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J. Nascimento DVM MS C.F. Raphael DVM MS A.F.C. Andrade DVM M.A. Alonso DVM MS E.C.C. Celeghini DVM MS PhD R.P. Arruda DVM MS PhD 《Journal of Equine Veterinary Science》2008,28(6):351-358
The advantages of using cryopreserved semen in equine reproduction are well known. During cryopreservation, spermatozoa undergo many changes that lead to a decrease in fertility. There is no agreement on the ideal sperm dose and concentration to maximize fertility rates. Thus, the objectives of this experiment were to evaluate sperm motion by computer-assisted analysis (CASA), sperm membrane integrity and function with fluorescence probes of cryopreserved sperm at three concentrations: 100 (C100), 200 (C200) and 400 × 106 sperm/mL (C400), and two straw volumes (0.50 and 0.25 mL). There was no interaction between sperm concentration and storage volume (P > .05). Sperm motion characteristics were influenced by concentration (C100 > C200 > C400; P < .05). Curvilinear velocity (VCL) in 0.25-mL straws had higher average values (P < .05). Membrane integrity and function were not changed by straw volume (P > .05). However, sperm concentration changed the percentage of cells with intact plasma membrane (C100 > C200 > C400; P < .05) and the percentage of cells with high mitochondrial membrane potential (C100 = C200; P > .05 and C400 < C100 and C200; P < .05). According to this experiment, the best freezing method was that involving 100 × 106 sperm/mL, regardless of straw volume. 相似文献
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Gabriela Bertaiolli Zoca Eneiva Carla Carvalho Celeghini Guilherme Pugliesi Carla Patricia Teodoro de Carvalho Mayra Elena Ortiz D'Avila Assumpção Adriano Felipe Perez Siqueira Leticia Zoccolaro Oliveira Renata Lançoni Rubens Paes de Arruda 《Reproduction in domestic animals》2021,56(6):872-883
This study aimed to evaluate the effect of seminal plasma on bovine sperm cryopreservation and to assess the integrity of plasma and acrosomal membranes, mitochondrial potential, remodelling of F-actin cytoskeleton and sperm chromatin fragmentation during the cooling, equilibrium and freezing/thawing stages. Six ejaculates collected from seven Nelore bulls (n = 42) were used in this study. Each ejaculate was divided into two aliquots (with seminal plasma = SP group; without seminal plasma = NSP group) and packed to a final concentration of 50 × 106 sperm per straw. Statistical analyses were performed using SAS software (version 9.3), and p ≤ .05 was considered significant. A time effect was observed for all sperm characteristics (p < .05), except for chromatin fragmentation (p > .05). The presence of seminal plasma better preserved the acrosomal integrity (SP = 75.2% and NSP = 71.7%; p < .05) and also provided lower F-actin remodelling during cryopreservation process (SP = 29.9% and NSP = 32.4%; p < .05). Regarding to the cryopreservation stages, it was observed that cooling step induced higher remodelling of F-actin than the equilibrium and freezing/thawing stages (56.3%, 32.2% and 23.9%, respectively; p < .05). The equilibrium step had minor influence on overall sperm characteristics while the freezing/thawing stage was responsible for the highest percentage of damage in plasma membrane (−65.2%), acrosomal membrane (−34.0%) and mitochondrial potential (−48.1%). On the other hand, none of the cryopreservation stages affected chromatin integrity. It was concluded that the presence of seminal plasma provides increased acrosomal integrity and reduced remodelling of F-actin cytoskeleton. Higher F-actin remodelling is observed after the cooling step while the freezing/thawing step is most damaging to sperm membranes and mitochondrial potential during bovine sperm cryopreservation. 相似文献
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Addition of Seminal Plasma to Post‐thawing Equine Semen: What is the Effect on Sperm Cell Viability?
