Academic literature on the topic 'Sperm maturation'

The epididymis is an organ that performs all the biochemical changes responsible for sperm maturation. During ageing, histological alterations in the epididymis and decreased protein synthesis have been found. This might affect the sperm maturation process. The aim of this study was to determine if the changes in the epididymis during ageing might cause alterations in sperm maturation. Wistar rats of 3–4months old (young) and 18–21months old (old) were used. The testosterone concentration was determined and the epididymides were dissected and divided in three regions: caput, corpus, and cauda. The tissues were used for histological processing and sperm extraction. Testosterone concentration decreased 34% in the old animals compared to the young ones. The distribution of mannose, sialic acid, and N-acetylglucosamine in the glycocalyx of the sperm membrane of old animals was different from that of young animals. The same occurred with phosphatidylserine externalisation and protein phosphorylation at tyrosine residues. Epididymis histology in old animals showed tubular and cellular degeneration. Our results suggest that ageing affects maturational markers, likely due to alterations in the epididymis as a result of the testosterone decrease associated with ageing.

Mammalian sperm must undergo two post-testicular processes to become fertilization-competent: maturation in the male epididymis and capacitation in the female reproductive tract. While caput epididymal sperm are unable to move and have not yet acquired fertilization potential, sperm in the cauda epididymis have completed their maturation, can move actively, and have gained the ability to undergo capacitation in the female tract or in vitro. Due to the impossibility of mimicking sperm maturation in vitro, the molecular pathways underlying this process remain largely unknown. We aimed to investigate the use of caput epididymal ligation as a tool for the study of sperm maturation in mice. Our results indicate that after seven days of ligation, caput sperm gained motility and underwent molecular changes comparable with those observed for cauda mature sperm. Moreover, ligated caput sperm were able to activate pathways related to sperm capacitation. Despite these changes, ligated caput sperm were unable to fertilize in vitro. Our results suggest that transit through the epididymis is not required for the acquisition of motility and some capacitation-associated signaling but is essential for full epididymal maturation. Caput epididymal ligation is a useful tool for the study of the molecular pathways involved in the acquisition of sperm motility during maturation.

Abstract After leaving the testis, mammalian sperm undergo a sequential maturation process in the epididymis followed by capacitation during their movement through the female reproductive tract. These phenotypic changes are associated with modification of protein phosphorylation and membrane remodeling, which is requisite for sperm to acquire forward motility and induce fertilization. However, the molecular mechanisms underlying sperm maturation and capacitation are still not fully understood. Herein, we show that PPP3R2, a testis-specific regulatory subunit of protein phosphatase 3 (an isoform of calcineurin in the testis), is essential for sperm maturation and capacitation. Knockout of Ppp3r2 in mice leads to male sterility due to sperm motility impairment and morphological defects. One very noteworthy change includes increases in sperm membrane stiffness. Moreover, PPP3R2 regulates sperm maturation and capacitation via (i) modulation of membrane diffusion barrier function at the annulus and (ii) facilitation of cholesterol efflux during sperm capacitation. Taken together, PPP3R2 plays a critical role in modulating cholesterol efflux and mediating the dynamic control of membrane remodeling during sperm maturation and capacitation.

Despite many fundamental similarities between the gametes of marsupials and placental mammals, the regulation and timing of prefertilization gamete maturation are quite different. The marsupial acrosome is remarkably stable and an acrosome reaction (AR) is not induced by reagent effective for the sperm of placental mammals. The ultrastructure of the marsupial sperm AR is essentially similar to that of placental mammals, however, whether an equatorial segment (ES) persists to serve as the site of sperm-egg membrane fusion is unclear. Diacylglycerol induction of the AR suggests that the sperm of Australian species lack an ES, yet an ES-like region appears to be involved in fertilization in the opossum Monodelphis. The marsupial oocyte, unlike those of placentals, continues to grow throughout follicular life and major cytoplasmic maturation events occur late in oocyte development. Cortical granules only become evident shortly before ovulation and mature dark granules may only appear after ovulation. Further, the zona pellucida (ZP) changes in character and function during the peri-ovulatory period. In vitro fertilization has been achieved for an opossum but not for any Australian marsupial, owing to failure of sperm-ZP binding. Requirement for a sperm maturation process is likely, but capacitation treatments used for placental sperm in vitro have been ineffective. Since it is now feasible to experimentally manipulate marsupial gametes in vitro major advances in our understanding of their function can be expected.

