Reduction of centrifugation force in discontinuous percoll gradients increases in vitro fertilization rates without reducing bovine sperm recovery

Guimarães, Antônio Carlos Galarça; Leivas, Fábio Gallas; Santos, Francielli Weber; Schwengber, Eduardo Brum; Giotto, Angelo Bertani; Machado, C.I.U.; Gonçalves, Cibele Garcia Moreira; Folchini, Natália Picoli; Brum, D. S.

doi:10.1016/j.anireprosci.2014.02.016

Cited by 23 publications

(15 citation statements)

References 35 publications

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“…As expected, centrifugation increased production of ROS, corroborating the findings of Guimarães et al. (), who centrifugated bovine semen samples on a continuous Percoll density gradient. Similar results have been reported for human semen which requires the use of centrifugation to select faster spermatozoa, but an increase in ROS levels is observed after the procedure (Shekarriz, DeWire, Thomas, & Agarwal, ).…”

Section: Discussionsupporting

confidence: 89%

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Albuquerque¹,

Morais

Reis

et al. 2017

Reprod Domestic Animals

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Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer-assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.

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Section: Discussionsupporting

confidence: 89%

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Albuquerque¹,

Morais

Reis

et al. 2017

Reprod Domestic Animals

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“…Regardless of sperm selection method used, centrifugation is required to concentrate the sperm sample in IVP. Although several studies examine the effects of the sperm selection method on sperm characteristics or report possible damage caused by centrifugation (DELL 'AQUA JÚNIOR et al, 2006;MACHADO et al, 2009;GUIMARÃES et al, 2014), there are no studies evaluating the effect of the second centrifugation performed during selection by Percoll gradients. In this study, we were able to prove that a lower duration (1 min) and force (2200 x g) in the second centrifugation of Percoll gradient did not reduce the sperm recovery rate.…”

Section: Discussionmentioning

confidence: 99%

“…A discontinuous Percoll gradient was created by layering 300 µL of each solution into a microtube, starting with the 90% Percoll solution at the bottom of the tube followed by the 60% and 30% solutions. On the top of the gradient, 200 µL of thawed semen was layered, and the tubes were centrifuged for 5 min at 2200 x g (GUIMARÃES et al, 2014). In Experiment I, the pellets were suspended in 300 µL of Talp-Fert medium and centrifuged at 9000 x g or 2200 x g for 1 min or 3 min, depending on the treatment.…”

Section: In Vitro Embryo Production (Ivp)mentioning

confidence: 99%

“…Other methods of sperm selection, such as the swimup method reduce the use of centrifugation, but this technique is still necessary to concentrate the sample. Studies report that high centrifugal forces often promote higher sperm recovery (WAITE et al, 2008;LEN et al, 2010;GLORIA et al, 2016) Some studies suggested that a reduction in the force and duration of centrifugation could reduce sperm damage and improve the sperm selection protocol using Percoll gradient (GUIMARÃES et al, 2014;KIM et al, 2013). However, these studies evaluated only the first centrifugation for sperm selection disregarding the second centrifugation for sperm concentration.…”

Section: Introductionmentioning

confidence: 99%

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Use of a colloid to optimize centrifugation in the selection of bovine sperm for IVF

Gonçalves

Leivas

Míssio

et al. 2018

SCA

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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. Key words: Bovine embryo. Cushion centrifugation. In vitro production. Percoll gradients. Sperm recovery. ResumoEsse 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 ...

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“…[5,15]. Filtration is the least common procedure, but it is worth noting that filtration is only applicable to samples of good quality, and because of the complexity this procedure is not applicable in veterinary [15,16].…”

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confidence: 99%

AN INCREASE IN THE VIABILITY OF RAM SPERM WHEN CULTURED IN MODIFIED MEDIUM FOR in vitro FERTILIZATION

Belyaev¹,

Gvozdetskii²,

Kanibolotskaya³

et al. 2017

S-h. biol.

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A b s t r a c tModern livestock necessitates controlled breeding, one of the main methods of which is a long-term preservation of animal reproductive qualities at cell level. In recent years, a considerable progress has been achieved in the development of techniques of animal sperm preparation for in vitro fertilization (IVF). In these, the main purpose is to obtain population of motile sperm with a suitable morphology capable of in vivo or in vitro oocyte fertilization. However, these methods are not always effective because of the lack of standards for the procedure. We first in Russia carried out a study on the viability of ram sperm cultured in the modified IVF medium. The semen was sampled from North Caucasian rams, 2-3 years of age, according to a routine technique, used for sheep and goats' artificial insemination, and domestic State Standards GOST 32222-2013. Sperm was evaluated on organoleptic and microscopic parameters, and cultured for capacitation to render the spermatozoa competent to fertilize an oocyte according to the prototype procedure, described by A.P. Gandhi (2000), and the method, developed by us. In the modified procedure, the sperm is brought to glucose-yolk-citrate diluent (GYC) and then transferred to medium SOF wash, prepared without glucose and supplemented with glutamine with 6 mg/ml bovine serum albumin, 0.2 mg ml caffeine and 50 mg/ml heparin. The developed technique increased the activity of sperm by 1.3 points compared to the commonly used method, the number of live semen increased by 43.7 %, their vitality increased 1.6 times and the number of spermatozoa with rectilinear movements was 1.6 times higher. Thus, the developed technique significantly improves ram sperm qualitative and quantitative characteristics and allows us to recommend this method of animal sperm maturation for in vitro fertilization procedure.

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Reduction of centrifugation force in discontinuous percoll gradients increases in vitro fertilization rates without reducing bovine sperm recovery

Cited by 23 publications

References 35 publications

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Use of a colloid to optimize centrifugation in the selection of bovine sperm for IVF

AN INCREASE IN THE VIABILITY OF RAM SPERM WHEN CULTURED IN MODIFIED MEDIUM FOR in vitro FERTILIZATION

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