Nuclear DNA Fragmentation in Boar Spermatozoa: Measurement Methods and Reproductive Performance Implications.

The aim of this research was to compare the different techniques to measure sperm nuclear DNA fragmentation (sDF) and to check its relations to boar reproductive value, classical spermiogram parameters, and reproductive results of the doses in sows. Sperm chromatin stability assay (SCSA), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and sperm chromatin dispersion test (SCD, Halomax®) results were compared, finding a statistically significant correlation only between SCSA and TUNEL results. The fertility direct boar effect (DBE) index, calculated from the whole productive life of the boar, was not correlated (p > 0.05) with sDF (measured by any technique). Total or progressive sperm motility was not correlated with sDF, while it found a positive correlation between TUNEL measure and abnormal acrosomes (%) and between SCD measure and total sperm morphological abnormalities (%). No significant correlations were obtained between fertility or prolificacy results and sDF results with the different techniques. However, in the case of total born and SCSA measure, the correlation was close to significance (r partial = -0.095; p = 0.066), appointing to a tendency; as SCSA increases, the number of total piglets born decreases. In conclusion, although the different techniques for the sDF seem not to target exactly the same DNA events and the relationship between their values and the reproductive results and the classical spermiogram results is still to be elucidated, the studied sDF techniques may offer extra information that could be useful for the management of AI studs.

Photoperiod and Melatonin Supplementation: Variable Effects on the Quality of Chilled Dog Semen.

The addition of melatonin in seminal extenders due to its antioxidant properties and its beneficial role in sperm preservation has been previously described, especially in seasonal species. The aim of this study was to study a potential seasonal effect based on photoperiod duration when adding a physiological concentration of melatonin in the canine ejaculate. A total of 24 ejaculates were obtained from 10 healthy dogs during the increasing photoperiod (from December 21 to June 21), whereas 12 ejaculates were collected from five healthy individuals during the decreasing photoperiod (from June 22 to December 20). Each ejaculate was separated into two aliquots, and one of them remained as a control, whereas melatonin (100 pM) was added to the other one (C and M treatment groups, respectively). Diluted semen was refrigerated at 5°C. On days 0, 1, 2, 3, and 6, sperm motility analyses were performed using a CASA system and hypoosmotic swelling test (HOST), osmotic resistance test (ORT), and flow cytometry analysis. No effect of melatonin on motility was detected in either photoperiod. Negative effects of melatonin were found for acrosomal defects, apoptosis, and viability in the decreasing photoperiod. The addition of melatonin to sperm in the decreasing photoperiod could create such a high level that it would cause the described negative effects. We found a beneficial effect of melatonin in the increasing photoperiod on acrosomal defects and apoptosis during 0-6 days. Melatonin treatment also increased viability in the short term (days 1 and 2) for both photoperiods. Also, melatonin can provide certain beneficial effects on mitochondrial activity in the medium term (days 2 and 3) in the decreasing photoperiod.

Eggshell membrane treatment for the healing of superficial open wounds in mice / Membrana de cáscara de huevo para la curación de heridas superficiales en ratones

Introduction: Eggshell membranes offer beneficial properties for tissue regeneration and their biomedical applications are important. Objective: To demonstrate the effectiveness of hen unfertilized eggshell membranes in the treatment of superficial open wounds in mice compared to the conventional procedure. Materials and methods: 15-mm linear wounds were made on the back of 10 male CF-1 mice. The mice were divided into four groups. One group did not receive any treatment and the other three were given conventional treatment, eggshell membranes dressing, and eggshell membranes powder. The evolution of the wounds was registered by photographs. We measured wounds length and healing time and calculated the length reduction rate and the percentage of healing. The healing percentages were analyzed by ANOVA with a post hoc Dunnett test (p<0.05). Results: The treatments with eggshell membranes and membrane powder had a woundlength reduction rate of 1.009 and 1.020 mm/day, greater than the conventional treatment, of 0.852 mm/day, and a healing time of 12 days, i.e., less than the 16-day conventional treatment. The statistical analysis showed significant differences between eggshell membrane treatments and the conventional treatment. Conclusions: Eggshell membrane dressing and eggshell membrane powder applied to open superficial wounds in mice were more effective than the conventional treatment in regenerating injured tissue in mice.

