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

Comparison of different sucrose-based extenders for stallion sperm vitrification in straws
(Reproduction Domestic Animals, 2018) Consuegra González Cesar; Crespo Castejón, Francisco; Dorado Martín, Jesús; Ortiz Jaraba, Isabel; Diaz Jiménez, María; Pereira Aguilar, Blasa; Hidalgo Prieto, Manuel
Vitrification of sperm is based on high-speed freezing by direct exposure to liquid nitrogen using non-permeable cryoprotectants, mainly disaccharides; yet, the concentration of cryoprotectants has a species-specific effect on the sperm cell. The aim of this study was to assess different sucrose concentrations for stallion sperm vitrification. Semen samples (n = 9) were collected from three stallions, centrifuged and resuspended to a concentration of 50 × 106 sperm/ml in a base extender (INRA96 + 1% of bovine serum albumin) with three different sucrose concentrations (Molar): 20 mM (S1), 100 mM (S2), or 200 mM (S3). Then, sperm were filled in covered 0.25 ml straws and directly plunged into liquid nitrogen. For warming, 0.25 ml straw was pulled out the covering straw and immersed in 3 ml of INRA96 at 43°C, with gentle pipetting to accelerate the melting. Total (TM, %) and progressive sperm motility (PM, %) were analysed using computer-assisted sperm analysis. Plasma (PMI, %) and acrosome membrane integrity (AIS, %) were assessed under epifluorescence microscopy. Post-warmed sperm parameters were compared between treatments by ANOVA. S2 showed significantly higher values in comparison with S1 and S3 for TM (S2 = 54.7 ± 5.5a ; S1 = 29.1 ± 3.3b ; S3 = 28.6 ± 3.0b ; p < 0.001) and PM (S2 = 31.3 ± 3.8a ; S1 = 18.5 ± 2.6b ; S3 = 17.7 ± 2.9b ; p < 0.01), respectively. No significant differences were found among treatments for PMI (S2 = 70.3 ± 5.2; S1 = 67.4 ± 4.3; S3 = 70.0 ± 3.7) neither for AIS (S2 = 57.1 ± 3.9; S1 = 53.9 ± 4.2; S3 = 57.0 ± 7.9). In conclusion, a concentration of 100 mM sucrose is recommended for stallion sperm vitrification in straws.
Project number: 36
Modelo docente de palpación del aparato reproductor en bovino como alternativa al uso de materiales de prácticasblanco
(2023) Sánchez Calabuig, María Jesús; Blanco Murcia, Francisco Javier; Moreno Gonzalo, Javier; Fominaya García, Hernán Luis; Zanitoni, Morgane; Serres Dalmau, María Consolacion; Crespo Castejón, Francisco; Domínguez Gimbernat, Mónica; Gutiérrez Cepeda, Luna; Mejías López, Elena; Sánchez Calabuig, María Jesús
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.
Stallion sperm selection prior to freezing using a modified colloid swim-up procedure without centrifugation
(Animal Rerpoduction Science, 2017) Hidalgo Prieto, Manuel; Ortiz Jaraba, Isabel; Dorado Martín, Jesús; Morrel, Jane M; Gosálvez Berenguer, Jaime; Consuegra González, Cesar; Diaz Jimenez,María; Pereira Aguilar, Blasa; Crespo Castejón, Francisco
The aims of this study were to: 1) develop a new method for stallion sperm selection using a modiﬁed swim-up procedure through a colloid and 2) evaluate its impact in good quality eja-culates from bad freezers in comparison to methods involving centrifugation such as single layer centrifugation and sperm washing. Ejaculates were processed before freezing using three dif-ferent procedures: sperm washing (SW), colloid single layer centrifugation (SLC) and a modiﬁed colloid swim-up (SU). After semen processing, sperm recovery rates were measured and sperm were frozen. Post-thaw sperm motility (assessed by computer-assisted sperm analysis), normal forms and plasma membrane integrity (evaluated under bright-ﬁeld and ﬂuorescence microscopy respectively), and DNA fragmentation (assessed by the Sperm-Halomax kit) were compared be-tween treatments. Sperm recovery rates were similar between SU and SLC but lower than SW. Sperm motility after thawing was lower in SU in comparison to SLC and SW, maybe due to the incomplete removal of seminal plasma before freezing. Sperm DNA fragmentation was lower in SU and SLC selection methods, particularly in SLC selected samples during the ﬁrst 6 h of in-cubation. The remaining sperm parameters assessed were similar among treatments. In conclu-sion, SLC is more suitable than SW and SU to process stallion semen prior to freezing, in parti-cular when sperm DNA damage is suspected. Further studies are needed in order to determine the potential beneﬁts of SU in samples where centrifugation is not necessary, such as epididymal sperm, ejaculate fractioning or post-thaw semen samples.
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.
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.
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.
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.