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Creating germplasm banks of wild species, such as the Iberian red Deer (Cervus elaphus hispanicus) can be challenging. One of the main difficulties is the obtention and cryopreservation of good-quality reproductive cells when the spermatozoa are obtained from epididymides after death. To avoid a loss of seminal quality during transport, developing alternative methods for cooling and freezing sperm samples under field conditions is necessary. The objective of this study was to evaluate the effects of different durations of equilibrium and different techniques of cooling and freezing on Iberian red deer epididymal sperm quality after thawing to optimize the processing conditions in this species. Three experiments were carried out: (I) evaluation of refrigeration in straws or tubes of 15 mL; (II) study of equilibration period (0, 30, 60, or 120 min); and (III) comparison of four freezing techniques (liquid nitrogen vapor in a tank (C), liquid nitrogen vapor in a polystyrene box (B), dry ice (DY), and placing straws on a solid metallic plate floating on the surface of liquid nitrogen (MP)). For all experiments, sperm motility and kinematic parameters, acrosomal integrity, sperm viability, mitochondrial membrane potential, and DNA integrity were evaluated after thawing. All statistical analyses were performed by GLM-ANOVA analysis. Samples refrigerated in straws showed higher values (p ≤ 0.05) for mitochondrial activity and lower values (p ≤ 0.05) for apoptotic cells. Moreover, the acrosome integrity showed significant differences (p ≤ 0.05) between 0 and 120 min, but not between 30 and 60 min, of equilibration. Finally, no significant differences were found between freezing in liquid nitrogen vapors in a tank or in a box, although there was a low quality after thawing when the samples were cryopreserved in dry ice or by placing straws on a solid metallic plate floating on the surface of liquid nitrogen. In conclusion, under field conditions, it would be possible to refr

[ES] El aumento de la producción de especies reactivas de oxígeno (ROS) es un elemento común en situaciones de estrés térmico ambiental y en procesos de criopreservación celular. Se ha observado que el estrés oxidativo ejerce un efecto negativo sobre la fisiología reproductiva del macho, provocando disfunción espermática y fallos en la fertilización. El objetivo general de este Trabajo Fin de Máster (TFM) ha sido identificar y localizar las enzimas liberadoras de sulfuro de hidrógeno (H2S) en testículos y espermatozoides epididimarios de pequeños rumiantes para dilucidar el posible uso del H2S como un nuevo agente antioxidante para preservar la calidad espermática. Para ello, se han seleccionado dos modelos animales (doméstico y silvestre): el morueco (Ovis aries) de raza ovina manchega y el ciervo ibérico (Cervus elaphus hispanicus). Los análisis histopatológicos y moleculares se realizaron empleando muestras testiculares y muestras espermáticas epididimarias procedentes de 9 moruecos y 3 machos adultos de ciervo ibérico, recogidas post mortem durante la época reproductiva (septiembre-octubre). En el primer y el segundo experimento se identificó, mediante los análisis de inmunofluorescencia e inmunocitoquímica, la presencia de las enzimas liberadoras de H2S en morueco y ciervo ibérico, localizándolas tanto en el epitelio germinal de secciones testiculares como en el espermatozoide (con mayor intensidad en la pieza intermedia). En el tercer experimento, se identificó mediante western blot la expresión de las proteínas en espermatozoides epididimarios de la especie ovina, no mostrando diferencias significativas entre individuos. Por último, en el cuarto experimento se describieron las diferencias intra e interespecíficas respecto a la intensidad de las enzimas liberadoras de H2S en testículo, identificando diferencias significativas entre individuos en el ovino así como diferencias entre especies, con una mayor expresión de todas las enzimas estudiadas (CBS, CTH y MS, [EN] The increase in the production of reactive oxygen species (ROS) is a common element in situations of environmental heat stress and in cellular cryopreservation processes. It has been observed that oxidative stress induce a negative effect on the reproductive physiology of the male, causing sperm dysfunction and failures in fertilization. The general objective of this Master's Thesis (TFM) has been to identify and locate hydrogen sulfide H2S-releasing enzymes in testes and epididymal spermatozoa of small ruminants to elucidate the possible use of H2S as a new antioxidant agent to preserve the sperm quality. For this, two animal models (domestic and wild) have been selected: the ram (Ovis aries) of the manchaga breed sheep and the Iberian deer (Cervus elaphus hispanicus). Histopathological and molecular analyzes were carried out using testicular samples and epididymal sperm samples from 9 rams and 3 adult males of Iberian deer, collected post-mortem during the reproductive season (September-October). In the first and second experiments, the presence of H2S-releasing enzymes in ram and Iberian deer was identified through immunofluorescence and immunocytochemistry analyses, locating them both in the germinal epithelium of testicular sections and in the sperm (with greater intensity in the middle piece). In the third experiment, the expression of the proteins in epididymal spermatozoa of the ovine species was identified by western blot, showing no significant differences between individuals. Finally, in the fourth experiment, intra- and interspecific differences were described regarding the intensity of H2S-releasing enzymes in the testicle, identifying significant differences between individuals in sheep as well as differences between species, with a greater expression of all the enzymes studied (CBS, CTH and MST) in the ram regarding the deer. Based on the results obtained in all the experiments of this TFM, the presence and location of H2S-releasing enzymes in te

