National Wildlife Research Institute IREC

Albacete, Spain

National Wildlife Research Institute IREC

Albacete, Spain
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Garcia-alvarez O.,National Wildlife Research Institute IREC | Maroto-Morales A.,National Wildlife Research Institute IREC | del Olmo E.,National Wildlife Research Institute IREC | Bisbal A.,National Wildlife Research Institute IREC | And 3 more authors.
Animal Reproduction Science | Year: 2012

The aim of this study was to evaluate the effect of semen collection method (artificial vagina compared to electroejaculation), season in which the semen was collected (breeding season compared to non-breeding season), freezing extender (Biladyl ®, Andromed ® and skim milk based extender) and pre-treatment procedure (washing compared to non-washing) on post-thaw semen quality in buck. Ejaculates from seven bucks of the Blanca-Celtibérica breed were collected by artificial vagina and electroejaculation during the breeding (July to December) and non-breeding season (January to June). Samples were split in two aliquots and one of them was washed. Three freezing extenders were evaluated on washing and non-washing sperm samples. Ejaculates collected by artificial vagina had a greater sperm quality after thawing, with greater values (P≤0.05) for SM (sperm motility), NAR (acrosome intact), YO-PRO-1-/PI- (intact spermatozoa), and Mitotracker+/YO-PRO-1- (spermatozoa with active mitochondria) and lower % DFI (DNA fragmentation index). Thawed sperm samples which were collected during the breeding season had greater values (P≤0.05) for NAR, intact spermatozoa and spermatozoa with active mitochondria, than those semen samples obtained during the non-breeding season. Semen freezing with Biladyl ® and Andromed ® resulted in a greater sperm quality (P≤0.05) after thawing in relation to milk-based extender. Washing procedure had no effect on sperm parameters assessed at thawing. Results from the present study suggest that the success of semen cryopreservation in Blanca-Celtibérica goat depends on semen collection method and season, as well as on the extender used. Thus, the post-thaw sperm quality will be greater (P≤0.05) when samples are collected by artificial vagina during the breeding season and when Biladyl ® or Andromed ® are used as freezing extenders. © 2012 Elsevier B.V.


Maroto-Morales A.,National Wildlife Research Institute IREC | Berlinguer F.,University of Sassari | Fernandez-Santos M.R.,National Wildlife Research Institute IREC | Fernandez-Santos M.R.,Institute of Regional Development IDR | And 7 more authors.
Theriogenology | Year: 2011

The aim of this work was to study the effect of storage temperature during the transport of ovaries on cleavage and blastocyst rates in Iberian red deer, because wild populations of this subspecies are usually far from laboratories. A total of 472 ovaries from 236 Iberian hinds were recovered and maintained in saline solution at 5-8 °C or 20-25 °C for 12 h. After storage, aspirated oocytes were matured with FSH/LH or EGF and the developed embryos were cultured with oviduct epithelial cells monolayer (OCM). A higher (P = 0.009) cleavage rate was obtained when the ovaries were stored at 5-8 °C. However, there were no differences between both storage temperatures in relation to the percentage of blastocysts obtained. Considering the management and production systems of Iberian red deer, this study provides important information about the ovary storage temperature during transport with the purpose of assuring an optimal in vitro embryo production. © 2011 Elsevier Inc.


Malo A.F.,Smithsonian Institution | Malo A.F.,Imperial College London | Martinez-Pastor F.,University of León | Martinez-Pastor F.,National Wildlife Research Institute IREC
Biology of Reproduction | Year: 2010

Mice (Peromyscus leucopus noveboracensis) from a captive-breeding program were used to test the effects of three genetic breeding protocols (minimizing mean kinship [MK], random breeding, and selection for docility [DOC]) and inbreeding levels on sperm traits and fertility. Earlier, in generation 8, one DOC replicate went extinct because of poor reproductive success. By generation 10, spermatozoa from DOC mice had more acrosome and midpiece abnormalities, which were shown to be strong determinants of fertility, as well as lower sperm production and resistance to osmotic stress. In addition, determinants of fertility, including male and female components, were assessed in a comprehensive manner. Results showed that the probability (P) of siring litters is determined by sperm number, sperm viability, and midpiece and acrosome abnormalities; that the P of siring one versus two litters is determined by tail abnormalities; and that the total number of offspring is influenced by female size and proportion of normal sperm, showing the relative importance of different sperm traits on fertility. On average, males with 20% normal sperm sired one pup per litter, and males with 70% normal sperm sired eight pups per litter. Interestingly, the proportion of normal sperm was affected by docility but not by relatively low inbreeding. However, inbreeding depression in sperm motility was detected. In the MK group, inbreeding depression not only affected sperm motility but also fertility: An increase in the coefficient of inbreeding (f ) of 0.03 reduced sperm motility by 30% and translated into an offspring reduction of three pups in second litters. A genetic load of 48 fecundity equivalents was calculated. © 2010 by the Society for the Study of Reproduction, Inc.