Rat sperm isolated from the caput and caudal epididymis and the vas deferens were subjected to multiple partition in aqueous two-phase systems. The technique was used to reveal heterogeneity of a sperm population with respect to particular surface properties. Sperm from all three regions gave broad distributions indicative of heterogeneous cell populations. Greatest heterogeneity was observed for cauda sperm with caput and was sperm producing similar distributions. Following multiple partition sperm from different regions of the distribution profiles were immunostained with three antibodies known to recognise maturation antigens. The results show that some antigens are acquired during epididymal transit whilst others are present throughout. The partition (surface heterogeneity) seen cannot therefore be explained solely by the distribution of the antigens recognised by 2D6, 6B2 and 3D5.

Beyond the haploid genome, mammalian sperm carry a payload of epigenetic information with the potential to modulate offspring phenotype. Morphologically mature sperm exit the testes, but cannot swim or interact with the oocyte without extensive remodeling during epididymal transit; this includes modifications to the lipid composition of the sperm membrane, gain of necessary proteins, and a dramatic shift in sperm RNA content. Epididymal maturation has also been linked to changes in the sperm methylome suggesting that the epididymis might play a broader role in shaping the sperm epigenome. First, we characterized the genome-wide methylation landscape in seven germ cell populations from throughout the male reproductive tract. Our data emphasize the stability of cytosine methylation in mammalian sperm, and identify a surprising, albeit transient, period during which sperm are associated with extracellular DNA. Second, given our interest in the small RNA repertoire of sperm we set out to address known bias in sequencing protocols by comparing several small RNA cloning protocols. We found a protocol recently developed by Kathleen Collins’ lab (OTTR) to be superior to commercially available kits in providing an accurate representation of tRNA fragment levels as compared to Northern blotting. These results not only provide a more accurate representation of tRNA fragments, but also more complexity than previously seen allowing us to reassess the true sperm small RNA content. Taken together, these results provide significant insight into the mechanisms and factors modulating sperm epigenomics during post-testicular sperm maturation.

Background: There is evidence to show that varicocele repair can improve conventional sperm parameters and sperm DNA integrity in infertile men with a clinical varicocele. Objective: To further examine the effect of varicocelectomy on sperm quality, specifically, sperm nuclear chromatin integrity, distribution of nuclear sulfhydryl groups and sperm maturation. Design, Setting and Participants: We prospectively evaluated a consecutive series of infertile men (n=29) presenting to Ovo clinic with one year or more history of infertility, a clinically palpable varicocele and abnormal semen parameters. Six sperm donors with normal sperm parameters served as controls. Surgical Procedure: Microsurgical sub-inguinal varicocelectomy. Outcome Measures: (1) Conventional sperm parameters, (2) aniline blue staining (AB is specific to histone lysines), (3) iodoacetamide fluorescein (IAF targets free protamine sulfhydryl groups) and (4) sperm chromatin structure assay (SCSA) with the results expressed as % DNA fragmentation index (%DFI) and percent high DNA stainability (%HDS) before and 4 months after microsurgical varicocelectomy. Results: The sperm %DFI, %HDS (a measure of chromatin compaction), % 5-IAF staining (diffuse head staining), % AB staining (dark blue) were all significantly lower in the control group compared to infertile men with varicocele (8 vs. 20%, 4.0 vs. 9.6%, 1.7 vs. 16.3%, and 2.5 vs. 13.5% respectively). The %5-IAF and %AB staining decreased significantly after surgery (from 16.3 to 5.4%, and from 13.5% to 5.4%, respectively). Similarly, the %HDS and %DFI also decreased significantly after surgery (from 10% to 6% and from 20% to 13%, respectively). The only notable relationships were between aniline blue staining and %HDS post varicocelectomy (r= 0.57, P <0.05), and both %IAF staining and %DFI were inversely correlated with motility (r=-0.44 and -0.43, respectively). Conclusion: The data show that varicocelectomy is associated with a consistent improvement in sperm DNA integrity and chromatin compaction using three different assays of sperm chromatin integrity (SCSA, IAF, Aniline Blue).
