Person:
Crespo Castejón, Francisco

First Name

Francisco

Last Name

Crespo Castejón

URI

https://hdl.handle.net/20.500.14352/80614

Affiliation

Universidad Complutense de Madrid

Faculty / Institute

Veterinaria

Department

Medicina y Cirugía Animal

Area

Medicina y Cirugía Animal

Identifiers

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Now showing 1 - 10 of 24

Simple and economic colloidal centrifugation protocols may be incorporated into the clinical equine sperm processing procedure
(Animal Reproduction Science, 2011) Gutiérrez Cepeda, Luna; Fernández, A; Crespo Castejón, Francisco; Gosálvez Berenguer, Jaime; Serres Dalmau, María Consolacion
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.
Shared Y chromosome repetitive DNA sequences in stallion and donkey as visualized using whole-genomic comparative hybridization
(European Journal of Histochemistry, 2010) Gosálvez Berenguer, Jaime; Crespo Castejón, Francisco; Vega Plá, Jose Luis; López Fernández, Carmen; Cortés Gutiérrez, Elvira; Devila Rodríguez, M. I.; Mezzanote, Roberto
The genome of stallion (Spanish breed) and donkey (Spanish endemic Zamorano-Leonés) were compared using whole comparative genomic in situ hybridisation (W-CGH) tech-nique, with special reference to the variability observed in the Y chromosome. Results show that these diverging genomes still share some highly repetitive DNA families localized in pericentromeric regions and, in the particular case of the Y chromosome, a sub-family of highly repeated DNA sequences, greatly expanded in the donkey genome, accounts for a large part of the chromatin in the stallion Y chromosome.
Localization of alkali-labile sites in donkey (Equus asinus) and stallion (Equus caballus) spermatozoa
(Theriogenology, 2014) Gosálvez Berenguer, Jaime; Gálvez, MJ; Acha Valls, Daniel; Gosálvez Berenguer, Jaime; Crespo Castejón, Francisco
The presence of constitutive alkali-labile sites (ALS) has been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization and comet assay in spermatozoa of donkey (Equus asinus) and stallion (Equus caballus). These results were compared with those obtained using a similar experimental approach using somatic cells. The relative abundance of ALS was of the order of four times more in spermatozoa than in somatic cells. Alkali-labile sites showed a tendency to cluster localized at the equatorial-distal regions of the sperm. The amount of hybridized signal in the ALS in the sperm of donkey (Equus asinus) was 1.3 times greater than in stallion (Equus caballus), and the length of the comet tail obtained in donkey sperm was 1.6 times longer than that observed in stallion (P < 0.05); however, these differences were not appreciated in somatic cells. In conclusion, ALS localization in sperm is not a randomized event and a different pattern of ALS distribution occurs for each species. These results suggest that ALS represents a species-specific issue related to chromatin organization in sperm and somatic cells in mammalian species, and they might diverge even with very short phylogenetic distances.
Fertilizing capacity of vitrified stallion sperm assessed utilizing heterologous IVF after different semen warming procedures
(Animal Rerpoduction Science, 2020) Consuegra González, Cesar; Crespo Castejón, Francisco; Dorado Martín, Jesús; María Díaz Jiménez; Sánchez Calabuig, María Jesús; Beltrán Breña, Paula; Pérez Cerezales, Serafín; Rizos, Dimitri; Hidalgo Prieto
The aim of this study was to evaluate the fertilizing capacity of frozen or vitriﬁed stallion sperm after assessing different warming procedures. In Experiment 1, different warming procedures were compared after sperm vitriﬁcation: immersion in extender at 43 ◦C (C), or in a water bath at 37 ◦C/30 s (W37), 43 ◦C/10 s (W43) or 60 ◦C/5 s (W60). With the W60 treatment, there were greater values (P < 0.05) for VCL (83.93 ± 3.6 μm/s) and ALH (3.00 ± 0.2 μm) than freezing and with the C group, and greater values (P < 0.001) for PM (35.33 ± 2.5 %) than with the W43 treatment. In Experiment 2, the fertilizing capacity of vitriﬁed and frozen sperm was assessed utilizing heterologous IVF procedures, using cattle oocytes. Vitriﬁcation resulted in greater values (P < 0.05) than freezing for the number of bound sperm (1.36 ± 0.3 and 0.69 ± 0.2, respectively). There were no differences between frozen or vitriﬁed sperm in pronuclear formation (26 hours post-insemination - hpi; 14.08 ± 4.2 % and 22.78 ± 4.8 %, respectively) or cleavage rate (32.77 ± 4.3 % and 39.66 ± 4.6 %, respectively). In conclusion, vitriﬁed stallion sperm warmed in a water bath at 60 ºC had the capacity to penetrate cattle oocytes, leading to pronuclear formation and hybrid embryo cleavage after heterologous IVF.
Sex-sorted bovine spermatozoa and DNA damage: II
(Theriogenology, 2011) Gosálvez Berenguer, Jaime; Ramírez, Miguel Ángel; López Fernández, Carmen; Crespo Castejón, Francisco; Evans, K.; Kjelland, M. E.; Moreno, Juan F.