Introducción. Las membranas de la cáscara de huevo presentan propiedades beneficiosas para la regeneración de tejidos y sus aplicaciones biomédicas son importantes. Objetivo. Demostrar la efectividad de las membranas de la cáscara de huevo no fecundado de gallina en el tratamiento de heridas abiertas superficiales en ratones, en comparación con el procedimiento convencional. Materiales y métodos. Se hizo una herida superficial lineal de 15 mm en la espalda de 10 ratones albinos machos. Los ratones se dividieron en cuatro grupos, uno no recibió ningún tratamiento y los otros tres sí: uno, tratamiento convencional, otro, con membranas de huevo directamente aplicadas a la herida y, el otro, con membranas en forma de polvo. La evolución de las heridas se registró en fotografías y se calculó la tasa de reducción de la longitud de la herida, así como el tiempo y el porcentaje de curación. Los porcentajes de curación se analizaron con ANOVA y la prueba de Dunnett (p<0,05). Resultados. Con los tratamientos con membranas de huevo y polvo de membrana, se logró una tasa de reducción de longitud de 1.009 y 1.020 mm/día, respectivamente, y un tiempo de curación de 12 días, en tanto que, con el tratamiento convencional, la tasa de reducción fue de 0,852 mm/día y la curación se dio en 16 días. El análisis estadístico mostró diferencias significativas entre los tratamientos con membrana de huevo y el tratamiento convencional. Conclusiones. Las membranas de la cáscara de huevo aplicadas de forma directa y en polvo resultaron más efectivas que la aplicación del procedimiento convencional en el tratamiento de heridas abiertas superficiales en ratones.

Influence of Non-conventional Sperm Quality Parameters on Field Fertility in Ovine.

The prediction of the fertilizing ability of a seminal dose continues to be a primary aim in the field of artificial insemination (AI). To achieve this goal, in this study we have included the evaluation of some non-conventional sperm quality markers. A total of 3,906 ewes from 52 different farms were inseminated with 357 refrigerated seminal doses obtained from 45 mature Rasa Aragonesa rams. The same samples were used for sperm quality analysis including membrane integrity, capacitation status, oxygen consumption and apoptotic-like markers such as phosphatidylserine translocation (PS), plasmalemma disorganization/mitochondrial membrane potential, caspase activation and DNA damage. Seminal doses from the breeding (B) season presented higher percentages of intact membrane (IM), non permeant (NP) membrane with high mitochondrial membrane potential (ΔΨm) and IM without PS translocation spermatozoa than those from the non-breeding (NB) season. Therefore, we can conclude that there were less spermatozoa showing apoptotic-like features in the seminal doses from the B than the NB season, although these differences did not affect field fertility. Only the percentage of intact membrane, non-capacitated (IM-NC) spermatozoa showed a significant correlation with in vivo fertility (P = 0.005) and fecundity (P = 0.007) values obtained after cervical AI when all data were evaluated. When the data were sorted by season and distance to the farms where AI was performed, the correlation between the percentage of IM-NC spermatozoa and reproductive parameters increased in the NB season and progressively with remoteness from the farms. Some other sperm parameters, like NP with high ΔΨm, IM sperm without active caspases and DNA-intact spermatozoa, also showed significant correlations with the reproductive parameters in the sorted data. Moreover, the increment in both the percentage of IM-NC and DNA-intact spermatozoa would increase the probability of obtaining a fertility higher than the mean (>52%), as revealed by a multiple logistic regression analysis. In conclusion, we have identified two seminal markers-the percentage of intact membrane, non-capacitated spermatozoa, and DNA intact spermatozoa-which could be used as a test to discard males in AI programs, which is highly important from an economic point of view and can contribute to achieving satisfactory fertility rates.

