Antimicrobial peptides and proteins as alternative antibiotics for porcine semen preservation.

BACKGROUND: Antimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture, are promoting AMR while also encouraging the research and employment of alternative drugs. The addition of antibiotics to the cell media is strongly recommended in sperm preservation, being gentamicin the most used for boar semen. Because of its continued use, several bacterial strains present in boar semen have developed resistance to this antibiotic. Antimicrobial peptides and proteins (AMPPs) are promising candidates as alternative antibiotics because their mechanism of action is less likely to promote AMR. In the present study, we tested two AMPPs (lysozyme and nisin; 50 and 500 µg/mL) as possible substitutes of gentamicin for boar semen preservation up to 48 h of storage. RESULTS: We found that both AMPPs improved sperm plasma membrane and acrosome integrity during semen storage. The highest concentration tested for lysozyme also kept the remaining sperm parameters unaltered, at 48 h of semen storage, and reduced the bacterial load at comparable levels of the samples supplemented with gentamicin (p > 0.05). On the other hand, while nisin (500 µg/mL) reduced the total Enterobacteriaceae counts, it also decreased the rapid and progressive sperm population and the seminal oxidation-reduction potential (p < 0.05). CONCLUSIONS: The protective effect of lysozyme on sperm function together with its antimicrobial activity and inborn presence in body fluids, including semen and cervical mucus, makes this enzyme a promising antimicrobial agent for boar semen preservation.

N-thiocarboxyanhydrides, amino acid-derived enzyme-activated H2S donors, enhance sperm mitochondrial activity in presence and absence of oxidative stress.

BACKGROUND: Hydrogen sulfide (H2S) donors are crucial tools not only for understanding the role of H2S in cellular function but also as promising therapeutic agents for oxidative stress-related diseases. This study aimed to explore the effect of amino acid-derived N-thiocarboxyanhydrides (NTAs), which release physiological H2S levels in the presence of carbonic anhydrase, on porcine sperm function during short-term incubation with and without induced oxidative stress. For this purpose, we employed two H2S-releasing NTAs with release half-lives (t1/2) in the range of hours that derived from the amino acids glycine (Gly-NTA) or leucine (Leu-NTA). Because carbonic anhydrase is crucial for H2S release from NTAs, we first measured the activity of this enzyme in the porcine ejaculate. Then, we tested the effect of Gly- and Leu-NTAs at 10 and 1 nM on sperm mitochondrial activity, plasma membrane integrity, acrosomal status, motility, motile subpopulations, and redox balance during short-term incubation at 38 °C with and without a reactive oxygen species (ROS)-generating system. RESULTS: Our results show that carbonic anhydrase is found both in spermatozoa and seminal plasma, with activity notably higher in the latter. Both Gly- and Leu-NTAs did not exert any noxious effects, but they enhanced sperm mitochondrial activity in the presence and absence of oxidative stress. Moreover, NTAs (except for Leu-NTA 10 nM) tended to preserve the sperm redox balance against the injuries provoked by oxidative stress, which provide further support to the antioxidant effect of H2S on sperm function. Both compounds also increased progressive motility over short-term incubation, which may translate into prolonged sperm survival. CONCLUSIONS: The presence of carbonic anhydrase activity in mammalian spermatozoa makes NTAs promising molecules to investigate the role of H2S in sperm biology. For the first time, beneficial effects of NTAs on mitochondrial activity have been found in mammalian cells in the presence and absence of oxidative stress. NTAs are interesting compounds to investigate the role of H2S in sperm mitochondria-dependent events and to develop H2S-related therapeutic protocols against oxidative stress in assisted reproductive technologies.

Habitat-dependent Culicoides species composition and abundance in blue tit (Cyanistes caeruleus) nests.

