Centrifugal force assessment in ram sperm: identifying species-specific impact

[EN] Background: Centrifugation is routinely employed in handling the ejaculates of some species, but it is not part of the commonly used protocols in ram. However, the development and implementation of new assisted reproductive technologies, alternative preservation models based on washing sperm from a cellular ageing-accelerating substance such as the seminal plasma, and basic studies in spermatology is associated with the use of centrifugation. This requires a specific evaluation of the centrifugation protocols considering the species-specific relationship with the potential damage produced by this procedure. No previous studies have determined the effect of different centrifugation forces on ram sperm. Therefore, we aimed to assess the performance of three centrifugal forces (600×g, 3000×g, and 6000×g for 10 min at room temperature) and their effects on ram sperm motility and functionality. Results: Sperm motility and functionality parameters were assessed at 0 h and after 2 h of incubation at 37 °C. As expected, a higher cell packaging degree was obtained at high centrifugation forces (P ≤ 0.0001). Cell packaging was unstable at all centrifugal forces. Thus, there was a high cell resuspension rate after less than 2 min. Regarding sperm quality, there was a change in movement pattern of 3000×g and 6000×g centrifuged sperm after 2 h of incubation at 37 °C, characterized by an increase in rapid progressive motility, linearity, straightness, and beat frequency, and a decrease in medium progressive motility, curvilinear velocity, path velocity, and head lateral amplitude. Non-significant differences were obtained among the different treatments concerning the total viability. However, we observed a significant increase (P ≤ 0.05) in the percentage of viable apoptotic sperm in the samples centrifuged at 6000×g at 0 h. Conclusions: Centrifugal forces equal to or greater than 3000×g induced some deleterious effects in ram sperm quality, and lower forces did not provide a successful cell packaging degree.SIThis work was financially supported by MINECO (AGL2017-83098-R) and the University of León. Marta Neila-Montero was supported by MEC (fellowship FPU17/04142) and Marta F Riesco and Rafael Montes-Garrido by Junta de Castilla y León (research contract LE253P18 and fellowship ORDEN EDU/556/2019, respectively)

Frequency of Semen Collection Affects Ram Sperm Cryoresistance

[EN] The improvement of frozen-thawed sperm quality has been mostly approached from the view of cryopreservation protocol optimization in terms of cryoprotectant solutions, freezing-thawing rates and antioxidant supplementation, while the impact of sperm collection frequency remains unknown in rams. In this work, a multiparametric study was carried out in cooled and frozen-thawed semen to evaluate sperm quality after different semen collection frequencies during a month: zero sperm collection (0 CW), four sperm collections per week (4 CW), and ten sperm collections per week (10 CW). Traditional analyses have been applied, in combination with novel technologies related to redox balance. Frozen-thawed semen quality showed a significant decrease (p < 0.05) in 0 CW and 10 CW in comparison to 4 CW, concerning motility and kinetics parameters. However, apoptosis showed a significant increase (p < 0.05) in 10 CW in comparison to 0 CW and 4 CW. The employment methods related to redox balance provided us with the definitive probe to ensure the influence of collection frequency on balance redox after thawing. Specifically, glutathione peroxidase (GPX) and superoxide dismutase (SOD) activity showed a significant decrease (p < 0.05) in 10 CW compared to 0 CW and 4 CW. The characterization of alternative strategies to sperm cryopreservation based on consideration of male sexual regimes, could improve the quality of frozen-thawed sperm.SIThis work was supported by MINECO (AGL2017-83098-R), the Junta de Castilla y León (LE253P18) and Desafio Universidad-Empresa—TCUE 2020 (MejorIA). C.P.-M. was supported by MINECO (PRE2018-086400), M.F.R. and R.M.-G. was supported by Junta de Castilla y León and FSE (research contract LE253P18 and fellowship ORDEN EDU/556/2019, respectively), M.N.-M. was supported by MEC (fellowship FPU17/04142)

