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 rams.

Vasectomy and Photoperiodic Regimen Modify the Protein Profile, Hormonal Content and Antioxidant Enzymes Activity of Ram Seminal Plasma.

This work aimed to determine the contribution of the testis and epididymis and the effect of the photoperiodic regimen on ram seminal plasma (SP). Semen was collected from 15 mature rams located in an equatorial (Colombian Creole and Romney Marsh, eight intact and two vasectomized) or a temperate climate (Rasa Aragonesa, three intact and two vasectomized). SP proteins were analyzed by Bradford, SDS-PAGE and difference gel electrophoresis (DIGE).

Melatonin membrane receptors MT and MT are expressed in ram spermatozoa from non-seasonal breeds.

In mammals, many melatonin biological functions are mediated through its interaction with the membrane receptors MT and MT. We have previously reported their presence in ram spermatozoa from males located in temperate climates, but there is no information on their presence in spermatozoa from rams in areas with an equatorial photoperiod (12L:12D). Thus, we have investigated the existence and cellular distribution of melatonin receptors in spermatozoa from three sheep breeds in Colombia (Colombian Creole, Hampshire, and Romney Marsh) during dry and rainy seasons, using indirect immunofluorescence and western blot.

Does Melatonin Exert Its Effect on Ram Sperm Capacitation Through Nitric Oxide Synthase Regulation?

Nitric oxide (NO·), synthesized from L-arginine by nitric oxide synthase (NOS), is involved in sperm functionality. NOS isoforms have been detected in spermatozoa from different species, and an increment in NOS activity during capacitation has been reported. This work aims to determine the presence and localization of NOS isoforms in ram spermatozoa and analyse their possible changes during in vitro capacitation.

OpenCASA: A new open-source and scalable tool for sperm quality analysis.

In the field of assisted reproductive techniques (ART), computer-assisted sperm analysis (CASA) systems have proved their utility and potential for assessing sperm quality, improving the prediction of the fertility potential of a seminal dose. Although most laboratories and scientific centers use commercial systems, in the recent years certain free and open-source alternatives have emerged that can reduce the costs that research groups have to face. However, these open-source alternatives cannot analyze sperm kinetic responses to different stimuli, such as chemotaxis, thermotaxis or rheotaxis.

Changes in melatonin concentrations in seminal plasma are not correlated with testosterone or antioxidant enzyme activity when rams are located in areas with an equatorial photoperiod.

In temperate climates, photoperiod and melatonin regulate ram reproduction, modulating hormonal secretions, sperm quality, and seminal plasma composition. Information on the effect of an equatorial photoperiod (12L:12D) on ram reproduction, however, is scarce, and no data on hormonal concentrations and antioxidant enzyme activity in seminal plasma have been reported. Thus, the variation was investigated of melatonin and its relationship with testosterone and antioxidant enzyme activity in the seminal plasma of three sheep breeds in Colombia, when there was a consistent photoperiod during two dry and two rainy seasons per year.

Melatonin reduces cAMP-stimulated capacitation of ram spermatozoa.

The presence of melatonin receptors on the surface of ram spermatozoa has led to speculation about melatonin having a role in sperm functionality. The aim of this study was to elucidate the mechanism through which melatonin regulates ram sperm capacitation induced by a cocktail containing cAMP-elevating agents. Cocktail samples capacitated in the presence of 1µM melatonin showed lower percentages of capacitated spermatozoa (chlortetracycline staining; P<0.

Melatonin affects the motility and adhesiveness of in vitro capacitated boar spermatozoa via a mechanism that does not depend on intracellular ROS levels.

This work sought to address the effects of melatonin during in vitro capacitation (IVC) and progesterone-induced acrosome exocytosis (IVAE) in boar spermatozoa. With this purpose, two different experiments were set. In the first one, IVC and IVAE were induced in the absence or presence of melatonin, which was added either at the start of IVC or upon triggering the IVAE with progesterone.

Role of melatonin on embryo viability in sheep.

Melatonin is a natural hormone synthesised in the pineal gland, the activity of which is regulated by day-night perception and dictates seasonal rhythms in reproduction in ovine species. Exogenous melatonin, administered via subcutaneous implants, is used to prolong the breeding season of ewes and can increase the proportion of pregnant ewes (fertility rate) and litter size. The increased proportion of ewes that become pregnant and the number of lambs born per lambing among melatonin-treated sheep may be caused by increased embryo survival, through enhanced luteal function, reduced antiluteolytic mechanisms, or improved embryo quality.

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.

Melatonin MT₁ and MT₂ Receptors in the Ram Reproductive Tract.

Some melatonin functions in mammals are exerted through MT₁ and MT₂ receptors. However, there are no reports of their presence in the reproductive tract of the ram, a seasonal species. Thus, we have investigated their existence in the ram testis, epididymis, accessory glands and ductus deferens.

Melatonin receptors MT1 and MT2 are expressed in spermatozoa from several seasonal and nonseasonal breeder species.

Melatonin is a ubiquitous and multipurpose molecule, and one of its roles is to regulate reproduction in some seasonal mammals. Our group has previously reported the variation in the melatonin levels in ram seminal plasma along the year and identified MT1 and MT2 receptors in ram spermatozoa. The objective of this study was to elucidate whether the presence of melatonin receptors (MT1 and MT2) in the sperm plasma membrane, and melatonin in the seminal plasma is related to seasonal breeding.