Wild birds are hosts of Culicoides from as early on as the nesting stage when constrained to their nests. However, the environmental factors which determine the abundance and composition of Culicoides species within each bird nest are still understudied. We sampled Culicoides from Eurasian blue tit (Cyanistes caeruleus) nests found in 2 types of forests located in southern Spain. Firstly, we monitored the abundance of Culicoides species in bird nests from a dry Pyrenean oak deciduous forest and a humid mixed forest comprising Pyrenean and Holm oaks throughout 2 consecutive years. During the 3rd year, we performed a cross-fostering experiment between synchronous nests to differentiate the role of rearing environment conditions from that of the genetically determined or maternally transmitted cues released by nestlings from each forest. We found 147 female Culicoides from 5 different species in the birds' nests. The abundance of Culicoides was higher in the dry forest than in the humid forest. Culicoides abundance, species richness and prevalence were greater when the nestlings were hatched later in the season. The same pattern was observed in the cross-fostering experiment, but we did not find evidence that nestling's features determined by the forest of origin had any effect on the Culicoides collected. These results support the notion that habitat type has a strong influence on the Culicoides affecting birds in their nests, while some life history traits of birds, such as the timing of reproduction, also influence Culicoides abundance and species composition.

The Interaction of NO and H2S in Boar Spermatozoa under Oxidative Stress.

Various recent studies dedicated to the role of nitric oxide (NO) and hydrogen sulfide (H2S) in somatic cells provide evidence for an interaction of the two gasotransmitters. In the case of male gametes, only the action of a single donor of each gasotransmitter has been investigated up until today. It has been demonstrated that, at low concentrations, both gasotransmitters alone exert a positive effect on sperm quality parameters. Moreover, the activity of gaseous cellular messengers may be affected by the presence of oxidative stress, an underlying condition of several male reproductive disorders. In this study, we explored the effect of the combination of two donors SNP and NaHS (NO and H2S donors, respectively) on boar spermatozoa under oxidative stress. We applied NaHS, SNP, and their combination (DD) at 100 nM concentration in boar spermatozoa samples treated with Fe2+/ascorbate system. After 90 min of incubation at 38 °C, we have observed that progressive motility (PMot) and plasma membrane integrity (PMI) were improved (p < 0.05) in DD treatment compared to the Ctr sample under oxidative stress (CtrOX). Moreover, the PMot of DD treatment was higher (p < 0.05) than that of NaHS. Similar to NaHS, SNP treatment did not overcome the PMot and PMI of CtrOX. In conclusion, for the first time, we provide evidence that the combination of SNP and NaHS surmounts the effect of single-donor application in terms of PMot and PMI in porcine spermatozoa under oxidative stress.

Comparative Study of Semen Parameters and Hormone Profile in Small-Spotted Catshark (Scyliorhinus canicula): Aquarium-Housed vs. Wild-Captured.

Several chondrichthyan species are threatened, and we must increase our knowledge of their reproductive biology in order to establish assisted reproductive protocols for ex situ or in situ endangered species. The small-spotted catshark (Scyliorhinus canicula) is one of the most abundant shark species of the Mediterranean coast and is easy to maintain in aquaria; therefore, it is considered an ideal reproductive model. This study aimed to compare S. canicula male reproductive function in aquarium-housed (n = 7) and wild-captured animals, recently dead (n = 17). Aquarium-housed animals had lower semen volume (p = 0.005) and total sperm number (p = 0.006) than wild-captured animals, but similar sperm concentrations. In terms of sperm parameters, aquarium-housed sharks showed higher total sperm motility (p = 0.004), but no differences were observed regarding sperm viability, mitochondrial membrane potential, or membrane integrity. A morphometric study pointed to a significantly longer head (p = 0.005) and acrosome (p = 0.001) in wild-captured animals. The results of the spermatozoa morphological study of S. canicula were consistent with previous results obtained in other chondrichthyan species. With regard to sex hormones, testosterone levels were significantly lower in aquarium-housed animals (p ≤ 0.001), while similar levels of 17ß-estradiol and progesterone were found. In short, the present study provides evidence of good in vitro semen quality in S. canicula housed in an aquarium, underlining their excellent potential for application in reproductive technologies for this and other chondrichthyan species.

Protein Identification of Spermatozoa and Seminal Plasma in Bottlenose Dolphin (Tursiops truncatus).