Contexte: Il y a la preuve(l'évidence) pour montrer que la réparation de varicocele peut améliorer des paramètres de sperme conventionnels et l'intégrité d'ADN de sperme dans des hommes infertiles avec varicocele clinique. Objectif: Examiner l'effet de varicocelectomy sur la qualité de sperme, spécifiquement, le sperme l'intégrité chromatin nucléaire, la distribution de groupes sulfhydryl nucléaires et la maturation de sperme. Schéma, environnement et participants : Nous avons éventuellement évalué une série consécutive d'hommes infertiles (n=29) présentant à la clinique Ovo avec un an ou plus d'histoire d'infertilité, varicocele cliniquement palpable et des paramètres de sperme anormaux. Six donneurs de sperme avec des paramètres de sperme normaux ont servi de contrôles. Intervention chirurgicale: Microchirurgie sous-inguinale varicocelectomie.Mesures des résultats: (1) Des paramètres de sperme conventionnels, (2) l'aniline bleu teintant(tachant) (d'AB est spécifique à histone lysines), (3) iodoacetamide fluorescein (libère protamine sulfhydryl des groupes) et (4) le sperme chromatin l'essai de structure (SCSA) avec les résultats (DFI) de fragmentation d'ADN de % et le pour cent haut ADN stainability (%HDS) auparavant et 4 mois après varicocelectomy microchirurgical Résultats: Le pourcentage de spermatozoïdes avec le sperme de DFI%, HDS%, positif de 5-IAF coloration, une coloration positive AB (bleu foncé) étaient significativement inférieure dans le groupe témoin par rapport aux hommes infertiles ayant une varicocèle (8 vs 20%, 4,0 vs 9,6%, 1,7 vs 16,3%, et 13,5 vs 2,5% respectivement). Le pourcentage de spermatozoïdes avec positifs 5-IAF coloration et AB nucléaire positif diminué de façon significative après la chirurgie (de 16,3 à 5,4%, et de 13,5% à 5,4%, respectivement). Les HDS% et DFI% également diminué de façon significative après la chirurgie (de 10% à 6% et de 20% à 13%, respectivement). Les seules relations entre les notables étaient coloration au bleu d'aniline et varicocélectomie après HDS (r = 0,57, P <0,05), et les deux taches IAF et DFI% ont été inversement corrélée avec la motilité (r = -0,44 et de -0,43, respectivement).Conclusion: Les données montrent que varicocélectomie est associée à une amélioration constante de l'intégrité d'ADN de sperme et compaction de la chromatine en utilisant trois différents dosages cytochimiques de l'intégrité chromatine des spermatozoïdes (SCSA, l'IAF, le bleu d'aniline).