Evaluation of Lasting Effects of Heat Stress on Sperm Profile and Oxidative Status of Ram Semen and Epididymal Sperm.

Higher temperatures lead to an increase of testicular metabolism that results in spermatic damage. Oxidative stress is the main factor responsible for testicular damage caused by heat stress. The aim of this study was to evaluate lasting effects of heat stress on ejaculated sperm and immediate or long-term effects of heat stress on epididymal sperm.

Induced lipid peroxidation in ram sperm: semen profile, DNA fragmentation and antioxidant status.

Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period.

New Insights into the Phylogeny and Gene Context Analysis of Binder of Sperm Proteins (BSPs).

Seminal plasma (SP) proteins support the survival of spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, resulting in higher fertilizing ability. Among SP proteins, BSP (binder of sperm) proteins are the most studied, since they may be useful for the improvement of semen diluents, storage and subsequent fertilization results. However, an updated and detailed phylogenetic analysis of the BSP protein superfamily has not been carried out with all the sequences described in the main databases.

Effect of seminal plasma proteins on the motile sperm subpopulations in ram ejaculates.

It has been proposed that seminal plasma proteins (SPP) support survival of ram spermatozoa, exerting a dual effect, both capacitating and decapacitating. In this study, changes in motility patterns of ram spermatozoa capacitated in the presence of epidermal growth factor (EGF) were evaluated. Clustering procedures were used to determine the presence of sperm subpopulations with specific motion characteristics.

New evidence of melatonin receptor contribution to ram sperm functionality.

The present study analysed the involvement of melatonin, acting via its receptors (MT and MT), in ram sperm functionality. Indirect immunofluorescence assays revealed no changes in the distribution or intensity of MT receptors, whereas different subpopulations were established for MT receptors in control, in vitro capacitated and acrosome-reacted ram spermatozoa. Chlortetracycline staining revealed the following correlations between the pattern of staining for MT receptors in: (1) non-capacitated (NC) sperm rate and the proportion of spermatozoa with equal immunostaining intensity in the acrosome and post-acrosome (r=0.

Oligomycin A-induced inhibition of mitochondrial ATP-synthase activity suppresses boar sperm motility and in vitro capacitation achievement without modifying overall sperm energy levels.

Incubation of boar spermatozoa in a capacitation medium with oligomycin A, a specific inhibitor of the F0 component of the mitochondrial ATP synthase, induced an immediate and almost complete immobilisation of cells. Oligomycin A also inhibited the ability of spermatozoa to achieve feasible in vitro capacitation (IVC), as measured through IVC-compatible changes in motility patterns, tyrosine phosphorylation levels of the acrosomal p32 protein, membrane fluidity and the ability of spermatozoa to achieve subsequent, progesterone-induced in vitro acrosome exocytosis (IVAE). Both inhibitory effects were caused without changes in the rhythm of O2 consumption, intracellular ATP levels or mitochondrial membrane potential (MMP).

The effect of exogenous melatonin during the non-reproductive season on the seminal plasma hormonal profile and the antioxidant defence system of Rasa Aragonesa rams.

The aim of this study was to analyze the effect of melatonin implants, during the non-reproductive season, on the content of melatonin, testosterone and 17-β estradiol levels, and the antioxidant enzymes glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase of seminal plasma in Rasa Aragonesa rams. Five rams were implanted with melatonin, and four others were used as a control group. Seminal plasma was separated from ejaculates collected one week before melatonin treatment until 21 weeks after melatonin placement, and the hormonal levels and the antioxidant enzyme activity were determined.

Characterization of the cDNA and in vitro expression of the ram seminal plasma protein RSVP14.

In previous studies we have shown that seminal plasma (SP) proteins can prevent and repair cold-shock membrane damage to ram spermatozoa. Three proteins of approximately 14, 20 and 22kDa, mainly responsible for this protective ability, were identified in ram SP. They are exclusively synthesized in the seminal vesicles and, consequently, named RSVP14, RSVP20 and RSVP22.

Identification and immunolocalisation of melatonin MT(1) and MT(2) receptors in Rasa Aragonesa ram spermatozoa.

The reproductive seasonality of sheep suggests that melatonin receptors may be present in ram spermatozoa. The present study confirms the presence of melatonin MT(1) and MT(2) receptors. The MT(1) receptor was detected using immunocytochemistry, with four sperm subpopulations identified based on the following labelling patterns: (1) one small subpopulation with labelling over the entire head and tail; (2) one of two main subpopulations that exhibited reactivity at the equatorial, post-acrosomal, neck and tail regions; (3) another main subpopulation with equatorial and tail labelling only; and (4) a subpopulation in which staining was detected only in the tail.

A novel epidermal growth factor-dependent extracellular signal-regulated MAP kinase cascade involved in sperm functionality in sheep.

Sperm capacitation is characterized by a series of significant biochemical and biophysical modifications. Unlike the case with most other mammalian species, ram spermatozoa are difficult to capacitate in vitro. We have already suggested that unusually high levels of intracellular phosphodiesterases would account for cAMP levels that are too low to initiate tyrosine phosphorylation of flagellar proteins that are indicative of capacitation.

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.