Proteins play an important role in many reproductive functions such as sperm maturation, sperm transit in the female genital tract or sperm-oocyte interaction. However, in general, little information concerning reproductive features is available in the case of aquatic animals. The present study aims to characterize the proteome of both spermatozoa and seminal plasma of bottlenose dolphins (Tursiops truncatus) as a model organism for cetaceans. Ejaculate samples were obtained from two trained dolphins housed in an aquarium. Spermatozoa and seminal plasma were analyzed by means of proteomic analyses using an LC-MS/MS, and a list with the gene symbols corresponding to each protein was submitted to the DAVID database. Of the 419 proteins identified in spermatozoa and 303 in seminal plasma, 111 proteins were shared by both. Furthermore, 70 proteins were identified as involved in reproductive processes, 39 in spermatozoa, and 31 in seminal plasma. The five most abundant proteins were also identified in these samples: AKAP3, ODF2, TUBB, GSTM3, ROPN1 for spermatozoa and CST11, LTF, ALB, HSP90B1, PIGR for seminal plasma. In conclusion, this study provides the first characterization of the proteome in cetacean sperm and seminal plasma, opening the way to future research into new biomarkers, the analysis of conservation capacity or possible additional applications in the field of assisted reproductive technologies.

Impact of Oxidative Stress on Male Reproduction in Domestic and Wild Animals.

Oxidative stress occurs when the levels of reactive oxygen species (ROS) overcome the antioxidant defenses of the organism, jeopardizing several biological functions, including reproduction. In the male reproductive system, oxidative stress not only impairs sperm fertility but also compromises offspring health and survival, inducing oxidative damage to lipids, proteins and nucleic acids. Although a clear link between oxidative stress and male fertility disorders has been demonstrated in humans and laboratory rodents, little information is available about the implications of impaired redox homeostasis in the male fertility of domestic and wild animals. Therefore, this review aims to provide an update regarding the intrinsic and extrinsic factors that are associated with oxidative stress in the male reproductive system and their impact on the reproductive performance of domestic and wild animals. The most recent strategies for palliating the detrimental effects of oxidative stress on male fertility are reviewed together with their potential economic and ecological implications in the livestock industry and biodiversity conservation.

Plant Extracts as Alternative Additives for Sperm Preservation.

Sperm preservation is a crucial factor for the success of assisted reproductive technology (ART) in humans, livestock, and wildlife. Irrespective of the extender and the storage conditions used, semen handling and preservation negatively affect sperm quality. Moreover, oxidative stress, which often arises during semen storage, significantly reduces sperm function and compromises the sperm fertilizing ability by inducing oxidative damage to proteins, lipids, and nucleic acids. Plant extracts have recently emerged as a cheap and natural source of additives to preserve and enhance sperm function during semen storage. The present work provides an update on the use of these natural compounds as alternative additives for sperm preservation in 13 animal species, including humans. A detailed description of the effects of 45 plant species, belonging to 28 families, on sperm function during semen storage is presented. The plant material and extraction method employed, dosage, possible toxic effects, and antimicrobial properties are provided.

Oxidative status of blue tit nestlings varies with habitat and nestling size.

Oxidative status has been proposed as an important ecological and evolutionary force given that pro-oxidant metabolites damage molecules, cells and tissues, with fitness consequences for organisms. Consequently, organisms usually face a trade-off between regulating their oxidative status and other physiological traits. However, environmental stressors and the availability of dietary-derived antioxidants vary according to local conditions and, thus, organisms inhabiting different habitats face different oxidative pressures. Still, there is little information on how different environmental conditions influence the oxidative status of animals inhabiting terrestrial environments. In this work, we examined the variation in oxidative status in the blue tit (Cyanistes caeruleus), a bird species with hatching asynchrony. Specifically, we examined the oxidative status of the largest and the smallest nestlings in the brood, inhabiting four forests differing in food availability and ectoparasite prevalence. We measured lipid peroxidation (malondialdehyde; MDA) as a marker of oxidative damage, total antioxidant capacity (Trolox-equivalent antioxidant capacity; TEAC) and antioxidant enzymatic activity (catalase, glutathione S-transferase, glutathione peroxidase) in blood samples. The glutathione peroxidase (GPX) activity differed among the forests, being the highest in the pine forest and the lowest in a mixed oak (Quercus) forest in the most humid area. Lipid peroxidation was higher in larger nestlings, suggesting higher oxidative damage with an increasing growth rate. Neither brood size, laying date, nor ectoparasites were related to the oxidative status of nestlings. These results suggest that nest rearing conditions might shape the oxidative status of birds, having consequences for habitat-dependent variation in regulation of oxidative status.