Introduction: Mature spermatozoa recognise and bind hyaluronic acid (HA) in the extracellular matrix via Hyaluronic Acid Binding Proteins (HABPs). HA-binding may be important in supporting the sperms’ progress in the female genital tract. Information on sperm HABPs is limited, however and the current study investigated their expression in human and bovine sperm. Materials and methods: The ability of differential density gradient centrifugation (DDGC) processed human sperm to bind HA was evaluated. DDGC sperm populations (90% and 45% fractions) and sperm interacting with HA were assessed for DNA fragmentation and chromatin compaction using acridine orange (AO) and aniline blue (AB) staining, respectively. Additionally, the effect of capacitation on HA-binding was assessed. Expression of HABPs in bovine and human sperm was also investigated alongside changes in their distribution following capacitation and the acrosome reaction. Proteomics was used to monitor changes in the human sperm proteome (including HABPs) following capacitation and the acrosome reaction. Following a protein panning procedure, proteomics was also used to help identify HABPs in both HA-binding and non-binding fractions. The presence of HA-binding motifs (BX7B and Link module, common in several well-known HABPs) was also evaluated in both fractions. Increasing the sensitivity of HABP isolation and detection was attempted using biotinylation of sperm surface proteins alongside HA-affinity chromatography. Results: Sperm recovered from 90% fractions had significantly higher HA-binding ability, lower levels of DNA fragmentation and higher levels of chromatin compaction. The reverse was true for sperm from the 45% fractions. HA-binding showed higher efficiency at selecting sperm with higher DNA integrity and chromatin compaction than DDGC. Capacitation enhanced HA binding, which may reflect associated changes in sperm HABPs. Immunocytochemistry localised CD44 to the acrosome and equatorial segment of non-capacitated human sperm and on the anterior acrosome and post-acrosomal sheath of non-capacitated bovine sperm. CD44 was more confined to the equatorial segment following capacitation in human sperm and to the post-acrosomal sheath of bovine sperm. Acrosomal labelling of CD44 was highly reduced after the acrosome reaction. Hyaluronic acid-TRITC (tetramethylrhodamine isothiocyanate) labelled the sperm membrane and the neck region, particularly strongly after capacitation and in acrosome reacted bovine sperm. Qualitative and quantitative characteristics of 121 proteins altered by capacitation and the acrosome reaction is also reported. Differentially regulated proteins are involved in sperm metabolism, energy production, cell signaling, sperm-oocyte recognition and sperm motility. Following capacitation, mass spectrometry (MS) detected an increase in cilia and flagella associated protein 20, containing two potentially HA-binding BX7B motifs. Analysis of the proteomics data from the panning fractions resulted in the identification of 28 proteins in HA-binding fractions with PDBsum and SAS server highlighting structural similarities between ZPBP2 (found only in HA-binding fractions) and the HA-binding domain of CD44. Data also showed that 50% of binding (including ADAM32 and cilia-and flagella associated protein 20 amongst others) and 34.5% of non-binding proteins contained BX7B motifs, indicating a likely selective enrichment of putative HABPs by the panning technique. Western blot analysis confirmed these results. Isolation of HABPs from bovine sperm using HA affinity chromatography followed by SDS PAGE revealed distinct protein bands of molecular masses 58 and 78 kDa, possibly corresponding to CD44 and RHAMM, respectively, were detected. Other protein bands detected at 16, 27, 38, 42 and 47 may be novel HABPs, including ZPBP2 (molecular mass 38 kDa). Conclusion: In conclusion, ZPBP2, ADAM32, Midkine and cilia and flagella associated protein 20 may have HA binding properties. This is also the first study which has brought together compelling evidence for the relationship between excising methods of enriching good quality sperm (DDGC) and HA-binding. Sperm DNA fragmentation and chromatin compaction status, therefore, reflects this relationship and supports claims for a positive sperm quality benefit for HA-based sperm selection particularly for ICSI, where the choice of sperm is more critical. In this regard, the use of more specific, anti-HABP-based methods for sperm selection (initially targeting RHAMM, CD44 and SPAM1) should also be considered.

