Use of specific mitochondrial complex inhibitors to investigate mitochondrial involvement on horse sperm motility and ROS production.

Equine spermatozoa highly rely on oxidative phosphorylation for their energy management. The present work aimed to characterize the role of mitochondria on horse sperm motility and ROS production by incubating spermatozoa with specific inhibitors of the different mitochondrial complexes. Equine spermatozoa were incubated 1 h and 3 h at 37 °C with: complex I inhibitor rotenone (5 μM, ROT), complex II inhibitor dimethyl-malonate (10 mM, DMM), complex III inhibitor antimycin A (1.

Role of exogenous antioxidants on the performance and function of pig sperm after preservation in liquid and frozen states: A systematic review.

In situations where an excessive generation of reactive oxygen species overwhelms antioxidant capacity, a harmful effect on sperm function is exerted. Antioxidants are molecules capable of minimizing this detrimental effect, which is important in pig sperm due to the high content of polyunsaturated fatty acids in their plasma membrane. The present systematic review aims at evaluating whether supplementing semen extenders (for liquid storage at 17 °C) or freezing and/or thawing media (for cryopreservation) with antioxidants influences sperm quality and functionality parameters, and in vitro/in vivo fertility outcomes.

Different approaches for assessing sperm function.

Different approaches can be used to assess sperm function in different conditions, i.e. sperm storage, freezing-thawing or activation by induction of capacitation and acrosome reaction.

Improvement of fertilization by a tannin rich vegetal extract addition to frozen thawed boar sperm.

Boar spermatozoa are very susceptible to cryopreservation injuries and, for this reason, pig remains one of the few species in which fresh semen is still preferred to thawed one for routine artificial insemination (AI). The present work evaluated the effect of supplementing boar sperm thawing medium with Silvafeed SP (SSP), a mixture of Chestnut and Quebracho wood extracts (60/40 w/w) rich in polyphenols (92.4% tannin content) on fertilization (IVF) and on the following sperm parameters: sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function and lipid peroxidation (assessed by flow cytometry) and capacitation status (immunolocalization of tyrosine phosphorylated proteins).

Sperm function and mitochondrial activity: An insight on boar sperm metabolism.

In this study boar sperm mitochondrial activity was studied and deepened in order to delineate the main metabolic strategies used by boar sperm to obtain energy and to link them to sperm function. Boar spermatozoa were collected, diluted at 30 × 10 spz/mL and incubated for 1 h with: Rotenone (ROT), complex I inhibitor, Dimethyl-malonate (DMM), complex II inhibitor, antimycin A (ANTI), complex III inhibitor, oligomycin (OLIGO), ATP synthase inhibitor, Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), uncoupling agent, 2-deoxy-glucose (2DG), glucose agonist, and Dimethyl sulphoxide (DMSO) as control vehicle. Viability and mitochondrial membrane potential (Sybr14/PI/JC1 staining) and sperm motility (using CASA system) were assayed after incubation.

Combined effects of resveratrol and epigallocatechin-3-gallate on post thaw boar sperm and IVF parameters.

Frozen-thawed boar semen suffer a fertility decrease that negatively affects its widespread use. In recent years supplementing frozen-thawed boar sperm with different antioxidants gave interesting and promising results; the aim of the present work was to study the effect of supplementing boar sperm thawing medium for 1 h with combination of epigallocatechin-3-gallate (EGCG, 50 μM) and Resveratrol (R, 2 mM), on boar sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function, lipid peroxidation and DNA integrity (assessed by flow cytometry), protein tyrosine phosphorylation (assessed by immunofluorescence) and on in vitro fertilization (IVF). Our results demonstrate that sperm motility is negatively affected by R (alone or associated with EGCG, p < 0.

Biological effects of polyphenol-rich extract and fractions from an oenological oak-derived tannin on in vitro swine sperm capacitation and fertilizing ability.

Although excessive ROS levels induce sperm damage, sperm capacitation is an oxidative event that requires low amounts of ROS. As the antioxidant activity of the ethanol extract (TRE) of a commercial oenological tannin (Quercus robur toasted oak wood, Tan'Activ R) and its four fractions (FA, FB, FC, FD) has been recently reported, the present study was set up to investigate the biological effects of TRE and its fractions in an in vitro model of sperm capacitation and fertilization. Boar sperm capacitation or gamete coincubation were performed in presence of TRE or its fractions (0, 1, 10, 100 μg/ml).

Implementing an open-access CASA software for the assessment of stallion sperm motility: Relationship with other sperm quality parameters.