This study examined the dynamic response of Spermatozoa DNA Fragmentation after sex selection in bulls using a MoFlo® SX (Beckman Coulter, Miami FL) spermatozoa sorter. The dynamic response of spermatozoa DNA fragmentation refers to the changing values of SDF, i.e., rate of SDF (rSDF), when analyzed periodically over a set incubation time at 37 °C. A dynamic assessment of SDF using non-sorted and sex-sorted spermatozoa samples during 72 h of incubation at 37 °C was performed. Results showed a reduced DNA longevity in sex-sorted frozen-thawed spermatozoa, with spermatozoa DNA damage appearing between 24 h and 48 h. The baseline SDF level was higher in conventional frozen-thawed than in sex-sorted frozen-thawed spermatozoa samples; while the reverse occurred for the rSDF. The afore-mentioned result produced a crossover point between both dynamic tendencies of SDF for sex-sorted versus conventional samples. We defined this crossover point as the Crossover Positioning Time (CPT) or the time (in hours) where both curves crossover after a period of spermatozoa incubation at 37 °C. The point at which the CPT occurs could be used as an indicator of the rSDF for individual bulls after X- and Y-chromosome bearing spermatozoa selection. CPT values produced a window of SDF ranging between 24 h and 48 h in the present experiment. It is proposed that higher values for CPT are indicative of bulls presenting chromatin that is more resistant to the external stressors affecting spermatozoa DNA after spermatozoa sorting.
Seasonal variations in sperm DNA fragmentation and pregnancy rates obtained after artificial insemination with cooled-stored stallion sperm throughout the breeding season (spring and summer)
(Theriogenology, 2020) Crespo Castejón, Francisco; Quiñones Pérez, Carlota; Ortiz Jaraba, Isabel; Diaz Jimenez,María; Consuegra González Cesar; Pereira Aguilar Blasa; Dorado Martín Jesús; Hidalgo Prieto Manuel
The aim of this study was to assess seasonal variations during different periods of the breeding season (spring and summer) on stallion sperm DNA fragmentation and in vivo fertility associated with cooled-stored semen samples. Ejaculates were collected from eleven stallions and assessed for sperm motility (assessed by computer-assisted sperm analysis) and plasma membrane integrity (evaluated under fluorescence microscopy). Sperm DNA fragmentation (evaluated by the Sperm Chromatin Dispersion test) was assessed in cooled-stored semen at 5 °C for up to 24 h. Artificial insemination was performed throughout the breeding season. Mares were inseminated with cooled-stored semen (up to 24 h) every other day until ovulation. Pregnancy rates per cycle were determined detecting the embryonic vesicle by ultrasonography fifteen days after ovulation. Values (mean ± SD) for progressive sperm motility were significantly higher (P < 0.05) in spring (53.57 ± 9.97%) in comparison to summer (41.37 ± 10.81%). No significant differences in plasma membrane integrity were found between seasons (P > 0.05). Sperm DNA fragmentation was significantly lower (P < 0.01) in spring in comparison to summer after 0h (4.81 ± 1.87% vs. 8.77 ± 5.78%), 6h (9.00 ± 3.19% vs. 18.73 ± 8.22%) and 24h (14.6 ± 4.13% vs. 30.14 ± 9.85%) of cooled-storage. Pregnancy rates per cycle were also significantly higher (P < 0.01) in spring (50%) in comparison to summer (37%). There was a moderate negative relationship between positive pregnancies and sperm with fragmented DNA (r = - 0.619; P < 0.001). Semen samples associated with moderate fertility levels (Pregnancy rate < 50%) showed a higher percentage of sperm with fragmented DNA compared to samples obtaining higher fertility levels. In conclusion, seasonal variations were found during the breeding season, obtaining lower sperm DNA fragmentation and higher pregnancy rates in spring. Additionally, samples with the highest proportion of sperm with fragmented DNA showed the lowest fertility levels throughout the breeding season.
Comparison of different mathematical models to assess seasonal variations in the longevity of DNA integrity of cooled-stored stallion sperm.
(Andrología, 2020) Rizos, Dimitri; Hidalgo Prieto; Hidalgo Prieto, Manuel; Consuegra González, Cesar; Diaz Jiménez, María; Dorado Martín, Jesús; Crespo Castejón, Francisco