de Andrade AF Zaffalon FG Celeghini EC Nascimento J Tarragó OF Martins SM Alonso MA Arruda RP 《Reproduction in domestic animals》2011,46(4):682-686
Effect of seminal plasma addition after thawing on viability or cryocapacitation is not definitively established. This experiment was performed to verify the effect of adding seminal plasma, autologous or homologous (from an animal with good semen freezability). Five ejaculates from each of four stallions with proven fertility were collected and cryopreserved. The semen was subsequently thawed and divided into the following three treatment groups: no seminal plasma addition after semen thawing (NOSP); the addition of homologous seminal plasma after semen thawing (HSP) and the addition of autologous seminal plasma after semen thawing (ASP). The addition of 20% of seminal plasma led to an increase in the cell population that simultaneously show plasma and acrosomal membrane integrity (p < 0.05). The addition of seminal plasma did not alter the total motility, the amount of cells with mitochondrial membrane potential or the sperm velocities (average path velocity, straight-line velocity and curvilinear velocity). However, the beat/cross-frequency, straightness and linearity were reduced in ASP and HSP groups (p < 0.05). Unexpectedly, the addition of homologous seminal plasma reduced the proportion of cells with progressive motility (p < 0.05) and the addition of autologous seminal plasma reduced the amplitude of the lateral head displacement (p < 0.05). Based on the increase in the cell populations that had the plasma and acrosomal membrane integrity simultaneously identified in this study, we proposed that the addition of seminal plasma (autologous or homologous) into post-thawed semen before insemination could increase semen fertility. 相似文献
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Letícia Zoccolaro Oliveira Rubens Paes de Arruda André Furugen Cesar de Andrade Eneiva Carla Carvalho Celeghini Ricarda Maria dos Santos Marcelo Emílio Beletti Rogério Fonseca Guimarães Peres Clara Slade Oliveira Vera Fernanda Martins Hossepian de Lima 《Livestock Science》2012,146(1):38-46
In order to provide information that may help researchers to understand the main cause(s) of differences in bull fertility frequently observed in field trials, this study aimed to investigate conception rates as well as several in vitro sperm characteristics of different sires of unknown fertility utilized in a Timed-AI (TAI) program. Suckled Nelore cows submitted to the same TAI protocol were allocated into eight breeding groups of approximately 120 animals each. Frozen semen doses from three Angus bulls and three different batches from each bull were utilized. Approximately 100 doses from each batch were used in TAI. Sires, batches and AI technicians were equally distributed across breeding groups. Cows were examined for pregnancy diagnosis 40 d after TAI. For in vitro sperm analyses, the same thawing procedure was repeated in the laboratory to mimic field conditions. The following in vitro sperm characteristics were assessed: computerized motility, thermal resistance, plasma and acrosomal membrane integrity, lipid peroxidation, morphology, morphometry and chromatin structure. No effect of breeding group, body condition score, AI technician and sire was observed. However, some significant differences among bulls were detected in laboratory analyses. Semen from sire presenting numerically lower (P > 0.05) pregnancy/AI also presented lower (P < 0.05) values in all sperm characteristics analyzed in thermal resistance test at 4 h (Total Motility, Progressive Motility, Average Path Velocity, Straight-Line Velocity, Curvilinear Velocity, Amplitude of Lateral Head Displacement, Beat Cross Frequency, Straightness, Linearity, and Percentage of Rapidly Moving Cells), higher (P < 0.05) Major and Total Defects in sperm morphological test, lower (P < 0.05) Length, Ellipticity and Fourier parameter (Fourier 0) in sperm morphometric analysis as well as higher (P < 0.05) chromatin heterogeneity. It was concluded that, although no bull effect was observed in the field experiment, the sire that presented numerically lower pregnancy/AI also presented lower semen quality according to the laboratory analyses performed. 相似文献
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AFC de Andrade RP de Arruda ECC Celeghini J Nascimento SMMK Martins CF Raphael AS Moretti 《Reproduction in domestic animals》2007,42(2):190-194