Setting an open-access computer assisted sperm analysis (CASA) may benefit the evaluation of motility in mammalian sperm, especially when economic constraints do not allow the use of a commercial system. There have been successful attempts to develop such a device in Zebra fish sperm and the system has been used in very few studies on mammalian spermatozoa. Against this background, the present study aimed at developing an open-access CASA system for mammalian sperm using the horse as a model and based upon the Image J software previously established for Zebra fish sperm.

Alkaline phosphatase added to capacitating medium enhances horse sperm-zona pellucida binding.

Alkaline phosphatase (AP) is present in equine seminal plasma and spermatozoa, but its functional role is not fully understood yet. Being that, sperm-oocyte interaction in equine species has been reported to be enhanced at a slightly basic pH, this work aimed at verifying whether exogenous alkaline phosphatase exerts any role on stallion spermatozoa and sperm-oocyte interaction at different pHs (7.4; 8.

Porcine circovirus type 2 detection in in vitro produced porcine blastocysts after virus sperm exposure.

This study was aimed at assessing the capability of semen experimentally infected with porcine circovirus type 2 (PCV2) to produce porcine blastocysts PCR positive for PCV2. Embryos were obtained from in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes or by parthenogenesis. Sperm suspension was exposed to PCV2b and utilized for IVF.

Characterization of alkaline phosphatase activity in seminal plasma and in fresh and frozen-thawed stallion spermatozoa.

Alkaline phosphatase (AP) has been studied in several situations to elucidate its role in reproductive biology of the male from different mammalian species; at present, its role in horse sperm physiology is not clear. The aim of the present work was to measure AP activity in seminal plasma and sperm extracts from freshly ejaculated as well as in frozen-thawed stallion spermatozoa and to verify whether relationship exists between AP activity and sperm quality parameters. Our data on 40 freshly ejaculated samples from 10 different stallions demonstrate that the main source of AP activity is seminal plasma, whereas sperm extracts contribution is very low.

Sex-sorting of boar spermatozoa does not influence the localization of glucose transporters.

Sex-sorting damages spermatozoa function, shortening their lifespan and fertility. This study used an immunofluorescence technique to investigate the effect of sex-sorting on the localization of glucose transporters (GLUTs) in boar spermatozoa. GLUTs are trans-membrane proteins responsible for glucose transport within cells.

Effects of resveratrol on vitrified porcine oocytes.

Vitrified MII porcine oocytes are characterized by reduced developmental competence, associated with the activation of the apoptotic pathway. Resveratrol (R), a polyphenolic compound present in several vegetal sources, has been reported to exert, among all its other biological effects, an antiapoptotic one. The aim of this study was to determine the effects of R (2  µM) on the apoptotic status of porcine oocytes vitrified by Cryotop method, evaluating phosphatidylserine (PS) exteriorization and caspases activation.

GLUTs and mammalian sperm metabolism.

Mammalian cells use glucides as a substrate that can be catabolized through glycolitic pathways or oxidative phosphorylation, used as a source of reducing potential, or used for anabolic aims. An important role in supplying cells with energy is played by different membrane proteins that can actively (sodium-dependent glucose transporters) or passively (glucose transporters; GLUT) transport hexoses through the lipidic bilayer. In particular, GLUTs are a family of 13 proteins that facilitate the transport of sugars and have a peculiar distribution in different tissues as well as a particular affinity for substrates.

Daidzein does affect progesterone secretion by pig cumulus cells but it does not impair oocytes IVM.

Daidzein, an isoflavone abundant in soybeans and other legumes, displays estrogen like properties. This study was aimed at evaluating the effect of daidzein (1 and 10 microM) on nuclear and cytoplasmic maturation of pig oocytes and on steroidogenic activity of cumulus cells. Daidzein supplementation during IVM had no effect on nuclear maturation and on fertilization traits.

Cortisol determination in hair and faeces from domestic cats and dogs.

The present study explored the feasibility of a hair cortisol assay in domestic cats (Felis silvestris catus) and dogs (Canis familiaris) as a valid and reliable alternative to existing non-invasive techniques for monitoring the hypothalamic-pituitary-adrenal (HPA) axis activity. To this aim, 56 new hair growth samples and 870 faecal samples from 27 domestic cats and 29 domestic dogs were collected and cortisol content was assessed. A significant positive association was observed in both species between the concentrations of cortisol determined in hair and faeces.

Leptin receptor in boar spermatozoa.

Leptin is active in both metabolism and reproduction. In fact, it seems to exert an inhibitory action on gonadal functions by reducing testosterone production. The presence of leptin in human and boar seminal plasma and in human spermatozoa has been demonstrated; recently, leptin receptors (Ob-R) have been localized in human spermatozoa, thus suggesting a possible action of this hormone even on these cells.

Characterization and differential expression of vascular endothelial growth factor isoforms and receptors in swine corpus luteum throughout estrous cycle.