Spermatogenic Activity and Sperm Traits in Post-Pubertal and Adult Tomcats (Felis catus): Implication of Intra-Male Variation in Sperm Size.

Tomcats are considered to be adults at 1 year of age, although many reach sexual maturity at an earlier age. Nevertheless, we still know little about whether the spermatogenic activity and sperm quality of mature under one-year-old tomcats differ from those of tomcats that are over one-year-old. This study aims to evaluate the spermatogenic activity, sperm traits, and their relationships in mature tomcats at two different ages. Sixteen tomcats showing complete spermatogenesis and spermatozoa in their epididymal caudae were used and classified according to their age as post-pubertal (<1 year old) or adult (˃1 year old). Our results show that adult cats have higher epididymal sperm concentration and lower coefficient of variation in sperm head width and ellipticity than post-pubertal cats. However, they do not differ in their testicular and epididymal mass, spermatogenesis, and sperm traits such as motility, mitochondrial activity, morphology, morphometry, as well as plasma membrane, acrosome, and DNA integrity. Reduced intra-male variation of sperm head ellipticity is associated with higher testis mass, epididymis mass, and sperm concentration. Interestingly, low intra-male variation in sperm head size is associated with increased Sertoli cell function and reduced post-meiotic germ cell loss. These findings increase our knowledge about feline reproductive physiology and provide new insights into the functional significance of low intra-male variation in sperm size and shape in tomcats.

Characterization of Bottlenose Dolphin (Tursiops truncatus) Sperm Based on Morphometric Traits.

Bottlenose dolphin (Tursiops truncatus) males follow many reproductive strategies to ensure their paternity. However, little is known about the sperm traits, including morphometric features, that contribute to their reproductive success. Our aim was to study dolphin sperm morphometry (a total of 13 parameters) in two adult males to evaluate (i) presumptive sperm subpopulations, (ii) the correlation of sperm morphometry with testosterone levels and (iii) the effect of refrigerated storage on the sperm morphometry. Sperm populations were classified into four principal components (PCs) based on morphometry (>94% of cumulative variance). The PCs clustered into two different sperm subpopulations, which differed between males. Furthermore, the levels of serum testosterone were positively correlated with the length of the midpiece but negatively correlated with head width and the principal piece, flagellum and total sperm lengths. Most of the sperm morphometric parameters changed during the storage period (day 1 vs. day 7), but only the principal piece length was affected by the storage temperature (5 °C vs. 15 °C). This is the first study to identify dolphin sperm subpopulations based on morphometry and the influence of serum testosterone and refrigeration on sperm morphometry.

Divergent effect of fast- and slow-releasing H2S donors on boar spermatozoa under oxidative stress.

Hydrogen sulphide (H2S) is involved in the physiology and pathophysiology of different cell types, but little is known about its role in sperm cells. Because of its reducing properties, we hypothesise that H2S protects spermatozoa against the deleterious effects of oxidative stress, a condition that is common to several male fertility disorders. This study aimed i) to determine the total antioxidant capacities of Na2S and GYY4137, which are fast- and slow-releasing H2S donors, respectively, and ii) to test whether H2S donors are able to protect spermatozoa against oxidative stress. We found that Na2S and GYY4137 show different antioxidant properties, with the total antioxidant capacity of Na2S being mostly unstable and even undetectable at 150 µM. Moreover, both H2S donors preserve sperm motility and reduce acrosome loss, although the effects were both dose and donor dependent. Within the range of concentrations tested (3-300 µM), GYY4137 showed positive effects on sperm motility, whereas Na2S was beneficial at the lowest concentration but detrimental at the highest. Our findings show that Na2S and GYY4137 have different antioxidant properties and suggest that both H2S donors might be used as in vitro therapeutic agents against oxidative stress in sperm cells, although the optimal therapeutic range differs between the compounds.