The purpose of this study was to validate a technique for simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in boar spermatozoa, using an association of fluorescent probes: Propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and JC-1. Three ejaculates from each of four different boars, all showing motility >or=80% and abnormal morphology 相似文献
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ECC Celeghini RP de Arruda AFC de Andrade J Nascimento CF Raphael 《Reproduction in domestic animals》2007,42(5):479-488
This experiment was performed to develop and validate practical techniques for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function in bovine spermatozoa using associations of fluorescent probes. Four protocols of fluorescent probes association were defined: protocol 1: propidium iodide (PI), fluorescein isothiocyanate‐conjugated Pisum sativum agglutinin (FITC‐PSA) and rhodamine 123; protocol 2: PI, FITC‐PSA and MitoTracker Green FM (MITO); protocol 3: PI, Hoechst 33342 (H342), FITC‐PSA and CMXRos; and protocol 4: PI, H342, FITC‐PSA and JC‐1. Three ejaculates from each of the four bulls (n = 12) were utilized, showing sperm motility ≥80% and abnormal morphology ≤10%. The semen was diluted in Modified Tyrode’s medium (TALP) (25 × 106 spermatozoa/ml) and split into two aliquots, one sample was flash‐frozen in liquid nitrogen and thawed. Samples for three treatments were prepared with the following ratio of fresh semen : flash‐frozen semen: 100 : 0, 50 : 50 and 0 : 100. Samples were stained in all four protocols and evaluated by epifluorescence microscopy. Protocol 1 did not result in a satisfactory stain, so it could not be validated. Protocols 2, 3 and 4 were validated and showed high determination coefficient to plasma membrane integrity (R2 = 0.95, 0.93 and 0.92, respectively), acrosome integrity (R2 = 0.95, 0.92 and 0.91, respectively) and mitochondrial function (R2 = 0.84, 0.93 and R2 = 0.93, respectively). These techniques are efficient for the simultaneous integrity evaluation of plasma and acrosomal membranes and mitochondrial function in bovine spermatozoa. However, JC‐1 has an advantage over MITO and CMXRos, as it separates two cell populations with high and low mitochondrial membrane potential. 相似文献
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Shirley Andrea Florez-Rodriguez Rubens Paes de Arruda Maíra Bianchi Rodrigues Alves Fernanda Jordão Affonso Henrique Fulaneti Carvalho Kleber Menegon Lemes Renata Lançoni André Furugen Cesar de Andrade Eneiva Carla Carvalho Celeghini 《Journal of Equine Veterinary Science》2014
The aim of this study was to assess extenders for cooling equine semen at 5°C and to be used in assisted reproductive technologies (ARTs). Four ejaculates were obtained from each of four stallions. Gel-free semen was diluted in three different extenders: (1) SMK, an opaque skim milk–based extender; (2) SMT, a skim milk (65%) and Tyrode medium (35%); and (3) BSAG, a clear extender containing 1% bovine serum albumin. Samples were packaged (10 mL; 50 × 106 sperm/mL) and stored in a cooling device at 5°C for 12 hours. Analyses were done at 0, 4, 8, and 12 hours after cooling. Semen was analyzed for sperm motility characteristics using a computer-assisted sperm analysis, for plasma membrane and acrosome integrity and mitochondrial membrane potential, using fluorescent probes (propidium iodide, Hoechst 33342, fluorescein isothiocyanate–conjugated Pisum sativum agglutinin, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide [JC-1]). Morphology was evaluated with differential interference contrast microscopy and sperm chromatin integrity by the toluidine blue technique. Data were analyzed by analysis of variance and by Tukey test, with time as a repeated measure (SAS, 1998), when P < .05 was significant. In general, milk-based extenders (SMT and SMK) showed improved maintenance of semen quality compared with BSAG. Finally, the addition of skim milk to equine semen extender for cooling at 5°C for 12 hours seems to play a crucial role in sperm preservation. Although, optically clearer extenders are desired for use in ARTs, such as sperm sexing, the milk-free extender (BSAG) is less efficient for cooled-stored equine semen. 相似文献
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