ProAKAP4 as Novel Molecular Marker of Sperm Quality in Ram: An Integrative Study in Fresh, Cooled and Cryopreserved Sperm

[EN] To improve artificial insemination protocols in ovine species it is crucial to optimize sperm quality evaluation after preservation technologies. Emerging technologies based on novel biomolecules and related to redox balance and proteins involved in sperm motility such as ProAKAP4 could be successfully applied in ram sperm evaluation. In this work, a multiparametric analysis of fresh, cooled, and cryopreserved ram sperm was performed at different complexity levels. Samples were evaluated in terms of motility (total motility, progressive motility, and curvilinear velocity), viability, apoptosis, content of reactive oxygen species, oxidation-reduction potential, and ProAKAP4 expression and concentration. As expected, cryopreserved samples showed a significant decrease of sperm quality (p < 0.05), evidencing different freezability classes among samples that were detected by ProAKAP4 analyses. However, in cooled sperm no differences were found concerning motility, viability, apoptosis, ROS content, and redox balance compared to fresh sperm that could explain the reported decrease in fertility rates. However, although the proportion of sperm ProAKAP4 positive-cells remained unaltered in cooled sperm compared to fresh control, the concentration of this protein significantly decreased (p < 0.05) in cooled samples. This altered protein level could contribute to the decrease in fertility rates of cooled samples detected by some authors. More importantly, ProAKAP4 can be established as a promising diagnostic parameter of sperm quality allowing us to optimize sperm conservation protocols and finally improve artificial insemination in ovine species.S

Multiparametric Study of Antioxidant Effect on Ram Sperm Cryopreservation—From Field Trials to Research Bench

The optimization of sperm cryopreservation protocols in ram is a feasible tool to reinforce artificial insemination technologies considering the desirable application of sperm by vaginal/cervical or transcervical deposition. Cryopreservation provokes different types of damage on spermatozoa and many of these detrimental effects are triggered by redox deregulation. For this reason, the antioxidant supplementation in sperm cryopreservation protocols to decrease reactive oxygen species (ROS) levels and to equilibrate redox status has been widely employed in different species. Despite this, more fertility trials are necessary to provide the definitive tool to ensure the antioxidant effectiveness on sperm quality. For this reason, in this work, we performed a multiparametric analysis of some previously tested antioxidants (crocin, GSH and Trolox) on ram sperm cryopreservation from field trials to sperm quality analyses focused on new strategies to measure redox balance. Attending to fertility trial, Trolox supplementation registered an improvement concerning to fertility (when we considered high fertility males) and multiple lambing frequency and other complementary and descriptive data related to lambing performance such as prolificacy and fecundity. This positive effect was more evident in multiple lambing frequency when we considered low fertility males than in global male analysis. In vitro analyses of sperm quality confirmed in vivo trials registering a positive effect on sperm viability and redox balance. In this study, we provided the definitive evidence that the role of trolox on redox balance maintenance has a direct effect on fertility parameters, such as prolificacy. The effectiveness of antioxidant treatments was tested, for the first time in ovine species, using an integrative and multiparametric approach combining in vivo and in vitro analyses and novel approaches, such as RedoxSYS. These types of strategies should be applied to improve sperm conservation methods and optimize AI technologies upgrading the correlation between in vitro and in vivo analyses.SIFunding: This work was financially supported by the Junta de Castilla y León (LE253P18) and MINECO (AGL2017-83098-R) project and the University of León.Acknowledgments: The authors acknowledge the JCyL-LE253P18 research contract and ORDEN EDU/556/2019 fellowship (Junta Castilla y León), FPU17/04142 (MCIU) and PRE2018-086400 (MEC) fellowships, Carlos Palacio Riocerezo, the ITRA-ULE technicians (Ainoa Jordán, Pedro de Vega and Cecilia Pérez), ANCHE and the staff from Ovigén