The Roles of NO and H2S in Sperm Biology: Recent Advances and New Perspectives.

After being historically considered as noxious agents, nitric oxide (NO) and hydrogen sulfide (H2S) are now listed as gasotransmitters, gaseous molecules that play a key role in a variety of cellular functions. Both NO and H2S are endogenously produced, enzymatically or non-enzymatically, and interact with each other in a range of cells and tissues. In spite of the great advances achieved in recent decades in other biological systems, knowledge about H2S function and interactions with NO in sperm biology is in its infancy. Here, we aim to provide an update on the importance of these molecules in the physiology of the male gamete. Special emphasis is given to the most recent advances in the metabolism, mechanisms of action, and effects (both physiological and pathophysiological) of these gasotransmitters. This manuscript also illustrates the physiological implications of NO and H2S observed in other cell types, which might be important for sperm function. The relevance of these gasotransmitters to several signaling pathways within sperm cells highlights their potential use for the improvement and successful application of assisted reproductive technologies.

Pharmacological Activity of Honeybush (Cyclopia intermedia) in Boar Spermatozoa during Semen Storage and under Oxidative Stress.

In recent decades, an increasing number of ethnopharmacological studies have been dedicated to medicinal plants from South African fynbos. Among these plants, honeybush (Cyclopia spp.) has become a popular tea, mainly due to its healthy properties and caffeine-free status. The antioxidant, antimutagenic, and antimicrobial properties of this plant have been reported in several cell types, but its effects on reproductive function are still unknown. Here, we assessed the effects of honeybush (Cyclopia intermedia) on boar sperm parameters under induced oxidative stress (Fe2+/ascorbate) and during five days of semen storage at 17 °C without oxidative stress. In both experiments, four concentrations (200, 50, 12.5, and 3.125 µg/mL) of fermented honeybush were tested. Our results show that honeybush enhances sperm parameters, and no toxic effects were observed at any of the tested extract concentrations. Interestingly, honeybush (12.5 µg/mL) improved the sperm motility and kinetic parameters, preserved the plasma membrane integrity, and reduced the lipid peroxidation in the samples exposed to Fe2+/ascorbate (p < 0.05). In the stored samples, positive effects of honeybush on sperm parameters (motility, kinetics, acrosome, and mitochondria) were observed from 48 h until 120 h of semen storage (p < 0.05). Our results clearly show the protective effects of honeybush on sperm samples, thus promoting its use as a natural source of antioxidants for boar semen.

High-energy diet enhances spermatogenic function and increases sperm midpiece length in fallow deer (Dama dama) yearlings.

Nutrition is a major factor involved in the sexual development of livestock ruminants. In the male, a high-energy diet enhances the reproductive function, but its effects on the underlying processes such as spermatogenic efficiency are not yet defined. Moreover, the possible changes in sperm size due to a supplemented diet remain poorly investigated. The main goal of this study was to evaluate whether a high-energy diet affects the spermatogenic activity, epididymal sperm parameters (concentration, morphology, morphometry and acrosome integrity) and blood testosterone levels in fallow deer yearlings. For this purpose, 32 fallow deer were allocated into two groups according to their diet: control (pasture) and experimental (pasture and barley grain) groups. Fallow deer from the experimental group showed a significant increase in the Sertoli cell function and sperm midpiece length, together with a higher testicular mass, sperm concentration and percentage of normal spermatozoa than the control group (p < 0.05). We also found a tendency for higher blood testosterone levels in the animals fed with barley grain (p = 0.116). The better sperm quality found in the experimental group may be related to their higher efficiency of Sertoli cells and to an earlier onset of puberty. The results of the present work elucidate the mechanisms by which dietary supplementation enhances the male sexual development and might be useful for better practices of livestock management in seasonal breeders.