[ES] En las últimas décadas, la anguila europea Anguilla anguila ha sufrido una disminución drástica de su población lo que ha llevado su inclusión como especie en peligro crítico en la lista roja UICN. Esta situación, junto con la gran importancia comercial de esta especie, evidencia la necesidad de tomar acciones como el desarrollo de la reproducción en cautividad y el control de la pesca. Una de las herramientas más interesantes para su uso en la biología de la conservación es la criopreservación de espermatozoides, que presenta varias ventajas para esta especie, incluyendo la sincronización de gametos, la selección de líneas genéticas o su uso para la creación de un criobanco. Sin embargo, el desarrollo de protocolos de criopreservación necesariamente requieren esperma de buena calidad. Además, se necesita un método preciso para evaluar la calidad del esperma tanto antes como después de la criopreservación. Sobre esta última cuestión, la motilidad de los espermatozoides de los peces se considera uno de los mejores biomarcadores para la evaluación de la calidad de los espermatozoides en los peces, y se puede estudiar de forma subjetiva u objetiva utilizando sistemas "computer assisted sperm analysis" (CASA-Mot). Primero, se realizó un experimento para evaluar la precisión y la exactitud de ambos métodos para estudiar la motilidad del esperma: el método subjetivo y la técnica objetiva que utiliza el sistema CASA-Mot. Además, se probó si el grado de experiencia de los técnicos en el caso del método subjetivo tiene un efecto en la precisión de la estimación de la motilidad y, por lo tanto, hay una influencia del personal del laboratorio en la evaluación de la motilidad del esperma. Aquí concluimos que tanto el método como la experiencia técnica eran factores clave para evaluar con precisión la motilidad del esperma en la anguila europea, por lo que se requiere el uso de CASA-Mot junto con material calificado para obtener resultados fehacientes. En segundo lugar, se evaluaron métodos alternativos para la maduración de los machos de anguila europeos probando dos tratamientos hormonales diferentes: OVI, una gonadotropina recombinante; y VET, una gonadotropina purificada a partir de orina femenina. Después de elegir el mejor tratamiento hormonal de los dos, se evaluó el efecto de tres dosis diferentes con el objetivo de obtener el mayor rendimiento al menor coste. Los resultados de este experimento apuntaron a OVI como el mejor tratamiento hormonal en una dosis semanal de 1.5 UI/g de pez, que proporciona la mayor rentabilidad, obteniendo esperma de alta calidad a menor precio. En un tercer experimento, y utilizando los conocimientos adquiridos en los dos primeros experimentos, se realizaron una serie de experimentos para estandarizar los protocolos de criopreservación de esperma de anguila europea disponibles en ese momento (utilizando DMSO o metanol como crioprotector). Los resultados apuntaron al protocolo que utiliza el metanol como el mejor de ellos dos en términos de motilidad, velocidad y viabilidad de los espermatozoides y la preservación de la integridad del ADN. Siguiendo este último método estandarizado, se realizó un cuarto experimento con el objetivo de mejorar el protocolo en términos de volumen (volúmenes de esperma más grandes) y de calidad espermática. Además, se desarrolló un protocolo simple de almacenamiento a corto plazo para complementar las opciones de preservar el esperma durante diferentes períodos de tiempo. De todas las condiciones de almacenamiento probadas, las diluciones 1/50 a 4 ºC mostraron los mejores resultados, manteniendo la motilidad en comparación con el control durante 3 días, y manteniendo cierta motilidad espermática (12%) después de 7 días. A partir del experimento de criopreservación, fue posible aumentar los volúmenes a 2 y 5 mL sin perder la calidad del esperma en comparación con volúmenes más pequeños, y mejorando las mot
[CAT] En les últimes dècades, l'anguila europea Anguilla anguila ha sofert una disminució dràstica de la seva població el que ha portat la seva inclusió com a espècie en perill crític en la llista vermella UICN. Aquesta situació, juntament amb la gran importància comercial d'aquesta espècie, evidencia la necessitat de prendre accions com el desenvolupament de la reproducció en captivitat i el control de la pesca. Una de les eines més interessants per al seu ús en la biologia de la conservació és la criopreservació d'espermatozoides, que presenta diversos avantatges per a aquesta espècie, incloent la sincronització de gàmetes, la selecció de línies genètiques o el seu ús per a la creació d'un criobanc. No obstant això, el desenvolupament de protocols de criopreservació necessàriament requereixen esperma de bona qualitat. A més, es necessita un mètode precís per avaluar la qualitat de l'esperma tant abans com després de la criopreservació. Sobre aquesta última qüestió, la motilitat dels espermatozoides dels peixos es considera un dels millors biomarcadors per