Albacete, Spain
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Anel-Lopez L.,Campus Universitario Sn | Garcia-Alvarez O.,Campus Universitario Sn | Maroto-Morales A.,Campus Universitario Sn | Iniesta-Cuerda M.,Campus Universitario Sn | And 4 more authors.
Animal Reproduction Science | Year: 2015

The potential protective effect of reduced glutathione (GSH) and trolox (TRX), an analogue of vitamin E, supplementation during in vitro culture (2. h, 39. °C) of electroejaculated frozen/thawed red deer sperm was investigated. Cryopreserved sperm were thawed and incubated with no additive (Control) and 1. mM or 5. mM of each antioxidant to find out whether these supplementations can maintain the sperm quality, considering the use of thawed samples for in vitro techniques such as in vitro fertilisation (IVF), sperm sex sorting or refreezing. The effect of GSH on sperm motility was positive compared to TRX which was negative (P<. 0.001). After 2. h of incubation at 39. °C, use of GSH improved motility while TRX supplementation reduced sperm motility compared with Control samples without antioxidant. Use of TRX at both concentrations (1 and 5. mM; TRX1 and TRX5) resulted in lesser percentages of apoptotic sperm (12.4 ± 1.1% and 11.7 ± 0.9%) than GSH1, GSH5 (15.2 ± 1% and 14.6 ± 1.1%) and Control samples (16.9 ± 1.2%) (P<. 0.001). Use of GSH at both concentrations (1 and 5. mM) resulted in greater mitochondrial activity as compared with findings for the Control, TRX1 and TRX5 groups. Results of this study indicate that GSH is a suitable supplement for electroejaculated red deer sperm. It would be necessary to conduct fertility trials (in vivo and in vitro), to assess whether GSH supplementation of thawed red deer sperm could improve fertility rates. © 2015 Elsevier B.V.


Ramon M.,CERSYRA | Perez-Guzman M.D.,CERSYRA | Jimenez-Rabadan P.,CERSYRA | Garcia-Alvarez O.,Campus Universitario Sn | And 5 more authors.
PLoS ONE | Year: 2013

Background: Sperm cryopreservation has become an indispensable tool in biology. Initially, studies were aimed towards the development of efficient freezing protocols in different species that would allow for an efficient storage of semen samples for long periods of time, ensuring its viability. Nowadays, it is widely known that an important individual component exists in the cryoresistance of semen, and efforts are aimed at identifying those sperm characteristics that may allow us to predict this cryoresistance. This knowledge would lead, ultimately, to the design of optimized freezing protocols for the sperm characteristics of each male. Methodology/Principal Findings: We have evaluated the changes that occur in the sperm head dimensions throughout the cryopreservation process. We have found three different patterns of response, each of one related to a different sperm quality at thawing. We have been able to characterize males based on these patterns. For each male, its pattern remained constant among different ejaculates. This latter would imply that males always respond in the same way to freezing, giving even more importance to this sperm feature. Conclusions/Significance: Changes in the sperm head during cryopreservation process have resulted useful to identify the ability of semen of males for freezing. We suggest that analyses of these response patterns would represent an important tool to characterize the cryoresistance of males when implemented within breeding programs. We also propose follow-up experiments to examine the outcomes of the use of different freezing protocols depending on the pattern of response of males. © 2013 Ramón et al.


Del Olmo E.,Campus Universitario Sn | Bisbal A.,Campus Universitario Sn | Garcia-Alvarez O.,Campus Universitario Sn | Maroto-Morales A.,Campus Universitario Sn | And 4 more authors.
Reproduction, Fertility and Development | Year: 2015

