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F J Peña

Publications and source records attributed to F J Peña.

At least 19 recordsLinked to original sources
PubMed2006

Detection of early changes in sperm membrane integrity pre-freezing can estimate post-thaw quality of boar spermatozoa.

A recently developed triple staining (SNARF-1/YO-PRO-1/ethidium homodimer) was used to assess early changes in boar sperm membrane integrity (MI) with the results of cryopreservation procedures and to seek for correlations among MI-spermatozoa in pre-freeze semen and its freezeability. Ejaculates from five boars were evaluated in the fresh and frozen-thawed (FT) state, and its freezeability defined as % of membrane intactness, MI% (MI%=% of FT-spermatozoa with intact membranes x 100 divided by the % of pre-freeze spermatozoa with intact membranes) estimated. Significant differences were found among boars for freezeability (MI%) and motility post-thaw (%). Interestingly, significant correlations were found between the percentage of YO-PRO-1-positive spermatozoa and freezeability (R=0.440, p<0.01), indicating this new triple staining can be used to safely disclose among ejaculates prior to freezing.

Animals↗
PubMed2006

In vitro evaluation of the quality and fertilizing capacity of boar semen frozen in 0.25 ml straws.

Twenty-two boar ejaculates were frozen in 0.25 ml straws using a controlled cooling rate, then evaluated in vitro in order to assess: (i) the extent to which a range of semen evaluation parameters accurately characterize sperm quality, (ii) the value of quality assessment in the characterization of long-term sperm survival and fertility and (iii) the suitability of the cryopreservation protocol used for yielding semen with good quality and fertilizing capacity. Motility with or without caffeine, plasma membrane integrity (PMI) evaluated with both propidium iodide (PI) and Hoechst 33258, and acrosome morphology were studied, the ejaculates being then classified into five quality groups. A thermoresistance test and a homologous in vitro fertilization test were applied to selected ejaculates of these groups. Caffeine-stimulated motility and PMI evaluated with PI provided better estimations of semen quality than the other tests of motility, PMI, or acrosome morphology, but this quality assessment could not reveal differences in fertilizing capacity or thermoresistance among ejaculates. Over 43% spermatozoa survived cryopreservation in 19 of the 22 ejaculates, with inter-boar and inter-ejaculate variability in the freezing success being observed. The fertilizing capacity, however, was seriously affected by the process regardless of the semen quality. It is concluded that caffeine-stimulated motility and PMI evaluated with PI give accurate information on sperm quality, but important aspects to the valuation of semen such as thermoresistance and fertilizing capacity are not revealed by this quality study. Moreover, the approach of selecting suitable protocols of cryopreservation does not appear to be sufficient for guaranteeing systematically good quality and fertilizing capacity in the frozen-thawed semen.

Acrosome↗
PubMed2006

A three-step statistical procedure to identify sperm kinematic subpopulations in canine ejaculates: changes after cryopreservation.

Strong evidences suggest that many different sperm subpopulations co-exist within the mammalian ejaculate. These subpopulations have been identified in a number of species; however, to the best of our knowledge, no data exist regarding the existence of sperm subpopulations within the canine ejaculate. Ejaculates were obtained by masturbation from four mongrels and processed using a standard freezing protocol. Motility data were analysed before and after cryopreservation using a computer assisted sperm analysis (CASA) system ISAS. On raw data, a principal component analysis (PCA) was performed to reduce the number of motility descriptors to a few informative variables, and then a K-means cluster procedure was performed and then a regression analysis to validate the clusters obtained in the second analysis. ANOVAs and chi-squared analyses were used to compare clusters and males. PCA revealed that two principal components represented more that the 88% of the variance with eigenvalues of 3.25 and 3.02, respectively. The clustering and discriminant analysis using curvilinear velocity and linear velocity as variables revealed the existence of 11 sperm subpopulations--four of them characterized by high velocities, two by medium values and five by low velocities. After freezing-thawing, nine subpopulations were found--four of high velocities, two of medium and three of low velocities. It is concluded that freezing-thawing not only impairs sperm motility but also produces changes in the sperm subpopulation structure in the canine ejaculate, that the evaluation of the sperm structure subpopulations is a better indicator of semen quality and freezeability than the use of mean values, and that two sperm motility quality indexes can be used to resume of the variables obtained from the CASA analysis.

Analysis of Variance↗
PubMed2006

Semen technologies in dog breeding: an update.