Bisphenol A Diglycidyl Ether (BADGE) and Progesterone Do Not Induce Ca2+ Signals in Boar Sperm Cells.

AIM: Exposure of boar sperm cells to Bisphenol A diglycidyl ether (BADGE) has been shown to lead to reproductive failure in sows, however, the mode of action is unknown. As we have recently shown that BADGE can interfere with Ca2 + signaling in human sperm cells through an action on CatSper, and as CatSper has been shown to be expressed in boar sperm cells, we hypothesized that a similar mechanism in the boar sperm cells could be responsible for the reproductive failure. METHODS: Direct effects of BADGE and the endogenous ligand of human CatSper, progesterone, on Ca2+ signaling in human and boar sperm cells were evaluated side-by-side using a Ca2+ fluorimetric assay measuring changes in intracellular Ca2+. Effects of BADGE on Ca2+ signaling in boar sperm were furthermore assessed by flow cytometry by an independent laboratory. RESULTS: The exact same solutions of BADGE and progesterone induced transient biphasic Ca2+ signals in human sperm cells, but failed to do so in both non-capacitated and capacitated boar sperm cells. BADGE also failed to induce transient biphasic Ca2+ signals in boar sperm cells in the flow cytometric assay. CONCLUSION: BADGE and progesterone failed to induce Ca2+ signals in boar sperm cells. This indicates that the signaling mechanisms leading to activation of CatSper differs between human and boar sperm cells, and suggests that the mode of action by which exposure of boar sperm cells to BADGE can lead to reproductive failure in sows does not involve effects on Ca2+ signaling.

Insemination of sows with seminal doses prepared by a two-step hypothermic dilution does not impair the reproductive performance at farm.

Reproduction in swine is mostly carried out through artificial insemination (AI). For this purpose, AI studs collect the ejaculates, analyse the sperm quality, dilute and package to produce seminal doses and ship them to sow farms to carry out the AI. Temperature is controlled during the process to avoid sperm damage. Semen is diluted in the extender in a one-step or a two-step process where the second can be isothermic (approximately 32°C) or hypothermic (room temperature 21-22°C). Both techniques are currently performed, and the latter could reduce time and costs, but the literature available comparing the processes is scarce and presents discrepancies. To date, there are no studies about its impact in fertility. This study compared hypothermic two-step dilution (HTSD) and isothermic two-step dilution (ITSD) in laboratory and field trial to elucidate whether HTSD has any effect. Ejaculates from 72 boars in nine AI studs were split and processed with both techniques using a high-performance extender and evaluated in laboratory. Four farms inseminated 345 sows with samples of four of these AI studs, and their fertility and prolificacy were registered. Results show no significant differences between doses prepared by HTSD and ITSD technique, having no impact in laboratory results (percentage of motile sperm, short hypoosmotic swelling test (sHOST) and short osmotic resistance test (sORT), viable sperm, damaged acrosomes, sperm under early apoptosis, high mitochondrial membrane potential (p > .1), fertility (92.2% versus 94.1%, p = .45) or farrowing rate (15.8 ± 0.3 versus 16.1 ± 0.3 p = .46). In conclusion, our results suggest that HTSD of semen on extender could be safely implemented in AI studs under the conditions tested.

Influence of nonylphenol from multilayer plastic films on artificial insemination of sows.