Dynamic assessment of sperm DNA fragmentation (SDF) has shown to give fuller understanding of stallion semen quality; however, there have been limited attempts to use this parameter to investigate seasonal changes in productive functions. The aims of this study were to: (a) establish a reliable mathematical model to describe the longevity of cooled-stored sperm DNA integrity; (b) to examine the effect of seasonal variations on SDF. Ejaculates were cooled to 5°C, and SDF was analysed after 0, 6 and 24 hr of storage. The coefficient of determination (R2) was calculated after fine-tuning linear (LIN), exponential (EXP) and second order polynomial (POL) models. R2 was significantly higher (p < .001) for POL than for LIN and EXP. The rate of DNA degradation was calculated using the slopes of POL equations. After assessing the rate of change of the POL functions, significant differences between the acceleration of DNA fragmentation were found (p < .01) among seasons, being higher for winter and summer than spring and autumn. In conclusion, DNA analysis of stallion sperm fits better to a second order polynomial mathematical model, being spring the best season to collect and process cooled stallion semen in order to maintain the DNA integrity of the stallion sperm.
Determining ACTB, ATP5B and RPL32 as optimal reference genes for quantitative RT-PCR studies of cryopreserved stallion semen
(Animal Rrepoduction Science, 2014) Sanmartín, M. L.; Pérez Rico, Almudena; Crespo Castejón, Francisco; Vega-Pla, J. L.; De Santiago, A.
Equine germplasm bank management involves not only the conservation and use of semen doses, in addition it can also be a resource to study stallion semen quality and after thaw-ing semen properties for reproductive purposes. A possible criterion to measure quality may be based on differential gene expression of loci involved during spermatogenesis and sperm quality maturation. The rapid degradation of sperm after thawing affects the integrity and availability of RNA. In this study we have analyzed genes expressed in equine cryopreserved sperm, which provided an adequate ampliﬁcation, speciﬁcity, and stability to be used as future reference genes in expression studies. Live spermatozoa were selected from cryopreserved semen straws derived from 20 stallions, through a discontinuous con-centration gradient. RNA puriﬁcation followed a combination of the organic and column extraction methods together with a deoxyribonuclease treatment. The selective ampliﬁ-cation of nine candidate genes was undertaken using reverse transcription and real-time polymerase chain reaction (qPCR) carried out in a one-step mode (qRT-PCR). Speciﬁcities were tested by melting curves, agarose gel electrophoresis and sequencing. In addition, gene stabilities were also calculated. Results indicated that ﬁve out of the nine candidate genes ampliﬁed properly (ˇ-Actin, ATP synthase subunit beta, Protamine 1, L32 ribosomal protein and Ubiquitin B), of which ˇ-Actin and the L32 Ribosomal protein showed the high-est stability thus being the most suitable to be considered as reference genes for equine cryopreserved sperm studies, followed by the ATP synthase subunit beta and Ubiquitin B.
Project number: 382
Elaboración de material docente para la enseñanza de la exploración física del caballo
(2022) Fores Jackson, Paloma; Gutiérrez Cepeda, Luna; Villaescusa Fernández, Alejandra; Villalba Orero, María; Ruiz de León Robledo, María de los Ángeles; Crespo Castejón, Francisco; Dominuez Gimbernat, Monica
El objetivo principal del presente proyecto fue incorporar una serie de recursos educativos virtuales que facilitaran e implementaran la enseñanza de la exploración física del caballo.
Effect of single-layer centrifugation or washing on frozen-thawed donkey semen quality: Do they have the same effect regardless of the quality of the sample?
(Theriogenology, 2015) Ortiz Jaraba, Isabel; Dorado Martín, Jesús; Morrell, Jane M; Crespo Castejón, Francisco; Gosálvez Berenguer, Jaime; Gálvez, MJ; Acha Valls, Daniel; Hidalgo Prieto, Manuel
The aims of this study were to determine the sperm quality of frozen-thawed donkey sperm samples after single-layer centrifugation (SLC) using Androcoll-E in comparison to sperm washing or no centrifugation and to determine if the effect on the sperm quality after SLC or sperm washing depends on the quality of the sample. Frozen-thawed sperm samples from Andalusian donkeys were divided into three aliquots, and they were processed using three different techniques after thawing: uncentrifuged diluted control (UDC), sperm washing (SW), and SLC. Afterward, sperm quality index was estimated by integrating all parameters (total and progressive sperm motility, membrane integrity, and DNA fragmentation) in a single value. The relationship between the sperm quality of thawed UDC samples and the effect on sperm parameters in SW and SLC-selected samples was assessed. Sperm quality index was significantly higher (P < 0.001) in SLC (0.8 ± 0.0) samples than that in UDC (0.6 ± 0.0) and SW (0.6 ± 0.0) samples, regardless of the sperm quality index after thawing of the sperm sample. In conclusion, SLC of frozen-thawed donkey spermatozoa using Androcoll-E-Small can be a suitable procedure for selecting frozen-thawed donkey sperm with better quality, in particular in those samples where an improvement in motility is needed.