Does Size Matter? Testicular Volume and Its Predictive Ability of Sperm Production in Rams

[EN] Over the years, testicular volume has been used to evaluate the reproductive capacity of rams and the effects of different factors related to reproductive performance. The aim of this study was to determine the most suitable tool and formula to calculate testicular volume under field conditions to guarantee a more accurate determination of sperm production. First, testicles from 25 rams (n = 50) were measured in vivo and postmortem using calipers and ultrasonography during the breeding season (BS). The accurate testicular volume (ATV) was calculated through water displacement. In addition, the sexual status of donor rams was evaluated during a period of four years in a reproduction center, and the three most crucial groups in terms of genetic value and seminal collections were studied in the second part of this experiment: ER-NBS (Elite rams during the non-breeding season), ER-BS-S (Elite rams with a standard frequency of seminal collection), and ER-BS-O (Elite rams with a high frequency of seminal collection). The total testicular volume (TTV), testosterone (T), and total spermatozoa obtained from two consecutive ejaculates in the same day (SPERM) were measured, and the relationship between SPERM and TTV and T was analyzed to predict SPERM. Although all published formulas revealed statistically significant differences (p ≤ 0.05) from the ATV, our proposed formula (ItraULE) (Testicular volume = L × W × D × 0.61) did not show significant differences. In the second part of the study, in the ER as a model donor ram for its high genetic value and high demand from farmers, TTV and T showed strong positive correlations with SPERM (r = 0.587, p = 0.007 NBS; r = 0.684, p = 0.001 BS-S; r = 0.773, p < 0.0001 BS-O). Moreover, formulas were established to predict SPERM in these practical scenarios. In conclusion, the use of ultrasonography and a new formula adapted to rams could improve the prediction of SPERM considering crucial factors such as season and semen collection frequencySIThis research was funded in part by Ministerio de Ciencia e Innovación, PID2021-122470OBI00/ AEI/10.13039/501100011033/FEDER, UE, Junta de Castilla y León, LE253P18, and Universidad de León. The APC was funded by Ministerio de Ciencia e Innovación, PID2021-122470OB-I00. R.M.-G. was supported by Junta de Castilla y León (through the Consejería de Educación and FSE PO 1420–CyL, fellowship ORDEN EDU/556/2019), M.N.-M. was supported by MEC (fellowship FPU17/04142), and C.P.-M. was supported by MINECO (PRE2018-086400

Morphological and ultrasonographic characterization of the three zones of supratesticular region of testicular artery in Assaf rams

[EN] To fully understand the histological, morphometrical and heamodynamic variations of different supratesticular artery regions, 20 mature and healthy Assaf rams were examined through ultrasound and morphological studies. The testicular artery images of the spermatic cord as shown by B-mode analysis indicated a tortuous pattern along its course toward the testis, although it tends to be less tortuous close to the inguinal ring. Doppler velocimetric values showed a progressive decline in flow velocity, in addition to pulsatility and vessel resistivity when entering the testis, where there were significant differences in the Doppler indices and velocities among the different regions. The peak systolic velocity, pulsatility index and resistive index were higher in the proximal supratesticular artery region, followed by middle and distal ones, while the end diastolic velocity was higher in the distal supratesticular region. The total arterial blood flow and total arterial blood flow rate reported a progressive and significant increase along the testicular cord until entering the testis. Histological examination revealed presence of vasa vasorum in the tunica adventitia, with their diameter is higher in the proximal supratesticular zone than middle and distal ones. Morphometrically, the thickness of the supratesticular artery wall showed a significant decline downward toward the testis; meanwhile, the outer arterial diameter and inner luminal diameter displayed a significant increase distally. The expression of alpha smooth muscle actin and vimentin was higher in the tunica media of the proximal supratesticular artery zone than in middle and distal ones.SIThe authors acknowledge the staff members and technicians of Comparative Anatomy and Pathology Department, and Animal Medicine and Surgery Department, Faculty of Veterinary Medicine, León University, Spain, and specially Professor P de Paz for their great help in the practical and laboratory parts of this study. Many thanks are extended to staff member of Anatomy and Embryology Department, Faculty of Veterinary Medicine, Sohag University, for their help with histological and morphometrical analyses. We are thankful and grateful for European Union for the financial support of this study through the project (ERASMUS+ KA107 2019/2020). This work was financially supported by the Junta de Castilla y León (LE253P18) and MINECO (AGL2017-83098-R) project and the University of León, and also by Sohag University, Egypt.This article was funded by Ministerio de Economía, Industria y Competitividad, Gobierno de España (AGL2017-83098-R) and Junta de Castilla y León (LE253P18)

