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 10

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.
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.
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.
Comparative Semen Microbiota Composition of a Stallion in a Taylorella equigenitalis Carrier and Non-Carrier State
(Animals (based), 2020) Quiñones Pérez, Carlota; Martinez Martinez, Amparao; Crespo Castejón, Francisco; Vega Plá, Jose Luis
Contagious equine metritis is receiving renewed attention due to the continuous detection of carriers in apparent agent-free farms. Interactions of Taylorella with the seminal microflora maybe the plausible cause behind these spontaneous changes of the carrier state. Accordingly, the aim of this study was to compare the differences in the seminal microbiome composition of one stallion in the contagious equine metritis carrier state and non-carrier state. Samples were cryopreserved after their extraction. Cell disruption was performed by high-speed homogenization in grinding media. Bacterial families were identified via V3 amplification of the 16S rRNA gene and Ion Torrent sequencing. Only bacterial families with relative abundance above 5% were taken into consideration. The positive sample contained a strong dominance of Corynebacteriaceae (37.75%) and Peptoniphilaceae (28.56%). In the negative sample, the Porphyromonadaceae (20.51%), Bacteroidaceae(19.25%) and Peptoniphilaceae (18.57%) families prevailed. In conclusion, the microbiome seminal composition varies when an individual carries Taylorella from when it is free of it. The wider differences were found in the Corynebacteriaceae, Porphyromonadaceae and Bacteroidaceae families. Due to the limitations of a single-case analysis, further studies are needed for a better understanding of the stallion seminal microflora interactions
Sex-sorted bovine spermatozoa and DNA damage: I. Static features
(Theriogenology, 2011) Gosálvez Berenguer, Jaime; Ramírez , Miguel Ángel; López Fernández, Carmen; Crespo Castejón, Francisco; Evans, K. M.; Kjelland, M. E.; Moreno, Juan Francisco
This study examined the static response of Spermatozoa DNA Fragmentation (SDF) after sex selection in bulls using a MoFlo® SX (Beckman Coulter, Miami FL) spermatozoa sorter to produce three different subpopulations: 1) Spermatozoa bearing X- chromosomes with a purity of 95%, 2) Spermatozoa bearing Y-chromosomes with a purity of 95%, and 3) non-viable spermatozoa. The static response of SDF refers to the baseline values observed for DNA damage when analyzed pre- and post sex-sorting. Results showed that while the baseline level SDF in pre-sorted bull spermatozoa samples ranged from 5.3% to 11% with an average of 7.9% ± 2.1%, the level of SDF obtained in X- and Y-chromosome sorted samples was much lower (3.1% ± 1.9%) and statistical differences were obtained after comparing both groups (P < 0.01). Spermatozoa containing a fragmented DNA molecule tend to be accumulated in the non-viable subpopulation. The baseline SDF level in X- and Y-chromosome sorted subpopulations is reduced, by 63% on average when compared to the values obtained in the neat semen sample. Different bulls exhibit unique SDF reduction efficiencies via the X- and Y-chromosome sex selection process.
Optimization of the Equine-Sperm Freeze Test in Purebred Spanish Horses by Incorporating Colloidal Centrifugation
(Animals (based), 2022) Bláquez Sarro, Juan Carlos; Gutiérrez Cepeda, Luna; Crespo Castejón, Francisco; Serres Dalmau, María Consolacion
The Purebred Spanish Horse, according to our clinical experience, is characterized by having a high number of stallions that do not meet the international commercial recommendations forequine-sperm cryopreservation. This means that artificial insemination with frozen semen from these stallions is less widespread than in other breeds. In this study, we investigated if the incorporation ofsingle-layer colloidal centrifugation prior to cryopreservation in clinical conditions could increase the number of ejaculates of Purebred Spanish stallions suitable for this processing, observing the influence of centrifugation and freezing extender protocol on post-thawed sperm motility. Using colloidal centrifugation, the percentage of ejaculates available to be frozen was increased from 35% (6/17) to71% (12/17), doubling the number of samples that could have been subjected to cryopreservation. We only found significant differences in linearity (LIN) and lateral head displacement (ALH) after5 min of incubation at 37 ◦C between colloidal and simple centrifugation processing techniques. No significant differences were found between the two different colloidal protocols in any of the variables considered. Colloidal centrifugation allowed us to obtain, from worse fresh-quality ejaculates, thawed sperm doses with similar quality to that of good-quality ejaculates. BotuCrio® produced, in general higher motility parameters and its characteristics than the other extenders analyzed, with significant differences found in comparison to Inra-Freeze® and Lac-Edta in both total (MOT) and progressive motility (PMOT) when using colloidal centrifugation and only in PMOT when applying simple centrifugation. Colloidal centrifugation optimized the efficiency of cryopreservation, as it allowed usto increase the number of ejaculates of Purebred Spanish Horses suitable to be frozen. Including these semen processing techniques in the freeze test could help to optimize equine-sperm cryopreservation protocols, especially when dealing with individuals or breeds for which initially low sperm quality prevents or limits their inclusion in sperm cryopreservation programs