The aim of the present study was to evaluate the effect of sperm reactive oxygen species (ROS) production and DNA changes on male fertility. For that purpose, six rams with significantly different pregnancy rates were used; these were classified as having high fertility, i.e. 59.4% average pregnancy rate, or low fertility, i.e. 23.1% average pregnancy rate. Sperm quality was assessed after a two-step process of sample thawing followed by an incubation of 2h, either in the freezing extender (37°C) or after dilution in synthetic oviductal fluid (SOF; 38°C, 5%CO2). Sperm viability (YO-PRO-1), ROS production (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein acetyl ester (CM-H2DCFDA)) and undamaged chromatin (sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling, chromomycin A3) were evaluated by flow cytometry. Although no significant differences in sperm viability were observed, our results showed increased ROS production during incubation in the freezing extender as well as in SOF medium. Comparison between fertility groups showed significant differences in ROS production after 2h of incubation for the two treatments. Regarding DNA integrity, our results showed no significant differences either between treatments and incubation times or fertility groups. Linear regression analysis showed that ROS production determined by CM-H2DCFDA was a good indicator parameter for in vivo male fertility of SOF-incubated samples, yielding a fair correlation between both parameters (r≤-0.92). These results indicate that detection of ROS production by CM-H2DCFDA and flow cytometry after 2h of incubation in SOF could be a useful procedure for predicting fertility of ram spermatozoa. © 2015 CSIRO.


Anel-Lopez L.,Campus Universitario Sn | Martinez-Rodriguez C.,University of León | Soler A.J.,Campus Universitario Sn | Fernandez-Santos M.R.,Campus Universitario Sn | And 2 more authors.
Animal Reproduction Science | Year: 2015

Single Layer Centrifugation is a useful technique to select sperm with good quality. The use of selection methods such as Androcoll could become an important tool to improve the quality of sperm samples and therefore to improve other artificial reproductive techniques such as sperm sex sorting, in vitro fertilization or AI. The aim of this study was to evaluate the effect of a Single Layer Centrifugation with Androcoll-S on the sperm quality of red deer sperm samples of two different origins, electroejaculated samples and epididymal samples obtained post-mortem, after thawing and after an incubation for 2. h at 37. °C. Sperm motility, viability, membrane permeability, mitochondrial activity, acrosomal status and DNA fragmentation were determined for all samples. The samples selected by Androcoll-S showed an improvement in sperm kinematics compared to unselected samples after thawing and after incubation. The same effect was observed in parameters such as viability, mitochondrial activity or acrosomal status which were improved after the selection. In contrast, no difference was found in DNA fragmentation between selected and unselected samples within the same sperm type. We conclude that sperm selection by SLC with Androcoll-S after thawing for red deer sperm of both types is a suitable technique that allows sperm quality in both types of sperm samples to be improved, thereby improving other assisted reproductive techniques. Further studies (IVF and in vivo fertilization) are required to determine whether this improvement can increase fertility, as has been shown for other species. © 2015 Elsevier B.V.


Garcia-Alvarez O.,Campus Universitario Sn | Maroto-Morales A.,Campus Universitario Sn | Ramon M.,Cersyra Centro Regional Of Seleccion Y Reproduccion Animal | Del Olmo E.,Campus Universitario Sn | And 5 more authors.
Reproduction, Fertility and Development | Year: 2014

The present study evaluated modifications occurring in thawed ram spermatozoa during incubation in different media that supported in vitro capacitation and fertilisation, and examines how these changes relate to IVF. Thawed sperm sampleswere incubated under capacitating (Cap) and non-capacitating (non-Cap) conditions for 0, 1 and 2 h and used in an IVF test. During incubation, changes related to membrane status and the motility pattern of spermatozoa were assessed, the latter being used to characterise sperm subpopulations. Asignificantly greater increase (P≤0.05) in the percentage of spermatozoa with higher membrane fluidity was observed in samples incubated with Cap medium from the beginning of incubation. In addition, changes over time in the distribution of the motile subpopulation were particularly evident when spermatozoa were incubated with Cap medium, with a noted increase in spermatozoa classified as 'hyperactivated like', with major changes occurring after 1 h incubation. Both characteristics (i.e. membrane fluidity and the percentage of the hyperactivated-like subpopulation) were significantly related with in vitro fertility, and only sperm samples incubated with the Cap medium were capable of fertilising oocytes. These results support the idea that changes in sperm membrane fluidity and motility pattern (i.e. an increase in hyperactivated spermatozoa) are needed for fertilisation to take place. © CSIRO 2014.