Aminoguanidine Protects Boar Spermatozoa against the Deleterious Effects of Oxidative Stress.

Aminoguanidine is a selective inhibitor of the inducible nitric oxide synthase (iNOS) and a scavenger of reactive oxygen species (ROS). Numerous studies have shown the antioxidant properties of aminoguanidine in several cell lines, but the in vitro effects of this compound on spermatozoa under oxidative stress are unknown. In this study, we tested the hypothesis that aminoguanidine may protect against the detrimental effects of oxidative stress in boar spermatozoa. For this purpose, sperm samples were incubated with a ROS generating system (Fe2+/ascorbate) with or without aminoguanidine supplementation (10, 1, and 0.1 mM). Our results show that aminoguanidine has powerful antioxidant capacity and protects boar spermatozoa against the deleterious effects of oxidative stress. After 2 h and 3.5 h of sperm incubation, the samples treated with aminoguanidine showed a significant increase in sperm velocity, plasma membrane and acrosome integrity together with a reduced lipid peroxidation in comparison with control samples (p < 0.001). Interestingly, except for the levels of malondialdehyde, the samples treated with 1 mM aminoguanidine did not differ or showed better performance than control samples without Fe2+/ascorbate. The results from this study provide new insights into the application of aminoguanidine as an in vitro therapeutic agent against the detrimental effects of oxidative stress in semen samples.

Rooibos (Aspalathus linearis) extract enhances boar sperm velocity up to 96 hours of semen storage.

Rooibos (Aspalathus linearis) is a native shrub from South African fynbos and has become very popular in the last decades for its antioxidant and medicinal attributes. Several studies have shown its beneficial properties in numerous cell lines, but to date, the in vitro effects of rooibos extract on sperm cells are still unknown. In this study, boar semen was supplemented with four concentrations both of fermented and unfermented rooibos extracts during 96 h of liquid storage at 17°C. The effects of rooibos extracts on sperm velocity, membrane integrity, and acrosomal status were evaluated at 2 h, 48 h, and 96 h of semen storage. Overall our results indicate that rooibos extract enhances sperm velocity, protects the acrosome structure, and tends to preserve the membrane integrity during semen storage. Although the unfermented rooibos showed higher total polyphenol content and total antioxidant capacity than the fermented one, the latter had better effects on sperm velocity leading to, for instance, an increase of 30% in the rectilinear velocity (VSL) at 48 h compared to the control group. Taking into account the different storage times, we established a suitable range of extracts concentrations to be used in boar semen. The rooibos extract ought to be considered as a powerful and natural source of antioxidants for the preservation of boar semen.

Integrity of head and tail plasmalemma is associated with different kinetic variables in boar sperm.

An intact and functional sperm plasmalemma has a major role in sperm motility and fertilizing capacity. Several techniques have been developed to evaluate the integrity of the sperm plasma membrane, but there are still some inconsistencies concerning the methods that are more closely associated with sperm function. In this study, the aim was to: i) evaluate the integrity of the boar sperm plasmalemma during 72h of semen storage at 17°C using four techniques: eosin/nigrosin (E/N), propidium iodide/carboxyfluorescein diacetate (PI/CFDA), hypo-osmotic swelling test (HOST), and combined HOST with eosin staining (HOST/E), ii) assess the correlations and the limits of consistency among these techniques, iii) and estimate the relationships with the acrosomal status and sperm kinetics. Results indicate that the integrity of the sperm plasmalemma decreases during 72h of storage, although significant differences were found only using the HOST and HOST/E techniques. Moreover, use of E/N and PI/CFDA results in greater values relative to the undamaged sperm membrane than use of HOST and HOST/E at any incubation time. Overall, results using all techniques were consistent and correlate except for findings with PI/CFDA and HOST, which was slightly below 95%. Moreover, values using the techniques for the evaluation of the integrity of the sperm head and tail membranes are positively associated with the acrosomal status and different kinetic variables with the tail integrity being related to rapid linear trajectories and the head integrity to rapid curvilinear trajectories. The results of this study provide new insights into the relevance of evaluating the boar sperm plasmalemma in the routine spermiogram.