Pure dog breeding is experiencing growing interest. Assisted reproductive technologies are each time demanded more by the small animal practitioner. In this paper different semen technologies are briefly reviewed under the perspective of the authors. In addition, new trends in the evaluation of canine ejaculates, developed in the laboratory of the authors are discussed. The existence of sperm subpopulations defined based both on kinematics and morphometric characteristics of the spermatozoa is now widely accepted by the scientific community. In the laboratory of the authors new, simpler and more straightforward statistical tools have been used to disclose the sperm subpopulation structure in the canine ejaculate. The study of the sperm subpopulation structure and its changes after cryopreservation has allowed us to gain an insight into the differences in freezeability among dogs, and also predict to a great extent the outcome of a cryopreservation procedure of a given semen sample. New techniques to evaluate early membrane changes have also been recently developed in our laboratory.

Animals↗
PubMed2005

Do different portions of the boar ejaculate vary in their ability to sustain cryopreservation?

Previous studies have shown sperm quality post-cryopreservation differs depending on the fraction of the seminal plasma boar spermatozoa are fortuitously contained in. As such, spermatozoa contained in the first 10 mL of the sperm-rich fraction (portion I) have better sustained handling procedures (extension, handling and freezing/thawing) than those contained in the ulterior part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction, portion II). However, those studies were performed using pooled samples. In the present study, individual ejaculates were used. Split ejaculates (portions I and II) from five boars were frozen and thawed using a conventional freezing protocol, followed by computer-assisted motility and morphology analysis (CASA and ASMA, respectively), as well as an Annexin-V assay for spermatozoa from each boar and ejaculate portion. Significant differences between portions were observed in all ASMA-derived variables, except in one boar. Also significant differences were observed between boars and ejaculate portions in sperm quality post-thaw. We identified, however, boars showing best results of motility and sperm membrane integrity post-thaw in portion I, while in other boar the best results was observed in portion II. It is concluded that the identification of the ejaculate portion more suitable to sustain cryopreservation in each individual boar may be a readily applicable and easy technique to diminish variation in sperm freezability among boars.

Animals↗
PubMed2005

Changes in tyrosine phosphorylation associated with true capacitation and capacitation-like state in boar spermatozoa.

Capacitation is defined as a series of events that render boar sperm competent to fertilize, either in vivo or in vitro. Moreover, preliminary stages of cryopreservation of spermatozoa involving cooling to 5 degrees C have been shown to induce capacitation-like changes in boar spermatozoa. Capacitation of boar spermatozoa is accompanied by protein phosphorylation, however the relationship between both processes is poorly understood. Capacitation status was assessed by chlortetracycline (CTC) staining. Changes in protein tyrosine phosphorylation were examined in pre-cleared whole cell lysates using a specific anti-phosphotyrosine monoclonal antibody. Our results in boar spermatozoa show a significant positive correlation between p32 tyrosine phosphorylation levels and percentage of capacitated (CTC pattern B) spermatozoa. Moreover, incubation of boar spermatozoa with two unrelated tyrosine kinase inhibitors induces a significant reduction in the percentages of capacitated and acrosome-reacted (AR) boar spermatozoa and a reduction in the p32 tyrosine phosphorylation. In our conditions, cooling boar spermatozoa to 5 degrees C and rewarming to 39 degrees C in a noncapacitating medium results in similar CTC staining patterns to those obtained after incubation of boar sperm for 1 or 4 hr at 39 degrees C in a capacitating medium. However, cooled-rewarmed fails to induce an increase in p32 tyrosine phosphorylation in boar spermatozoa. Moreover, CTC staining patterns of cooled-rewarmed spermatozoa do not change after incubation with a tyrosine kinase inhibitor. In conclusion, our results show a direct relationship between capacitation and tyrosine phosphorylation and suggest that p32 tyrosine phosphorylation levels could be used as a marker of the true capacitation changes observed in boar spermatozoa. Moreover, our results show that true capacitation and capacitation-like changes induced after cooling involve alternative intracellular tyrosine phosphorylation pathways in boar spermatozoa.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2005

A new and simple method to evaluate early membrane changes in frozen-thawed boar spermatozoa.

Detection of early changes in the sperm plasma membrane during cryopreservation is of utmost importance when designing freezing protocols and has previously been studied in the pig species using annexin-V detection of phosphatidylserine translocation. In the present study we designed a new assay to detect these changes in boar spermatozoa, based on the slight increase of sperm membrane permeability occurring during the early stages of cryoinjury, using the combination of three fluorescent probes, SNARF-1, YO-PRO-1 and ethidium homodimer. Four ejaculates from five different boars were frozen-thawed and flow cytometrically (FC) evaluated as paired samples. One of the samples was assayed using the annexin-V/propidium iodide staining and the other sample was evaluated using the new triple staining. Using this combination of probes, four sperm subpopulations were easily detected: viable, with stable membranes (SNARF-1 positive cells), and three with compromised membranes, one of YO-PRO-1+/Eth- cells, one ethidium homodimer+ spermatozoa and, finally spermatozoa stained both with YO-PRO-1 and ethidium homodimer (YO-PRO-1+/Eth+). The latter three categories corresponded to dead spermatozoa, but with different degree of membrane damage, being YO-PRO+/Eth- an earlier stage of membrane destabilization, (manifested by an increase in membrane permeability, while still maintaining membrane integrity) than YO-PRO+/Eth+. A method agreement analysis between both methods was performed revealing good agreement, although the percentage of live cells was 9.44% larger for the triple stain than the annexin-V assay. The new assay stained all sperm sub-populations present in the sample, making it especially suitable for both fluorescence microscopy and flow cytometry, facilitating the exclusion of debris and egg-yolk particles when using FC.