Corpus luteum (CL) undergoes growth and regression during each estrous cycle; these processes are accompanied by growth and regression of the luteal vascular bed. Vascular endothelial growth factor (VEGF) is the main regulator of angiogenesis, inducing endothelial cell proliferation, migration, vascular permeability, and vessel lumen formation. VEGF presents several isoforms that are produced by alternative splicing of the same mRNA transcript.

Sperm sorting procedure induces a redistribution of Hsp70 but not Hsp60 and Hsp90 in boar spermatozoa.

Heat shock proteins, besides their protective function against stresses, have been recently indicated as key factors for sperm fertilizing ability. Since sexing sperm by high-speed flow-cytometry subjects them to different physical, mechanical, and chemical stresses, the present study was designed to verify, by immunofluorescence and Western blot, whether the sorting procedure induces any modification in the amount and cellular distribution of heat shock proteins 60, 70, and 90 (Hsp60, Hsp70, Hsp90). Immunolocalization and Western blot quantification of both Hsp60 and Hsp90 did not reveal differences between unsorted and sorted semen.

Food deprivation stimulates the luteolytic capacity in the gilt.

The aims of this study were to study the effects of fasting on progesterone (P4) production in the pig and to verify whether fasting influences luteal expression of PGF(2alpha) receptor (FPr) and prostaglandin secretion. Superovulated prepubertal gilts were used; half of them were fasted for 72h starting on day 2 (F2) or 9 (F9) of the induced estrous cycle, respectively, while two groups (C2 and C9) served as respective controls. Plasma P4 and PGFM concentrations were determined by RIA while FPr mRNA expression in CLs collected at the end of fasting period was measured by real-time PCR.

Growth hormone expression and secretion in pig pituitary and median eminence slices are not influenced by the VGF protein.

Body homeostasis is maintained by a complex system that involves the brain and the periphery via many circulating hormones. In recent years the VGF protein has been indicated as an important peptide affecting the regulation of body composition. We examined the effects of VGF on growth hormone (GH) expression and secretion in porcine pituitary slices, incubated alone (group 1) or with stalk median eminence (SME) (group 2).

Heat shock protein 70, heat shock protein 32, and vascular endothelial growth factor production and their effects on lipopolysaccharide-induced apoptosis in porcine aortic endothelial cells.

Lipopolysaccharide (LPS) is a highly proactive molecule that causes in vivo a systemic inflammatory response syndrome and activates in vitro the inflammatory pathway in different cellular types, including endothelial cells (EC). Because the proinflammatory status could lead to EC injury and apoptosis, the expression of proinflammatory genes must be finely regulated through the induction of protective genes. This study aimed at determining whether an LPS exposure is effective in inducing apoptosis in primary cultures of porcine aortic endothelial cells and in stimulating heat shock protein (Hsp)70 and Hsp32 production as well as vascular endothelial growth factor (VEGF) secretion.

Immunolocalization of heat shock protein 70 (Hsp 70) in boar spermatozoa and its role during fertilization.

The presence and cellular distribution of heat protein 70 (Hsp70) in ejaculated, capacitated, and acrosome-reacted boar spermatozoa was evaluated by immunofluorescence and Western blot; the role of Hsp70 during fertilization was also studied. In freshly ejaculated spermatozoa, Hsp70 immunoreactivity is present in a well-defined triangular-shaped area in the equatorial segment that seems to correspond to the equatorial sub-segment. The distribution of the fluorescent signal changes in capacitated sperm, that exhibit different patterns probably in relation to the stage of capacitation of individual cells; after acrosome reaction Hsp70 immunoreactivity is localized on both a thick sub-equatorial band and a triangle in the equatorial segment.

Fasting influences steroidogenesis, vascular endothelial growth factor (VEGF) levels and mRNAs expression for VEGF, VEGF receptor type 2 (VEGFR-2), endothelin-1 (ET-1), endothelin receptor type A (ET-A) and endothelin converting enzyme-1 (ECE-1) in newly formed pig corpora lutea.

This study was designed to verify whether fasting influences vascular endothelial growth factor (VEGF) production and VEGF, VEGF receptor-2 (VEGFR-2) as well as endothelin (ET) system members (endothelin converting enzyme-1, ECE-1; ET-1; endothelin receptor type A, ET-A) mRNA expression in pig corpora lutea; furthermore, we wanted to assess whether fasting affects steroidogenesis in luteal cells. Eight prepubertal gilts were induced to ovulate and were randomly assigned to two groups: (A) n = 4, normally fed; and (B) n = 4, fasted for 72 h starting 3 days after ovulation. At the end of fasting, ovaries were removed from all the animals and corpora lutea (CLs) were collected.