Diagnosis value of fine needle aspiration cytology in testicular disorders of red deer (Cervus elaphus): a case report

We used fine needle aspiration cytology (FNAC) to diagnose Sertoli cell-only pattern and hypospermatogenesis in an Iberian red deer (Cervus elaphus hispanicus). Cytologic diagnosis was confirmed by histology and epididymal sperm analysis. We conclude that FNAC can be an important diagnostic tool in testicular diseases of wildlife.Eliana Pintus and José Luis Ros-Santaella are supported by grants CIGA 20145001 and 99830/1181/1822 (Czech University of Life Sciences).Peer Reviewe

Intramale variation in spermsize: functional significance in a polygynous mammal

Studies concerning the relationships between sperm size and velocity at the intraspecific level are quite limited and often yielded contradictory results across the animal kingdom. Intramale variation in sperm size may represent a meaningful factor to predict sperm velocity, due to its relationship with the level of sperm competition among related taxa. Because spermphenotype is under post-copulatory sexual selection, we hypothesized that a reduced intramale variation in sperm size is associated with sperm competitiveness in red deer. Our results show that low variation in sperm size is strongly related to high sperm velocity and normal sperm morphology, which in turn are good predictors of male fertility in this species. Furthermore, it is well known that the red deer show high variability in testicular mass but there is limited knowledge concerning the significance of this phenomenon at intraspecific level, even though it may reveal interesting processes of sexual selection. Thereby, as a preliminary result, we found that absolute testes mass is negatively associated with intramale variation in sperm size. Our findings suggest that sperm size variation in red deer is under a strong selective force leading to increase sperm function efficiency, and reveal new insights into sexual selection mechanisms.Czech University of Life Sciences CIGA 20145001. JLRS and EP were supported by the project CIGA 20145001 (Czech University of Life Sciences, Prague, Czech Republic).Peer Reviewe

Sperm flagellum volume determines freezability in red deer spermatozoa

The factors affecting the inter-individual differences in sperm freezability is a major line of research in spermatology. Poor sperm freezability is mainly characterised by a low sperm velocity, which in turn is associated with low fertility rates in most animal species. Studies concerning the implications of sperm morphometry on freezability are quite limited, and most of them are based on sperm head size regardless of the structural parts of the flagellum, which provides sperm motility. Here, for the first time, we determined the volumes of the flagellum structures in fresh epididymal red deer spermatozoa using a stereological method under phase contrast microscopy. Sperm samples from thirty-three stags were frozen and classified, as good freezers (GF) or bad freezers (BF) at two hours post-thawing using three sperm kinetic parameters which are strongly correlated with fertility in this species. Fourteen stags were clearly identified as GF, whereas nineteen were BF. No significant difference in sperm head size between the two groups was found. On the contrary, the GF exhibited a lower principal piece volume than the BF (6.13 μm3 vs 6.61 μm3, respectively, p = 0.006). The volume of the flagellum structures showed a strong negative relationship with post-thawing sperm velocity. For instance, the volume of the sperm principal piece was negatively correlated with sperm velocity at two hours post-thawing (r = -0.60; p<0.001). Our results clearly show that a higher volume of the sperm principal piece results in poor freezability, and highlights the key role of flagellum size in sperm cryopreservation success.JLRS was supported by I3P program (Spanish National Research Council, CSIC, Spain), post-doctoral fellowship no. 99830/1181/1822 (Czech University of Life Sciences, Prague, Czech Republic), and by the project CIGA 20145001 (Czech University of Life Sciences, Prague, Czech Republic).Peer Reviewe

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 two types of forest 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 two consecutive years. During the third 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.This study was partially funded by projects within the National Plan of the Spanish Ministry of Economy and Competition (CGL2014-55969-P and CGL2017-84938-P), the Spanish Ministry of Science and Innovation (PID2020-118205GB-I00), and the Andalusian government (A.RNM.48.UGR20), co-funded with FEDER funds from the European Union. JLRS and EP were supported by Erasmus+ grants from the European Union. JGB was supported by a FPU predoctoral contract from the Spanish Ministry of Education (FPU18/03034)

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

[EN] Comprehensive knowledge of chondrichthyan reproductive biology is crucial for the development of reproductive technologies. For that reason, a male reproductive evaluation was performed on the basis of a comparison of samples collected from wild-captured and aquarium-housed small-spotted catshark (Scyliorhinus canicula). Semen quality, sperm morphometry, and reproductive hormones were assessed. The results demonstrate good in vitro semen quality in aquarium-housed sharks, although there was lower plasma testosterone. 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 & LE; 0.001), while similar levels of 17 beta-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.Muñoz-Baquero, M.; Marco-Jiménez, F.; Garcia-Domínguez, X.; Ros-Santaella, JL.; Pintus, E.; Jiménez-Movilla, M.; García-Párraga, D.... (2021). Comparative Study of Semen Parameters and Hormone Profile in Small-Spotted Catshark (Scyliorhinus canicula): Aquarium-Housed vs. Wild-Captured. Animals. 11(10):1-14. https://doi.org/10.3390/ani11102884114111

Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments

P. 856-870Antioxidants could improve sperm media, extending the viability of spermatozoa and protecting their DNA. The protective ability of lipoic acid, melatonin, Trolox and crocin was tested on red deer spermatozoa incubated at 37°C. Cryopreserved spermatozoa were thawed and incubated with 1 mM or 0.1 mM of each antioxidant, with or without oxidative stress (100 μM Fe2+). Motility (CASA), viability, mitochondrial membrane potential and acrosomal status were assessed. Lipoperoxidation (malondialdehyde production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were checked at 4 h. Incubation alone increased ROS and decreased motility. Oxidative stress intensified these effects, increasing lipoperoxidation and DNA damage. Lipoic acid had little protective effect, whereas 1 mM melatonin showed limited protection. Trolox lowered ROS and lipoperoxidation both in oxidised and non-oxidised samples. In oxidised samples, Trolox prevented DNA and acrosomal damage, and ameliorated motility. Crocin at 1 mM showed similar results to Trolox, but noticeably stimulated motility and had no effect on lipoperoxidation. In a second experiment, a broader range of crocin and melatonin concentrations were tested, confirming the effects of crocin (positive effects noticeable at 0.5–0.75 mM), but showing an increase in lipoperoxidation at 2 mM. Melatonin was increasingly effective at 2.5 and 5 mM (ROS, lipoperoxidation and DNA status). Crocin seems a promising new antioxidant, but its particular effects on sperm physiology must be further studied, especially the consequences of motility stimulation and confirming its effect on lipoperoxidation. Melatonin might be useful at relatively high concentrations, compared to Trolox.S

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

17 Pág. Departamento de Reproducción animalProteins 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.This research was supported by the Spanish Ministry of Science and Innovation (PID2019-106380RB-I00/AEI/10.1303/501100011033) and PGC2018-094781-B-I00 (MCINN/AEI/FEDER, UE), and Oceanográfica Foundation.Peer reviewe

Morfometría espermática en ciervoibérico (Cervus elaphus hispanicus; Hilzheimer 1999): implicaciones biológicas y criobiológicas

Los resultados obtenidos en la presente tesis doctoral, ponen de manifiesto la implicación del volumen y longitud del flagelo espermático (espermatozoides epididimarios frescos) y sus estructuras (pieza intermedia, pieza principal y pieza terminal), en procesos tales como la supervivencia espermática (semen fresco) así como la congelabilidad de los espermatozoides. Se usaron los espermatozoides de 67 ciervos para la elaboración de la tesis. Hasta el momento, la mayoría de los estudios que usan la morfometría espermática para intentar predecir la congelabilidad de los espermatozoides en distintas especies animales, han estado centrados en el estudio de la cabeza espermática, sin contemplar el del flagelo. Así mismo, existen muchos trabajos que relacionan la forma y/o tamaño de los espermatozoides con la velocidad y longevidad espermática; sin embargo, muchos de ellos no tienen en cuenta ciertas longitudes espermáticas, como por ejemplo la pieza terminal del flagelo así como los volúmenes de las distintas estructuras del flagelo espermático.Peer Reviewe

Beyond testis size: links between spermatogenesis and sperm traits in a seasonal breeding mammal

Spermatogenesis is a costly process that is expected to be under selection to maximise sperm quantity and quality. Testis size is often regarded as a proxy measure of sperm investment, implicitly overlooking the quantitative assessment of spermatogenesis. An enhanced understanding of testicular function, beyond testis size, may reveal further sexual traits involved in sperm quantity and quality. Here, we first estimated the inter-male variation in testicular function and sperm traits in red deer across the breeding and nonbreeding seasons. Then, we analysed the relationships between the testis mass, eight parameters of spermatogenic function, and seven parameters of sperm quality. Our findings revealed that the Sertoli cell number and function parameters vary greatly between red deer males, and that spermatogenic activity co-varies with testis mass and sperm quality across the breeding and non-breeding seasons. For the first time in a seasonal breeder, we found that not only is the Sertoli cell number important in determining testis mass (r = 0.619, p = 0.007 and r = 0.248, p = 0.047 for the Sertoli cell number assessed by histology and cytology, respectively), but also sperm function (r = 0.703, p = 0.002 and r = 0.328, p = 0.012 for the Sertoli cell number assessed by histology and cytology, respectively). Testicular histology also revealed that a high Sertoli cell number per tubular crosssection is associated with high sperm production (r = 0.600, p = 0.009). Sperm production and function were also positively correlated (r = 0.384, p = 0.004), suggesting that these traits co-vary to maximise sperm fertilisation ability in red deer. In conclusion, our findings contribute to the understanding of the dynamics of spermatogenesis, and reveal new insights into the role of testicular function and the Sertoli cell number on testis size and sperm quality in red deer.EP and JLRS were supported by the CIGA grant no. 20145001 (Czech University of Life Sciences, Prague, Czech Republic).Peer Reviewe

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