Artificial insemination is common practice in mass livestock farming. Recently, it was shown that chemicals leaching from multilayer plastic bags affect the fertility of boars, although common quality tests did not show any impact on the sperm. It is not clear whether this incidence was a single case or whether it could be a systematic problem. Therefore, we studied six multilayer plastic bags. A total of 49 compounds were found, but most of them were at very low intensity. Nonylphenols in the range of 19-99 µg/g plastic were found. Migration tests using water and 10% ethanol as simulants, to mimic the behavior of semen with the extender, were performed. The most interesting migrants in terms of potential reprotoxicity were identified as nonylphenols. The identification in depth demonstrated the presence of 10 isomers of nonylphenol with a total concentration range between 16 to 58 µg/Kg simulant, among other migrants at lower concentration. The influence of these nonylphenols and their maximum tolerable concentration in direct contact with semen from boars was studied. Motility, viability, mitochondrial activity and acrosomes reacted were significantly affected at 10 mg/Kg of nonylphenols in contact with the sperm, but in vitro penetration rate was significantly decreased with only 2 mg/Kg. Insight into the mode of action is also provided.

A common surfactant used in food packaging found to be toxic for reproduction in mammals.

Migration from a multilayer plastic material intended for food contact showed that 2,4,7,9-tetramethyl-5-decyne-4,7-diol mixture (surfynol), used as a surfactant in the adhesive employed to build the multilayer, was transferred to water and other food simulants in contact with the plastic. When these multilayer plastics were used for containing seminal doses for artificial insemination, it was found that fertility was seriously damaged in terms of motility, acrosome integrity, mitochondrial activity and penetration capacity in the cells, thus affecting male fertility. Quantitative proteomic analysis of exposed germinal cells demonstrated the inhibition of key proteins involved in the fertilization capacity by affecting the cytoskeleton, sperm motility, the energy machinery and sperm defense mechanisms against oxidation, therefore confirming the surfactant-induced male infertility. These results open up new and interesting perspectives for the study of reprotoxicity caused by different chemicals common in our daily lives. SIGNIFICANCE: This paper demonstrates the toxicity for reproduction of a common surfactant used in food packaging and the scientific reasons why the sperm loses reproductive capacity in presence of this chemical. So, the surfactant affects the male fertility. The surfactant is present in many adhesives used either for building multilayer materials or to glue paper and plastic in food packaging. This is the first time that reprotoxicity is demonstrated for this compound. According to the theoretical approach Threshold of Toxicological Concern (TTC) the compound is highly toxic but experimental data did not exist so far. The study described in this paper and the results obtained open a door to further research in which male infertility caused by chemicals could be demonstrated.

c-Jun N-terminal kinase and p38 mitogen-activated protein kinase pathways link capacitation with apoptosis and seminal plasma proteins protect sperm by interfering with both routes†.

The mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38) signaling cascades are involved in triggering apoptosis in somatic cells. Given that spermatozoa are able to undergo apoptosis, we tested the hypothesis that these pathways might be functional in ram spermatozoa as two signal transduction mechanisms that contribute to the modulation of capacitation and apoptosis. Indirect immunofluorescence and western blot analysis evidenced the presence of JNK and p38 in ram spermatozoa. To verify the involvement of these enzymes in sperm physiology, we determined the effect of specific inhibitors of JNK or p38 on in vitro capacitation induced with either cAMP-elevating agents or epidermal growth factor (EGF). Both inhibitions reduced the EGF-induced capacitation with a decrease in the chlortetracycline capacitated-sperm pattern, protein tyrosine phosphorylation, phosphatidylserine externalization, caspase-3 and -7 activation, and the proportion of DNA-damaged spermatozoa. No significant changes were found in the high-cAMP capacitated samples. The addition of 3.4 mg/ml seminal plasma proteins (SPPs) to the EGF-containing samples, either alone or together with each inhibitor, resulted in a decreased proportion of capacitated sperm pattern, protein tyrosine phosphorylation, loss of plasma membrane integrity, and apoptotic alterations. Furthermore, SPPs significantly reduced the phosphorylation level of JNK and p38 MAPK (active forms). These findings show a relationship between capacitation and apoptosis, and represent a step forward in the knowledge of the SPP protective mechanism in spermatozoa.

New insights into the mechanisms of ram sperm protection by seminal plasma proteins.