Ramon M.,CERSYRA | Ramon M.,Campus Universitario Sn | Soler A.J.,Campus Universitario Sn | Ortiz J.A.,Medianilla SL Finca Las Lomas | And 4 more authors.
Biology of Reproduction | Year: 2013

Sperm design and velocity play key roles in influencing sperm performance and, therefore, can determine fertilization success. Several interspecific studies have demonstrated how these features correlate, and it has been hypothesized that selection may drive changes in these sperm traits. Here, we examine the association between sperm design and swimming velocity in a study conducted at an intraspecific level in Iberian red deer (Cervus elaphus hispanicus). We addressed how the structure of different sperm subpopulations, based on sperm morphometry and velocity, are interrelated and, in turn, how they associate with fertility. Our results show that males with high fertility rates have ejaculates with high percentages of spermatozoa exhibiting fast and linear movements and that these are highly correlated with a large proportion of spermatozoa having small and elongated heads. On the other hand, males with low fertility are characterized by a subpopulation structure in which slow and nonlinear as well as small and wide spermatozoa are predominant. These findings provide insight regarding how sperm size and velocity are interrelated and how they both are associated with fertility. © 2013 by the Society for the Study of Reproduction, Inc.


Beracochea F.,University of the Republic of Uruguay | Gil J.,University of the Republic of Uruguay | Sestelo A.,Jardin Zoologico de la Ciudad de Buenos Aires Fundacion Bioandina Argentina | Garde J.J.,Campus Universitario sn | And 3 more authors.
Animal Reproduction Science | Year: 2014

Pampas deer (Ozotoceros bezoarticus) is a native endangered species. Knowledge of the basic spermiogram characteristics and the morphometric descriptors is necessary to effectively develop sperm cryopreservation. In other species, sperm sub-population is related to sperm cryo-resistance. The objective was to provide a general description of the sperm, including sperm head morphometric descriptors, its repeatability, and the existence of sperm sub-populations. Sperm were obtained from adult males by electroejaculation during the breeding season. The motility score was 3.4±0.2 (mean±SEM) and progressive motility was 59.4±3.7%. Ejaculated volume was 413.9±51.0μl, the total number of sperm ejaculated was 321.2±55.4×106. Also, 63.3±3.1% of the sperm were morphologically abnormal and 23.7±2.3% had acrosome damage. The sperm head length was 7.6±0.01μm, width 4.4±0.01μm, area 28.1±0.07μm2 and the perimeter was 21.9±0.04μm. There was a positive relationship among morphometric descriptors and the motility score, overall motility and progressive motility. Also length (P=0.011), width (P=0.003), area (P=0.006) and perimeter (P=0.009) of sperm head obtained in two different collections were positively related. Overall, the low concentration, volume, overall quality and abnormal morphology, and wide variation of these variables may be a limitation for the development of sperm cryopreserved banks. There were three sperm sub-populations with different morphometric characteristics. The morphometric descriptors are maintained similarly among different collections. © 2014 Elsevier B.V.


PubMed | University of the Republic of Uruguay, Jardin Zoologico de la Ciudad de Buenos Aires Fundacion Bioandina Argentina, Campus Universitario sn and Instituto Nacional Of Investigacion Y Tecnologia Agraria Y Alimentaria Inia
Type: Journal Article | Journal: Animal reproduction science | Year: 2014