a l'avaluació de la qualitat dels espermatozoides en els peixos, i es pot estudiar de forma subjectiva o objectiva utilitzant sistemes "computer assisted sperm analysis" (CASA-Mot). Primer, es va realitzar un experiment per avaluar la precisió i l'exactitud de tots dos mètodes per estudiar la motilitat de l'esperma: el mètode subjectiu i la tècnica objectiva que utilitza el sistema CASA-Mot. A més, es va provar si el grau d'experiència dels tècnics en el cas del mètode subjectiu té un efecte en la precisió de l'estimació de la motilitat i, per tant, hi ha una influència del personal del laboratori en l'avaluació de la motilitat del esperma. Vam concloure que tant el mètode com l'experiència tècnica eren factors clau per avaluar amb precisió la motilitat de l'esperma en l'anguila europea, de manera que es requereix l'ús de CASA-Mot juntament amb material qualificat per obtenir resultats fefaents. En segon lloc, es van avaluar mètodes alternatius per a la maduració dels mascles d'anguila europeus provant dos tractaments hormonals diferents: OVI, un gonadotropina recombinant; i VET, un gonadotropina purificada a partir d'orina femenina. Després de triar el millor tractament hormonal dels dos, es va avaluar l'efecte de tres dosis diferents amb l'objectiu d'obtenir el major rendiment al menor cost. Els resultats d'aquest experiment van apuntar a OVI com el millor tractament hormonal en una dosi setmanal de 1.5 UI/g de peix, que proporciona la major rendibilitat, obtenint esperma d'alta qualitat a un preu millor. En un tercer experiment, i utilitzant els coneixements adquirits en els dos primers experiments, es van realitzar una sèrie d'experiments per estandarditzar els protocols de criopreservació d'esperma d'anguila europea disponibles en aquest moment (utilitzant DMSO o metanol com crioprotector). Els resultats van apuntar al protocol que utilitza el metanol com el millor d'ells dos en termes de motilitat, velocitat i viabilitat dels espermatozoides i la preservació de la integritat de l'ADN. Seguint aquest últim mètode estandarditzat, es va realitzar un quart experiment amb l'objectiu de millorar el protocol en termes de volum (volums d'esperma més grans) i de qualitat espermàtica. A més, es va desenvolupar un protocol simple d'emmagatzematge a curt termini per complementar les opcions de preservar l'esperma durant diferents períodes de temps. De totes les condicions d'emmagatzematge provades, les dilucions 1/50 a 4ºC van mostrar els millors resultats, mantenint la motilitat en comparació amb el control durant 3 dies, i mantenint certa motilitat espermàtica (12%) després de 7 dies. A partir de l'experiment de criopreservació, va ser possible augmentar els volums a 2 i 5 ml sense perdre la qualitat de l'esperma en comparació amb volums més petits.
[EN] In the last decades, the European eel Anguilla anguilla has suffered a drastic decrease in the recruitment in most areas of their distribution range, leading the species to be included as critically endangered in the IUCN list. This situation, together with the high commercial importance of the species, evidence the need of taking actions such as development of reproduction in captivity and control of fisheries based on the complexity of their life cycle. One of the most interesting tools for its use in conservation biology is the sperm cryopreservation, which presents several advantages for this species such as the synchronization of gametes, selection of genetic lines or cryobanking. However, the development of cryopreservation protocols necessarily requires good quality sperm, and it is also needed an accurate method to assess sperm quality both pre- and post-cryopreservation. On this last matter, fish sperm motility is considered one of the best quality biomarkers for sperm quality assessment in fish, and it can be evaluated subjectively or objectively using computer assisted sperm analysis (CASA-Mot) systems. First, an experiment was conducted to evaluate the precision and accuracy of both methods for assessing sperm motility: the subjective method and the objective technique using CASA-Mot system. Moreover, it was tested whether the degree of expertise of the technicians in the case of the subjective method, has an effect on the accuracy of the motility estimation, and therefore there is an influence of the laboratory staff on the sperm motility assessment. Here we concluded that both the method and the technician expertise were key factors in order to accurately assess sperm motility in European eel, so the use of CASA-Mot together with qualified stuff is required to obtain reliable results. Secondly, and alternative methods for European eel males maturation was evaluated by testing two different hormonal treatments: OVI, a recombinant ¿-choriogonadotropin; and VET, a human chorionic gonadotropin purified from female urine. After choosing the best hormonal treatment, the effect of three different doses was evaluated aiming for best performance and lowest cost