Animals↗
PubMed2005

Do computer-assisted, morphometric-derived sperm characteristics reflect DNA status in canine spermatozoa?

It is widely accepted that sperm morphology is a strong indicator of semen quality. As the sperm head mainly comprises the sperm DNA, it is have been proposed that subtle changes in sperm morphology may be related to abnormal DNA content. Semen from four mongrel dogs was used to investigate DNA quality by means of the sperm chromatin structure assay (SCSA), and for computerized sperm morphometry (CASMA). Each sperm head was measured for nine primary parameters [head area (A), head perimeter (P), head length (L), head width (W), midpiece width (w), midpiece area (a), distance (d) between the major axes of the head and midpiece, angle (theta) of divergence of the midpiece from the head axis] and four parameters of head shape [FUN1, L/W; FUN2, 4piA/P(2); FUN3, (L - W)/(L + W); FUN4, piLW/4A]. Significant differences were found in all CASMA-derived parameters among dogs (p < 0.001). Linear regression models including sperm head shape factors 1, 3 and 4 predicted the extent of DNA denaturation (p < 0.001). We conclude that the CASMA analysis can be considered a powerful tool to improve the spermiogram.

Animals↗
PubMed2005

Inhibition of phosphatidylinositol 3-kinase modifies boar sperm motion parameters.

Motility is the most widely used indicator of sperm quality. Besides modulation by the cAMP pathway little is known regarding the intracellular pathways that regulate boar sperm motility. Recently the role of phosphatidylinositol 3-kinase (PI3-K) in the regulation of human sperm motility has been described. Therefore, the aim of this study was to investigate the role of PI3-K in boar sperm kinematics by using the specific PI3-K inhibitor, LY294002. Boar sperm was incubated up to 1 h in non-capacitating medium in the presence or absence of the cAMP analog, 8Br-cAMP or the PI3-K inhibitor, LY294002 or both. Boar sperm incubated in capacitating medium was treated in the presence or absence of LY294002. First, we have clearly identified that PI3-K is present in whole lysates of boar spermatozoa. Inhibition of PI3-K significantly increased boar sperm straight-line velocity, circular velocity and average velocity without an effect on the percentage of progressively motile spermatozoa in both media. Inhibition of PI3-K induced the same effects on boar sperm velocities as activation of the cAMP/protein kinase A (PKA) pathway and treatment with the PI3-K inhibitor, LY294002 had neither summatory nor synergic effects on boar sperm motion parameters when treated simultaneously with the cAMP analog 8Br-cAMP. Our data suggest that PI3-K plays a negative role, regulating boar sperm motion parameters through a possible inhibition of the cAMP/PKA activating pathway, and since some Computer Aided Sperm Analysis (CASA)-derived parameters have been related to field fertility our results point to the possibility of modulating sperm motile quality by modifying the PI3-K cellular pathway.

Animals↗
PubMed2004

Effect of hyaluronan supplementation on boar sperm motility and membrane lipid architecture status after cryopreservation.

We investigated the effect of supplementing extended boar semen with different amounts of hyaluronan (HA) prior to freezing on post-thaw sperm characteristics. Using a split sample design, the effect of HA at a final concentration of 500 or 1000 microg/ml semen on post-thaw motility parameters, and membrane lipid architecture status assessed by merocyanine-540/YOPRO-1 and flow cytometry were evaluated. HA-supplementation improved motility parameters (P < 0.05 to P < 0.001) and decreased the percentage of hyperactivated spermatozoa (P < 0.05). HA-supplemented samples had more spermatozoa showing high lipid membrane stability as assessed with merocyanine-540. In conclusion, HA appeared to preserve post-thaw spermatozoa viability in vitro and maintained membrane stability after cryopreservation.

Animals↗
PubMed2004

Antioxidant supplementation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture.