Dominguez-Rebolledo A.E.,National Wildlife Research Institute IREC | Martinez-Pastor F.,University of León | Bisbal A.F.,National Wildlife Research Institute IREC | Ros-Santaella J.L.,National Wildlife Research Institute IREC | And 6 more authors.
Reproduction in Domestic Animals | Year: 2011

Contents: Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H2O2) levels on red deer spermatozoa after cryopreservation, and the role of male-to-male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200μm H2O2 for 2h at 37°C. Intracellular reactive oxygen species (H2DCFDA-CM) increased with H2O2 concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200μm. Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50μm H2O2 and above. In a second experiment, samples from seven males were submitted to 0 and 200μm H2O2 for 2h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H2O2 presence. We found that the kinematic parameters reflected male-to-male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H2O2 or after incubation with H2O2. Red deer spermatozoa are relatively resilient to H2O2 after thawing, but it seems to be a great male-to-male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols. © 2010 Blackwell Verlag GmbH.


Maroto-Morales A.,National Wildlife Research Institute IREC | Ramon M.,National Wildlife Research Institute IREC | Soler A.J.,National Wildlife Research Institute IREC | Esteso M.C.,University of León | And 3 more authors.
Theriogenology | Year: 2010

Sperm morphology has been identified as a characteristic that can be useful in the prediction of fertilizing capacity. The aim of the current study was to characterize ram sperm heads morphometrically as a basis for future studies on the relationship between sperm quality and male fertility. For this purpose, ejaculates from 241 mature rams (Ovis aries) belonging to 36 different dairy herds were used to evaluate sperm head morphometry by means of the Sperm-Class Analyzer. Sperm samples, collected by artificial vagina, were diluted in phosphate buffered saline (PBS) for the analysis. A microscope slide was prepared from single-diluted fresh sperm samples. Slides were air-dried and stained with Hemacolor. A minimum of 115 sperm heads were analyzed from each male. Each sperm head was measured for four primary parameters (area, perimeter, length, width), and four derived parameters of head shape were obtained. Significant differences in sperm head morphometry were found between rams (CV for morphometric parameters ranging from 0.9 to 10.1), and there were marked differences in the sperm morphometric composition of the ejaculates. For all parameters, within-animal CVs were greater than between-animal CVs. Within-animal CVs ranged from 4.2 to 10.6, showing the high degree of sperm polymorphism present in the sheep ejaculate. Significant differences in sperm head morphometry were found between rams belonging to the different herds (i.e., origin). An important part of the variability observed on morphometric parameters was due to the male itself, with an explained variance ranging from 3.6% for regularity to 34.0% for p2a (perimeter2/[4 × π × area]). The explained variance by the herd of origin of the males ranged from 0.6% for regularity to 10.8% for area. Our results suggest that a genetic component might be responsible for the observed sperm head morphometry differences between herds. © 2010 Elsevier Inc. All rights reserved.


Mata-Campuzano M.,University of León | Alvarez-Rodriguez M.,University of León | Alvarez M.,University of León | Anel L.,University of León | And 3 more authors.
Reproduction in Domestic Animals | Year: 2012

Contents: Thawed ram spermatozoa were incubated at 37°C in the presence of dehydroascorbic acid (DHA), TEMPOL (TPL), N-acetyl-cysteine (NAC) and rutin (RUT), at 0.1 and 1mm, in order to test their effects on sperm physiology. Cryopreserved spermatozoa from four rams were thawed, pooled, washed and incubated in TALP-Hepes with 1mm or 0.1mm of each antioxidant, performing a replicate with induced oxidative stress (Fe2+/ascorbate). Motility (CASA), viability and mitochondrial membrane potential (flow cytometry) were analysed at 2 and 4h. Lipoperoxidation (MDA production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were analysed at 4h. Antioxidants, except DHA 0.1mm, decreased motility and kinematic parameters, but had little effect on viability or mitochondrial activity. Except 1mm DHA, the antioxidants reduced ROS at 4h. Moreover, NAC 1mm, rutin and TEMPOL reduced ROS and DNA damage in the presence of oxidative stress. N-acetyl-cysteine, rutin 1mm and TEMPOL reduced lipoperoxidation in the presence of oxidative stress. However, DHA did not affect lipoperoxidation. At 1mm, DHA increased DNA damage in the absence of oxidative stress. Dehydroascorbic acid effects could arise from spermatozoa having a low capacity for reducing it to ascorbic acid, and it may be tested in the presence of other antioxidants or reducing power. Future research should focus in testing whether the inhibition of motility observed for NAC, rutin and TEMPOL is reversible. These antioxidants might be useful at lower temperatures (refrigerated storage or cryopreservation) when their protective effects could be advantageous. © 2012 Blackwell Verlag GmbH.