Ovine fertility by artificial insemination in the breeding season could be affected by intraseasonal variations in ram sperm proteomic profile

[EN] It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21st to July 20th) and at the end (November 20th to December 21st) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6–19 rams at two very distant points in the mating season (July as Early Breeding Season –EBS– and November as Late Breeding Season –LBS–). Rutinary assessments carried out in the ovine reproduction centers (testicular volume, libido, sperm production and mass motility) showed non-significant differences (P ≥ 0.05) between both studied times, as well as the ram ultrasonographic evaluation (Resistive and Pulsatility Index as Doppler parameters; and pixels mean gray level, and hypoechoic areas percentage and density as echotexture parameters). However, at level of sperm functionality, although sperm quality appeared non-significantly lower (P ≥ 0.05) in the EBS, we identified a significantly different (P 4, a P = 2.40e-07, and a q = 2.23e-06: Fibrous Sheath-Interacting Protein 2, Disintegrin and Metalloproteinase Domain-Containing Protein 20-like, Phosphoinositide-Specific Phospholipase C, Tektin 5, Armadillo Repeat-Containing Protein 12 Isoform X3, Solute Carrier Family 9B1, Radial Spoke Head Protein 3 Homolog, Pro-Interleukin-16, NADH Dehydrogenase [Ubiquinone] 1 Alpha Subcomplex Subunit 8, Testis, Prostate and Placenta-Expressed Protein, and Acyl Carrier Protein Mitochondrial. In conclusion, while our basic analyses on male and sperm quality showed similar results between the beginning and the end of the breeding season, on a proteomic level we detected a lower expression of sperm proteins linked to the energy metabolism, sperm-oocyte interactions, and flagellum structure in the EBS. Probably, this different protein expression could be related to the lower fertility rate of Assaf ewes after cervical artificial insemination at this time. More importantly, sperm proteins can be used as highly effective molecular markers in predicting sperm fertilization ability related to intraseasonal variations.S

Seed storage conditions change the germination pattern of clonal growth plants in Mediterranean salt marshes.

8 páginas, 4 tablas, 8 figuras.The effect of salinity level and extended exposure to different salinity and flooding conditions on germination patterns of three saltmarsh clonal growth plants (Juncus subulatus, Scirpus litoralis, and S. maritimus) was studied. Seed exposure to extended flooding and saline conditions significantly affected the outcome of the germination process in a different, though predictable, way for each species, after favorable conditions for germination were restored. Tolerance of the germination process was related to the average salinity level measured during the growth/germination season at sites where established individuals of each species dominated the species cover. No relationship was found between salinity tolerance of the germination process and seed response to extended exposure to flooding and salinity conditions. The salinity response was significantly related to the conditions prevailing in the habitats of the respective species during the unfavorable (nongrowth/nongermination) season. Our results indicate that changes in salinity and hydrology while seeds are dormant affect the outcome of the seed-bank response, even when conditions at germination are identical. Because these environmental-history-dependent responses differentially affect seed germination, seedling density, and probably sexual recruitment in the studied and related species, these influences should be considered for wetland restoration and managementFinancial support from the Spanish Ministry of the Environment (MMA, project 05/99) and the Junta de Andalucía (research group 4086)enabled us to carry out the present work.Peer reviewe

Seed storage conditions change the germination pattern of clonal growth plants in Mediterranean salt marshes.