Previous studies have shown sperm quality after cryopreservation differs depending on the fraction of seminal plasma the boar spermatozoa are contained in. Thus, spermatozoa contained in the first 10 ml of the sperm-rich fraction (portion I) withstand handling procedures (extension, handling and freezing/thawing) better than those contained in the latter part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction; portion II). The present study evaluated whether an exogenous antioxidant, the water-soluble vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), could, when added to the freezing extender in a split-sample design trial, improve the post-thaw viability and membrane quality of this particular portion of the ejaculate, with particular attention to the status of the plasma membrane. Using a split-sample design, the initial changes in the fluidity status of the sperm plasmalemma after thawing were measured by flow cytometry (FC) after loading with Merocyanine-540 and YO-PRO-1. The FC-derived data revealed a clear ejaculate portion-dependent effect of the antioxidant supplementation. While no beneficial effect of the antioxidant supplementation was visible in spermatozoa from portion I, more spermatozoa with intact membranes were observed in the supplemented samples of portion II, suggesting the protective effect of vitamin E is dependent of the portion of the boar ejaculate considered.

Animals↗
PubMed2003

Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate.

Antioxidant supplementation during cooling was assayed to improve the motility of frozen-thawed (FT) boar spermatozoa from two different fractions of the ejaculate, the first component of the sperm-rich fraction (Fraction I) and the rest of the bulk ejaculate (Fraction II). Using a split-sample design, addition of two different concentrations (100 and 200 microMl(-1)) of the water-soluble Vitamin E analogue Trolox (6-hydroxy -2,5,7,8-tetramethylchroman -2-carboxylic acid) was evaluated for an effect on sperm motility (measured both subjectively and by means of a computer assisted motility assessment (CASA)), and on mitochondrial membrane potential using flow cytometry after cell-loading with JC-1. The effect of the Vitamin E analogue was clearly dose-dependent and varied with the fraction of the ejaculate considered. Motility was significantly higher in Trolox-treated spermatozoa (200 microm), from either ejaculate fraction, albeit the effect was more evident in spermatozoa from Fraction II (P<0.05) for any Trolox-concentration. Antioxidant supplementation resulted, also dose-dependent, in a higher number of spermatozoa showing high mitochondrial activity as assessed by the JC-1 staining, in both ejaculate fractions. In the present trial, exogenous Trolox positively affected post-thaw sperm viability (as motility and mitochondrial membrane potential) in both fractions of the ejaculate. The magnitude of the effect appeared, however, to be dependent of the fraction of the ejaculate considered.

Animals↗
PubMed2003

Genital lesions in an outbreak of caprine contagious agalactia caused by Mycoplasma agalactiae and Mycoplasma putrefaciens.

This paper reports on the genital lesions observed in adult male and female goats from a commercial flock in the Extremadura region of southwestern Spain, following an outbreak of contagious agalactia syndrome caused by Mycoplasma agalactiae and M. putrefaciens. Although both species were isolated from several organs, M. putrefaciens was the only agent isolated from the genital lesions reported here, characterized by desquamative salpingitis and cystic catarrhal metritis in females and by testicular degeneration in males. Mycoplasma putrefaciens was isolated from the testes of only one of the males examined.

Animals↗
PubMed2001

Effect of vulvomucosal injection of D-cloprostenol at weaning and at insemination on reproductive performance of sows during the low fertility summer season under field conditions.

The aim of this study was to evaluate the hypothesis that the intravulvar injection of a PGF2alpha analogue at weaning just prior to insemination may minimise the effects of summer infertility in pigs. From July to September 1999, two groups of 30 sows were randomly formed each month. The experimental group comprised sows receiving 37.5 microg of a PGF2alpha analogue, D-cloprostenol, in 0.5 ml injected into the vulvar lips at weaning and at insemination. Group 2 sows received 0.5 ml of saline solution injected into the vulvar lips and served as controls. The percentage of sows in oestrus within 7 days after weaning in treated sows was 27.93% higher than in controls (P<0.001). Fertility for treated sows was also significantly increased by the treatment (P<0.01). However, neither the percentage of inseminated sows that farrowed nor litter size was affected by treatment.

Animals↗
PubMed1998

Effect of vulvomucosal injection of PGF2alpha at insemination on subsequent fertility and litter size in pigs under field conditions.

The aim of this study was to determine the effects of injecting 5 mg of PGF2alpha into the vulvar mucosa on the reproductive performance of sows maintained under field conditions. At an intensively managed piggery in northwest Spain, two experimental groups were formed randomly and observed throughout the year. The first group comprised sows receiving simultaneously, with every insemination, 5 mg of PGF2alpha injected into the vulvar lips. Group 2 sows received 1 ml of saline solution injected into the vulvar lips at insemination and served as the controls. The farrowing rates for each group were 78.46 and 54.39, while the litter sizes were 10.72 +/- 0.27 and 9.14 +/- 0.47 during the low fertility season (July-September). During the rest of the year (October-June), the farrowing rates were 83.91 and 80.93, while the litter sizes were 11.16 +/- 0.15 and 9.99 +/- 0.15. We conclude that injection of 5 mg of PGF2alpha into the vulvar lips at insemination is an effective method of compensating for the low fertility together with the decreased litter size of the summer months.

Animals↗