Pampas deer (Ozotoceros bezoarticus) is a native endangered species. Knowledge of the basic spermiogram characteristics and the morphometric descriptors is necessary to effectively develop sperm cryopreservation. In other species, sperm sub-population is related to sperm cryo-resistance. The objective was to provide a general description of the sperm, including sperm head morphometric descriptors, its repeatability, and the existence of sperm sub-populations. Sperm were obtained from adult males by electroejaculation during the breeding season. The motility score was 3.4 0.2 (mean SEM) and progressive motility was 59.4 3.7%. Ejaculated volume was 413.9 51.0 l, the total number of sperm ejaculated was 321.2 55.4 10(6). Also, 63.3 3.1% of the sperm were morphologically abnormal and 23.7 2.3% had acrosome damage. The sperm head length was 7.6 0.01 m, width 4.4 0.01 m, area 28.1 0.07 m(2) and the perimeter was 21.9 0.04 m. There was a positive relationship among morphometric descriptors and the motility score, overall motility and progressive motility. Also length (P=0.011), width (P=0.003), area (P=0.006) and perimeter (P=0.009) of sperm head obtained in two different collections were positively related. Overall, the low concentration, volume, overall quality and abnormal morphology, and wide variation of these variables may be a limitation for the development of sperm cryopreserved banks. There were three sperm sub-populations with different morphometric characteristics. The morphometric descriptors are maintained similarly among different collections.


PubMed | CERSYRA and Campus Universitario Sn
Type: | Journal: Animal reproduction science | Year: 2015

The potential protective effect of reduced glutathione (GSH) and trolox (TRX), an analogue of vitamin E, supplementation during in vitro culture (2h, 39C) of electroejaculated frozen/thawed red deer sperm was investigated. Cryopreserved sperm were thawed and incubated with no additive (Control) and 1mM or 5mM of each antioxidant to find out whether these supplementations can maintain the sperm quality, considering the use of thawed samples for in vitro techniques such as in vitro fertilisation (IVF), sperm sex sorting or refreezing. The effect of GSH on sperm motility was positive compared to TRX which was negative (P<0.001). After 2h of incubation at 39C, use of GSH improved motility while TRX supplementation reduced sperm motility compared with Control samples without antioxidant. Use of TRX at both concentrations (1 and 5mM; TRX1 and TRX5) resulted in lesser percentages of apoptotic sperm (12.41.1% and 11.70.9%) than GSH1, GSH5 (15.21% and 14.61.1%) and Control samples (16.91.2%) (P<0.001). Use of GSH at both concentrations (1 and 5mM) resulted in greater mitochondrial activity as compared with findings for the Control, TRX1 and TRX5 groups. Results of this study indicate that GSH is a suitable supplement for electroejaculated red deer sperm. It would be necessary to conduct fertility trials (in vivo and in vitro), to assess whether GSH supplementation of thawed red deer sperm could improve fertility rates.


PubMed | Swedish University of Agricultural Sciences, Campus Universitario Sn and University of León
Type: | Journal: Animal reproduction science | Year: 2015

Single Layer Centrifugation is a useful technique to select sperm with good quality. The use of selection methods such as Androcoll could become an important tool to improve the quality of sperm samples and therefore to improve other artificial reproductive techniques such as sperm sex sorting, in vitro fertilization or AI. The aim of this study was to evaluate the effect of a Single Layer Centrifugation with Androcoll-S on the sperm quality of red deer sperm samples of two different origins, electroejaculated samples and epididymal samples obtained post-mortem, after thawing and after an incubation for 2h at 37C. Sperm motility, viability, membrane permeability, mitochondrial activity, acrosomal status and DNA fragmentation were determined for all samples. The samples selected by Androcoll-S showed an improvement in sperm kinematics compared to unselected samples after thawing and after incubation. The same effect was observed in parameters such as viability, mitochondrial activity or acrosomal status which were improved after the selection. In contrast, no difference was found in DNA fragmentation between selected and unselected samples within the same sperm type. We conclude that sperm selection by SLC with Androcoll-S after thawing for red deer sperm of both types is a suitable technique that allows sperm quality in both types of sperm samples to be improved, thereby improving other assisted reproductive techniques. Further studies (IVF and in vivo fertilization) are required to determine whether this improvement can increase fertility, as has been shown for other species.

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