8 páginas, 4 tablas, 8 figuras.The effect of salinity level and extended exposure to different salinity and flooding conditions on germination patterns of three saltmarsh clonal growth plants (Juncus subulatus, Scirpus litoralis, and S. maritimus) was studied. Seed exposure to extended flooding and saline conditions significantly affected the outcome of the germination process in a different, though predictable, way for each species, after favorable conditions for germination were restored. Tolerance of the germination process was related to the average salinity level measured during the growth/germination season at sites where established individuals of each species dominated the species cover. No relationship was found between salinity tolerance of the germination process and seed response to extended exposure to flooding and salinity conditions. The salinity response was significantly related to the conditions prevailing in the habitats of the respective species during the unfavorable (nongrowth/nongermination) season. Our results indicate that changes in salinity and hydrology while seeds are dormant affect the outcome of the seed-bank response, even when conditions at germination are identical. Because these environmental-history-dependent responses differentially affect seed germination, seedling density, and probably sexual recruitment in the studied and related species, these influences should be considered for wetland restoration and managementFinancial support from the Spanish Ministry of the Environment (MMA, project 05/99) and the Junta de Andalucía (research group 4086)enabled us to carry out the present work.Peer reviewe

Doñana. Acta vertebrata. vol 24(1/2)

Alimentación de la lagartija colilarga Psammodromus algirus (L) (Sauria, Lacertidae), en el litoral de Huelva (SO EspañaLa alimentación de Myotis myotis Borkh, 1791 (Chiroptera, Vespertilionidae) en la cuenca del rio Guadix (sureste de España)Distribución y selección de hábitat de la garduña (Martes foina, Erxleben, 1777) en Vizcaya y Sierra Salvada (Burgos).Nuevo modelo de trampa para reducir el impacto de la pesca de cangrejos sobre los vertebrados en las marismas del GuadalquiviThe functions of song and the spatial pattern of song production in the rufous bush chat (Cercotrichas galactotes)Migración e invernada de las lavanderas cascadena Motacilla cinerea y blanca M. alba en la Penínula Ibérica e Islas BalearesAbundancia y reproducción de Glis glis (Linnaeus, 1766) (Rodentia, Gliridae) en el Pirineo occidental.Estatus de residencia, categorización trófica y abundancia de aves en el zoológico de La Plata, ArgentinaAvifauna reproductora y estructura del hábitat en la campiña y sierras Subbéticas de JaénDispersión de semillas de retama (Retama sphaerocarpa (L.) Boiss por el conejo (Oryctolagus cuniculus L.) en el centro de EspañaGuía para la identificación de restos óseos pertenecientes a algunos peces comunes en las aguas continentales de la Península Ibérica para el estudio de la dieta de depredadores ictiófagosDistribución y abundancia del corzo (Capreolus capreolus L. 1758) en la provincia de JaénAlimentación de las larvas de anuros en ambientes temporales del sistema del rio Paraná, Argentina.Mauremys leprosa como presa de Lutra lutraNota sobre la alimentación del lince ibérico en el Parque Natural de la Sierra de Andújar (Sierra Morena oriental)Presencia de Echinococcus granulosus (Cestoda) en un lobo ibérico (Canis lupusLa orientación de los nidos de paseriformes estepariosComparación de la dieta obtenida a partir de muestras estomacales y fecales del Tuco-tuco, Ctenomys mendocinus, en dos poblaciones de la precordillera de los Andes, ArgentinaEstructura genética y distribución de la variabilidad enzimática en poblaciones naturales de estornino negro (Sturnus unicolor)Estimación de la disponibilidad trófica para el quebrantahuesos (Gypaetus barbatus) en Cataluña (NE España) e implicaciones sobre su conservaciónPeer reviewe