC) in the queen increases the interest in handling fresh and cryopreserved feline semen. The ETC requires a small volume of the insemination dose with a high concentration, not easily reached with the actual frozen technique in this species. Centrifugation is widely used to concentrate spermatozoa for several purposes, but this process is detrimental to spermatozoa. This study verified the effects of conventional and cushioned centrifugation on fresh and cryopreserved feline spermatozoa. To this, semen was collected from 20 toms, grouped in seven pools and diluted. After dilution, the pools were divided into two aliquots, the first used for centrifugation on fresh semen, and the second, after freezing, on cryopreserved semen. Centrifugation regimens were: conventional at 500×g, conventional at 1000×g, and cushioned (iodixanol) at 1000×g. The sperm recovery rate was calculated for the three centrifugation regimens, and sperm kinematics, membrane and acrosome integrity, and plasma membrane stability on viable spermatozoa were assessed as endpoints. The data reported in this study showed that the centrifugation at 500×g resulted in negligible effects on both fresh and cryopreserved spermatozoa, but the lower recovery rate (62.4 ± 3.1 % and 60.2 ± 1.6 %, respectively) underlines the loss of a large proportion of spermatozoa, unfavourable in a species with small total sperm ejaculated. On the other hand, the centrifugation at 1000×g improved the recovery rate (86.9 ± 4.3 % and 89.8 ± 2.4 % in fresh and cryopreserved samples, respectively), but was more deleterious for feline spermatozoa, especially in cryopreserved samples (i.e. total motility of 40.7 ± 5.4 % compared with 57.2 ± 9.8 % in cryopreserved uncentrifuged samples, P < 0.05), resulting in artificial insemination doses of lower quality. The recovery rate in cushioned centrifugation appeared less efficient, likely due to the small volume of feline samples, which makes difficult the separation of sperm pellet and cushioned fluid. Interestingly, in cryopreserved samples centrifuged at 1000×g the number of viable spermatozoa with membrane destabilization (31.3 ± 3.2 %) was greater than uncentrifuged (4.1 ± 0.7 %, P < 0.05) and those centrifuged at 500×g (9.8 ± 1.3 %, P < 0.05), suggesting modifications induced by the cryopreservation amplifies centrifugation sublethal damage on feline spermatozoa. Cushioned centrifugation on cryopreserved samples showed kinematics similar to uncentrifuged samples, but higher viable spermatozoa with membrane destabilization (37.4 ± 3.4 % vs 4.1 ± 0.7 %; P < 0.05). In felines, g-force is crucial for sperm recovery rate during centrifugation, with better results at 1000×g; on the other hand, greater g-forces could have a significant impact on the quality of feline insemination dose, especially in cryopreserved samples.

Effect of plasma separation techniques and sperm selection on sperm recovery and viability of cooled pony stallion semen for 48h at 5°C / [Efeito das técnicas de separação de plasma e de seleção de esperma na recuperação e viabilidade do sêmen de garanhão pônei resfriado por 48h a 5°C]

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)

Method to obtain platelet-rich plasma from rabbits (Oryctolagus cuniculus ) / Método de obtenção de plasma rico em plaquetas de coelhos (Oryctolagus cuniculus)

Platelet-rich plasma (PRP) is a product easy and inxpesnsive, and stands out to for its growth factors in tissue repair. To obtain PRP, centrifugation of whole blood is made with specific time and gravitational forces. Thus, the present work aimed to study a method of double centrifugation to obtain PRP in order to evaluate the effective increase of platelet concentration in the final product, the preparation of PRP gel, and to optimize preparation time of the final sample. Fifteen female White New Zealand rabbits underwent blood sampling for the preparation of PRP. Samples were separated in two sterile tubes containing sodium citrate. Tubes were submitted to the double centrifugation protocol, with lid closed and 1600 revolutions per minute (rpm) for 10 minutes, resulting in the separation of red blood cells, plasma with platelets and leucocytes. After were opened and plasma was pipetted and transferred into another sterile tube. Plasma was centrifuged again at 2000rpm for 10 minutes; as a result it was split into two parts: on the top, consisting of platelet-poor plasma (PPP) and at the bottom of the platelet button. Part of the PPP was discarded so that only 1ml remained in the tube along with the platelet button. This material was gently agitated to promote platelets resuspension and activated when added 0.3ml of calcium gluconate, resulting in PRP gel. Double centrifugation protocol was able to make platelet concentration 3 times higher in relation to the initial blood sample. The volume of calcium gluconate used for platelet activation was 0.3ml, and was sufficient to coagulate the sample. Coagulation time ranged from 8 to 20 minutes, with an average of 17.6 minutes. Therefore, time of blood centrifugation until to obtain PRP gel took only 40 minutes...