Martinez-Pastor F.,University of León | Tizado E.J.,University of León | Garde J.J.,National Wildlife Research Institute IREC | Anel L.,University of León | de Paz P.,University of León
Theriogenology | Year: 2011

Computer-assisted sperm analysis (CASA) allows assessing the motility of individual spermatozoa, generating huge datasets. These datasets can be analyzed using data mining techniques such as cluster analysis, to group the spermatozoa in subpopulations with biological meaning. This review considers the use of statistical techniques for clustering CASA data, their challenges and possibilities. There are many clustering approaches potentially useful for grouping sperm motility data, but some options may be more appropriate than others. Future development should focus not only in improvements of subpopulation analysis, but also in finding consistent biological meanings for these subpopulations. © 2011 Elsevier Inc.


Castellanos P.,National Wildlife Research Institute IREC | Maroto-Morales A.,National Wildlife Research Institute IREC | Garcia-Alvarez O.,National Wildlife Research Institute IREC | Garde J.J.,National Wildlife Research Institute IREC | Mateo R.,National Wildlife Research Institute IREC
Bulletin of Environmental Contamination and Toxicology | Year: 2013

In vitro effects of lead (Pb) on ram (Ovis aries) spermatozoa were studied to establish a threshold level that affects sperm function. Spermatozoa were incubated between 15 and 180 min with Pb concentrations ranging from 0 to 5,000 ng/mL. Sperm motility, acrosome integrity, membrane functionality and sperm viability were all negatively affected by Pb and incubation time. Acrosome integrity was linearly affected by Pb levels at an incubation time of 30 min, and 50 ng/mL was the lowest Pb level producing such effect. These experimental conditions can be appropriate for in vitro studies of the mechanisms of action of Pb on spermatozoa. © 2013 Springer Science+Business Media New York.


PubMed | National Wildlife Research Institute IREC
Type: Comparative Study | Journal: Reproduction in domestic animals = Zuchthygiene | Year: 2011

Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H(2)O(2)) levels on red deer spermatozoa after cryopreservation, and the role of male-to-male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200 m H(2)O(2) for 2 h at 37 C. Intracellular reactive oxygen species (H(2)DCFDA-CM) increased with H(2)O(2) concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200 m. Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 m H(2)O(2) and above. In a second experiment, samples from seven males were submitted to 0 and 200 m H(2)O(2) for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H(2)O(2) presence. We found that the kinematic parameters reflected male-to-male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H(2)O(2) or after incubation with H(2)O(2) . Red deer spermatozoa are relatively resilient to H(2)O(2) after thawing, but it seems to be a great male-to-male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols.


PubMed | National Wildlife Research Institute IREC
Type: Journal Article | Journal: Theriogenology | Year: 2010

Sperm morphology has been identified as a characteristic that can be useful in the prediction of fertilizing capacity. The aim of the current study was to characterize ram sperm heads morphometrically as a basis for future studies on the relationship between sperm quality and male fertility. For this purpose, ejaculates from 241 mature rams (Ovis aries) belonging to 36 different dairy herds were used to evaluate sperm head morphometry by means of the Sperm-Class Analyzer. Sperm samples, collected by artificial vagina, were diluted in phosphate buffered saline (PBS) for the analysis. A microscope slide was prepared from single-diluted fresh sperm samples. Slides were air-dried and stained with Hemacolor. A minimum of 115 sperm heads were analyzed from each male. Each sperm head was measured for four primary parameters (area, perimeter, length, width), and four derived parameters of head shape were obtained. Significant differences in sperm head morphometry were found between rams (CV for morphometric parameters ranging from 0.9 to 10.1), and there were marked differences in the sperm morphometric composition of the ejaculates. For all parameters, within-animal CVs were greater than between-animal CVs. Within-animal CVs ranged from 4.2 to 10.6, showing the high degree of sperm polymorphism present in the sheep ejaculate. Significant differences in sperm head morphometry were found between rams belonging to the different herds (i.e., origin). An important part of the variability observed on morphometric parameters was due to the male itself, with an explained variance ranging from 3.6% for regularity to 34.0% for p2a (perimeter(2)/[4xpixarea]). The explained variance by the herd of origin of the males ranged from 0.6% for regularity to 10.8% for area. Our results suggest that a genetic component might be responsible for the observed sperm head morphometry differences between herds.

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