on the treatment. The results of this experiment pointed at OVI as the best hormonal treatment in terms on sperm quantity and quality in most of the weeks of treatment, and at a weekly dose of 1.5 IU/g fish, which also provide the greatest profitability, obtaining high quality sperm at a lower price. In a third experiment, and using the knowledge acquired in the two first experiments (using the OVI hormonal treatment and CASA-Mot to assess sperm quality), a series of experiments were conducted to standardize the European eel sperm cryopreservation protocols available at the moment (using DMSO or methanol as cryoprotectant). The results indicated that the protocol using methanol was the best of them two in terms of sperm motility and velocity, sperm viability and preservation of DNA integrity. Following this last standardized method, a fourth experiment was conducted aiming for improvement of the protocol in terms of volume (larger volumes) and sperm quality outcome. Moreover, a simple protocol for short-term storage was developed to complement the options to preserve sperm for different time periods. Of all the tested storing conditions, 1/50 dilutions at 4 ºC showed the best results, maintaining the motility compared to control for 3 days, and some sperm motility (12%) was still observed after 7 days. From the cryopreservation experiment, it was possible to scale up the cryopreserved volumes to 2 and 5 mL without losing sperm quality compared to lower volumes. Moreover, the protocol was further improved by supplementing the protocol with egg yolk as an additive, obtaining the highest cryopreserved sperm motilities (over 50%) ever reported in European eel.
Herranz Jusdado, JG. (2019). Improvement of techniques for sperm evaluation and cryobanking in European eel [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/130846
TESIS

The overall goal was to identify survival factors that can improve pregnancy success following insemination or embryo transfer in lactating dairy cows exposed to heat stress. First, we demonstrated that oocytes are actually damaged by elevated temperature in the summer. Then we tested two thermoprotective molecules for their effect on oocyte damage caused by heat shock. One molecule, ceramide was not thermoprptective. Another, insulin-like growth factor-1 (IGF) reduced the effects of heat shock on oocyte apoptosis and oocyte cleavage when added during maturation. We also used lactating cows exposed to heat stress to determine whether bovine somatotropin (bST), which increases IGF1 levels in vivo, would improve fertility in summer. Cows treated with bST received a single injection at 3 days before insemination. Controls received no additional treatment. Treatment with bST did not significantly increase the proportion of inseminated cows diagnosed pregnant although it was numerically greater for the bST group (24.2% vs 17.8%, 124–132 cows per group). There was a tendency (p =0.10) for a smaller percent of control cows to have high plasma progesterone concentrations (≥ 1 ng/ml) at Day 7 after insemination than for bST-treated cows (72.6 vs 81.1%). When only cows that were successfully synchronized were considered, the magnitude of the absolute difference in the percentage of inseminated cows that were diagnosed pregnant between bST and control cows was reduced (24.8 vs 22.4% pregnant for bST and control). Results failed to indicate a beneficial effect of bST treatment on fertility of lactating dairy cows. In another experiment, we found a tendency for addition of IGF1 to embryo culture medium to improve embryonic survival after embryo transfer when the experiment was done during heat stress but not when the experiment was done in the absence of heat stress. Another molecule tested, granulocyte-macrophage colony-stimulating factor (GM-CSF; also called colony-stimulating factor-2), improved embryonic survival in the absence of heat stress. We also examined whether heat shock affects the sperm cell. There was no effect of heat shock on sperm apoptosis (programmed cell death) or on sperm fertilizing ability. Therefore, effects of heat shock on sperm function after ejaculation if minimal. However, there were seasonal changes in sperm characteristics that indicates that some of the decrease in dairy cow fertility during the summer in Israel is due to using semen of inferior quality. Semen was collected from five representative bulls throughout the summer (August and September) and winter (December and January). There were seasonal differences in ion concentration in seminal plasma and in the mRNA for various ion channels known to be involved in acrosome reactions. Furthermore, the proportion of sperm cells with damaged acrosomes was higher in post-thaw semen collected in the summer than in its counterpart collected in winter (54.2 ± 3.5% vs. 51.4 ± 1.9%, respectively; P < 0.08Further examination is required to determine whether such alterations are involved in the low summer fertility of dairy cows.