O plasma rico em plaquetas (PRP) é um produto de fácil obtenção a baixo custo, destacando-se pelos seus fatores de crescimento na reparação tecidual. Para obtenção do PRP, a centrifugação do sangue total é realizada com tempos e forças gravitacionais específicas. Assim, o presente trabalho teve por objetivo estudar o método da dupla centrifugação para obtenção do PRP, a fim de avaliar a eficácia de aumento da concentração de plaquetas no produto final, a preparação de gel de PRP e otimizar o tempo de preparação da amostra final. Quinze coelhos Nova Zelândia Branco, fêmeas, foram submetidos à coleta de sangue para a preparação de PRP. As amostras foram separadas em dois tubos estéreis contendo citrato de sódio. Os tubos foram submetidos ao protoloco de dupla centrifugação, com a tampa fechada a 1600 revoluções por minuto (rpm) durante 10 minutos, resultando na separação dos glóbulos vermelhos, plaquetas e plasma contendo os leucócitos. Na sequência, foram destapados para pipetar o plasma e transferí-lo para outro tubo de estéril. O plasma foi novamente centrifugado a 2000pm durante 10 minutos, resultando em duas partes: a parte superior, que consistia em plasma pobre em plaquetas (PPP) e a parte inferior do botão de plaquetas. Parte PPP foi descartado de modo que apenas 1ml de PPP permaneceu no frasco juntamente com o botão de plaquetas. Este material foi agitado suavemente para promover a ressuspensão das plaquetas, o que resultou na produção de PRP. O protocolo de centrifugação dupla foi capaz de promover a concentração de plaquetas 3 vezes maior em relação à amostra de sangue inicial. O volume de gluconato de cálcio utilizado para a ativação das plaquetas foi de 0,3ml, e foi suficiente para coagular a amostra, e o tempo de coagulação variou de 8 a 20 minutos, com uma média de 17,6 minutos. O tempo da centrifugação do sangue até a obtenção do PRP gel levou apenas 40 minutos...

Contagem total e diferencial dos leucócitos presentes no leite de cabras sadias utilizando-se a coloração de verde de metil e pironina e a citocentrifugação / Total and differential leukocyte counts in the milk of healthy goats, using methyl green-pyronin stain and cytocentrifugation

As particularidades da secreção láctea do tipo apócrina, na espécie caprina, tornam necessárias técnicas específicas para a determinação da quantidade e da qualidade das células presentes no leite desta espécie. Dentre estas particularidades, pode-se citar a presença dos corpúsculos citoplasmáticos. De acordo com estas características, o objetivo do presente trabalho foi determinar a contagem total de leucócitos no leite de cabras sadias, por meio da contagem microscópica direta com verde de metil e pironina-Y, e a contagem celular automática por citometria de fluxo, assim como determinar os tipos leucocitários, através da técnica de citocentrifugação. Foram analisadas 102 de leite de cabras sadias, das raças Saanen, Parda Alpina e Toggenburg. O valor mediano obtido pela contagem microscópica direta e automática foi de 142.840 e 406.000 células somáticas/mL de leite, respectivamente. Os valores médios obtidos na citocentrifugação foram de 73,24 ± 18,35% de neutrófilos, 3,55 ± 3,06% de linfócitos e 24,33 ± 18,89% de monócitos e células epiteliais. De acordo com os resultados, pode-se concluir que o valor obtido pela coloração de verde de metil e pironina-Y é significativamente menor, sendo um importante método para a determinação da celularidade presente no leite de cabras, pois exclui os corpúsculos citoplasmáticos característicos desta espécie.