To provide new insights into the mechanisms through which seminal plasma proteins (SPP) are able to protect spermatozoa, we tested the hypothesis that apoptosis can contribute to the negative effect of refrigeration on ram spermatozoa, and that SPP prevent this damage. Having proved the presence of key constituents of apoptosis-related pathways in ram sperm protein extracts, we carried out a comparative analysis of the effects of the addition of SPP before refrigeration (15 °C, 30 min) and induced-apoptosis with betulinic acid or fibroblast-associated receptor ligand, assessing sperm quality parameters and apoptotic markers. The protective effect of SPP on plasma membrane integrity and potential, motility and mitochondrial inner membrane potential, and surface (cardiolipin content) was evidenced in refrigerated and induced-apoptosis samples. The addition of SPP resulted in lower values of phosphatidylserine externalization, DNA damage, and caspase activity. Therefore, apoptosis in fresh or refrigerated ram spermatozoa can occur due to activation of both the extrinsic and the intrinsic mediated pathway, and SPP might interfere with both pathways. The addition of SPP also resulted in higher proportions of viable, noncapacitated sperm and fertilizing ability (ZBA rate). This report demonstrates that SPP support survival of ram spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, and proposes the possibility that SPP might interfere with the extrinsic and the intrinsic apoptotic pathways. This opens new, interesting perspectives for the study of cellular regulatory mechanisms in spermatozoa that could be crucial for the improvement of ram semen preservation protocols.

Centrifugal countercurrent chromatography to elucidate surface differences of adipose tissue-derived stem cells.

The current methods of isolation of adipose tissue-derived stem cells result in a heterogeneous population that might interfere with their differentiation potential and makes it difficult to compare the results between different groups. Partition in aqueous two-phase systems is one of the few techniques that separate cells on the basis of surface properties, gentle enough to isolate fragile cell types in isotonic conditions without altering their structure, and can be easily scaled. In this study, stem cells isolated from human adipose tissue seeded and expanded in vitro were fractionated by using centrifugal countercurrent distribution in an aqueous two-phase system. The separated subpopulations revealed the high heterogeneity of adipose tissue-derived stem cell samples. Comparative partition analyses showed that aging induces a loss of heterogeneity, which is not due to a loss of cell viability associated to age. The phosphatidylserine externalization, an apoptotic feature, is the main factor in cell partition that results in a decreased hydrophobicity of the cell surface. This procedure may be suitable for separating adipose tissue-derived stem cell populations enriched in some functional and/or structural surface characteristics. The possibility of a very effective separation of different subpopulations in opposite phases would be an interesting development of the method.

Melatonin prevents capacitation and apoptotic-like changes of ram spermatozoa and increases fertility rate.

We recently demonstrated the presence of melatonin in ram seminal plasma and differences in its concentration in this fluid between the breeding and nonbreeding season. In this study, we investigate the hypothesis that in vitro treatment with melatonin affects ram sperm quality, and that this is reflected in the in vitro fertilization (IVF) results. Semen from nine rams was collected during the nonreproductive season and treated with 1 mum, 10 nm and 100 pm melatonin. Samples were incubated at 39 degrees C and 5% CO2, and motility, viability, capacitation status and phosphatidylserine (PS) translocation were assessed before and after melatonin addition, either 1 or 3 hr of incubation. Fertility rate of the melatonin-treated samples was determined by means of IVF. Although melatonin failed to affect both sperm kinematic parameters and viability, the exposure of ram spermatozoa to melatonin has a direct effect, decreasing capacitation and PS translocation at 1 mum, and increasing short-term capacitation at 100 pm, which caused an increased oocyte fertilization rate following IVF. Furthermore, cleavage rate of oocytes fertilized with 100 pm melatonin-treated spermatozoa was higher than that with 1 mum melatonin and control samples (P < 0.1). These results prove that melatonin has a direct effect on ram spermatozoa in the nonreproductive season, which can be explained, at least in part, by the melatonin capacity as a reactive oxygen species scavenger and antioxidant. These findings might help to select the optimal experimental conditions for IVF and to improve sperm preservation protocols.