Equivalent seminal characteristics in human and stallion at first and second ejaculated fractions
(Andrología, 2017) De la Torre, Javier; Sánchez Martín, P; Gosálvez Berenguer, Jaime; Crespo Castejón, Francisco
Sperm quality was assessed in normozoospermic human (n = 10) and Spanish breed stallion (n = 10) after sperm fractionation during ejaculation. The first ejaculated frac-tion was separated from the second. A third sample was reconstituted using equiva-lent proportion of both fractions (RAW). Fraction 1, Fraction 2 and RAW semen were incubated for 30 min at 37°C to homogenise the impact of iatrogenic damage be-tween both species. Sperm concentration, motility and sperm DNA damage were as-sessed in each fraction and RAW semen. The results showed two important facts: (i) spermatozoa confined at Fraction 1 exhibit superior parameters than those included at Fraction 2 in both species, and (ii) there is a certain level of concordance between spe-cies in the proportion of benefit observed when Fraction 1 is compared to RAW semen. Altogether, these results call into question whether the standard practice of whole ejaculate collection can be considered the best strategy when using male gam-etes for artificial insemination. In fact, the reconstituted RAW semen exhibits poorer semen characteristics than those found in Fraction 1.
DNA fragmentation in frozen sperm of Equus asinus: Zamorano-Leonés, a breed at risk of extinction
(Theriogenology, 2008) Cortés Gutiérrez, Elvira; Crespo Castejón, Francisco; Gosálvez Berenguer, Jaime; Dávila Rodríguez, M. I.; López Fernández, Carmen; Gosálvez, Antonio
The dynamics of sperm DNA fragmentation (sDF) and sperm viability were analyzed in frozen-thawed sperm samples of Equus asinus (Zamorano-Leonés), a breed at risk of extinction. Sperm DNA fragmentation was assessed using an adaptation of the sperm chromatin dispersion test developed for stallions in five different frozen samples. Sperm were thawed and incubated at different temperatures (37 °C, 25 °C, and 4 °C) and sDF was assessed at different times and compared. The mean sDF after thawing at the beginning of the experiment was 18.20 ± 14.77% and did not differ significantly from the results of a neutral comet assay (22.0 ± 19.34%). The tendency in the sDF of all donkeys indicated that sperm DNA is more sensitive to breakage when incubated at 37 °C than when incubated at 25 °C or 4 °C. Interestingly, the tendency was not the same when different animals were compared, and differences in sDF dynamics were established among individuals. sDF correlated negatively with sperm viability in some individuals but not in others. From a conservation perspective, sDF analysis may offer a new way to assess sperm quality in endangered breeds in order to identify and select the best semen samples for artificial reproduction purposes. In particular, we recommend for artificial insemination the use of semen samples with a slow increase in sDF with time after thawing.
The effect of two pre-cryopreservation single layer colloidal centrifugation protocols in combination with different freezing extenders on the fragmentation dynamics of thawed equine sperm DNA
(Acta Veterinaria Scandinávica, 2012) Gutiérrez Cepeda, Luna; Fernández, Alvaro; Crespo Castejón, Francisco; Ramírez, Miguel Ángel; Gosálvez Berenguer, Jaime; Serres Dalmau, María Consolacion
Variability among stallions in terms of semen cryopreservation quality renders it difficult to arrive at a standardized cryopreservation method. Different extenders and processing techniques (such us colloidal centrifugation) are used in order to optimize post-thaw sperm quality. Sperm chromatin integrity analysis is an effective tool for assessing such quality. The aim of the present study was to compare the effect of two single layer colloidal centrifugation protocols (prior to cryopreservation) in combination with three commercial freezing extenders on the post-thaw chromatin integrity of equine sperm samples at different post-thaw incubation (37°C) times (i.e., their DNA fragmentation dynamics). Results Post-thaw DNA fragmentation levels in semen samples subjected to either of the colloidal centrifugation protocols were significantly lower (p<0.05) immediately after thawing and after 4 h of incubation at 37°C compared to samples that underwent standard (control) centrifugation. The use of InraFreeze® extender was associated with significantly less DNA fragmentation than the use of Botu-Crio® extender at 6 h of incubation, and than the use of either Botu-Crio® or Gent® extender at 24 h of incubation (p<0.05). Conclusions These results suggest that single layer colloidal centrifugation performed with extended or raw semen prior to cryopreservation reduces DNA fragmentation during the first four hours after thawing. Further studies are needed to determine the influence of freezing extenders on equine sperm DNA fragmentation dynamics.