The characteristics of the apocrine milk secretion observed in goats make it necessary to use specific techniques to determine the quantity and quality of cells found in this kind of milk. Among these characteristics is the presence of cytoplasmic bodies. The objective of the present study was to determine total leukocyte counts in the milk of healthy goats using direct microscopy and methyl green-pyronin Y and automatic cell count by flow cytometry as well as to determine leukocyte types by means of cytocentrifugation. A total of 102 milk samples from healthy Saanen, Brown Alpine and Toggenburg goats were analyzed. The median value of direct microscopic and automatic counts was 142,840 and 406,000 somatic cells/mL of milk, and mean values obtained in cytocentrifugation were 73.24 ± 18.35% neutrophils, 3.55 ± 3.06% lymphocytes and 24.33 ± 18.89% monocytes and epithelial cells. According to the results obtained, it was concluded that methyl green-pyronin Y is an important method, perhaps the most precise one, for determining cell counts in goat milk, because it excludes cytoplasmic bodies. Moreover, the different cell types found in milk are important defenses of the mammary gland against mastitis-causing pathogens.

Platelet concentration of plateletrich plasma from dogs, obtained through three centrifugation speeds

The platelets release at least 4 growth factors (Platelet Derived Growth Factor. â1 and â2 Transforming Growth Factors and Insulin-like Growth Factor) which are responsible for the migration and activation of cells that will start the reparation of soft tissues and bones. The Platelet Rich Plasma is an autogenous source for Growth Factors, obtained by platelet concentration by centrifuging total blood. This study aimed the comparison of platelet concentrations in plasma centrifuged in three different centrifugation speeds (1300, 1600 e 3200rpm), for the production of platelet rich plasma. Blood was drowned from 15 dogs, 40ml of each, and these were divided into four groups and centrifuged at 800rpm. Then the first group was centrifuged at 1300rpm, the second at 1600rpm, the third at 3200rpm and the last was used as control, named plasma. The mean percentage increase in the platelet concentration for each technique was: 1300 –183%, 1600 – 210% and 3200 – 222%. But in centrifugation at 3200rpm, platelets presented altered morphology and different sizes in every sample studied, which was understood as severe cell damage. It was concluded that the best technique for the preparation of the platelet rich plasma in dogs consisted of the previous centrifugation of the blood at 800rpm for ten minutes, and then the plasma should be separated. This plasma is then submitted to a second centrifugation of 1600rpm for 10 minutes, and the platelet poor plasma is separated and discharged.

Plaquetas liberam ao menos quatro fatores de crescimento ( Fator de Crescimento derivado de Plaquetas, Fatores de transformação de crescimento â1 and â2 e Fator de crescimento semelhante a insulina) responsáveis pela migração e ativação de células que iniciarão os processos de reparação de tecidos moles e ossos. O Plasma Rico em Plaquetas é fonte autógena de fatores de crescimento, obtida pela concentração das plaquetas através de centrifugação de sangue total. Este estudo visa a comparação das concentrações plaquetárias no plasma obtidas por três diferentes velocidades de centrifugação (1300,1600 e 3200 rpm), para produção de Plasma Rico em Plaquetas. 40 ml de sangue total foram retirados de cada animal, divididos em quatro grupos, e centrifugados inicialmente a 800 rpm. A seguir, as amostras do primeiro grupo foram centrifugadas a 1300 rpm, as do Segundo a 1600 rpm, as do terceiro a 3200 rpm e as do quarto grupo foram usadas como controle, denominadas plasma. O aumento médio da porcentagem na concentração de plaquetas para cada técnica foi: 1300- 183%, 1600 - 210% e 3200 - 222%. No entanto, a centrifugação a 3200 rpm, as plaquetas apresentaram a morfologia alterada e tamanhos diferentes em cada amostra estudada, que foram compreendidas como danos celulares severos. Como conclusão deste estudo, obteve-se que a melhor técnica para a preparação do plasma rico em plaquetas de cães consiste na centrifugação precedente do sangue em 800 rpm por dez minutos, separando o plasma, sendo este submetido a uma segunda centrifugação de 1600 rpm por 10 minutos, separando e desprezando o plasma pobre em plaquetas.