The main drawback in developing in vitro embryo production (IVP) systems in wild species, such as the Iberian red deer, is that access to these animals is usually restricted, and long distances from the collection site to the laboratory are usually inevitable. Prolonged ovary storage is known to negatively influence the quality and developmental competence of the oocytes used for IVP. To overcome this issue, we evaluated the effect of adding a caspase-3 inhibitor, z-DEVD-fmk, to the in vitro maturation media to improve the quality and developmental potential of Iberian red deer oocytes. Oocytes were in vitro matured with and without z-DEVD-fmk, and the following parameters were analyzed: viability, early apoptosis, caspase-3 activity, DNA fragmentation, and relative abundance of mRNA transcript related to apoptosis. Moreover, oocyte maturation and blastocyst rates were also assessed. The results showed that z-DEVD-fmk decreased early apoptosis (inhibitor= 44.44 ± 3.6% vs. control= 60 ± 2.79%), DNA fragmentation (inhibitor= 57.83 ± 1.91% vs. control= 74.62 ± 1.91%), caspase-3 activity (inhibitor= 41.88 ± 3.42% vs. control= 67.10 ± 3.42%) and the relative abundance of TP53 and ITM2B transcripts, as well as increased the number of live (inhibitor= 41.48 ± 2.32% vs. control= 20 ± 1.8%) and in vitro-matured oocytes (inhibitor= 88.18 ± 1.99% vs. control= 74.01 ± 1.99%) rates. Nevertheless, the blastocyst production was not different between both experimental groups (inhibitor: 7.35 ± 2.30 vs. control: 13.77 ± 2.30). The supplementation of z-DEVD-fmk to the maturation medium improved the quality of Iberian red deer oocytes. Further research and alternative strategies are needed to evaluate if this inhibitor could still enhance the developmental potential of oocytes during prolonged ovarian transport.

Additional file 2. Table 1. Details of primers used for qPCR., Additional file 3. Figure 2. Depicted images of the cumulus cell populations in terms of (A) viability, apoptosis, and necrosis, (B) active mitochondria, and (C) intracellular levels of ROS and GSH using flow cytometry and analyzed in IDEAS software., Additional file 4. Figure 3. Depicted images of (A) a Control TUNEL-positive expanded sheep blastocyst stained with (a) Hoechst 33342 and (b) fluorescein; and (B) a Control TUNEL-negative expanded sheep blastocyst stained with (a) Hoechst 33342 and (b) fluorescein.

Deficient sperm motility is a frequent cause of the age-related male sub-/infertility. Since the protein sirtuin 1 (SIRT1) develops anti-aging action and participates in sperm motility and ATP synthesis in mitochondria, we investigated its role in the acquisition of hyperactivated motility during capacitation. For this, the dynamics of sperm subpopulations were studied, using males of Sirt1+/− heterozygous mutant mice. After 2 hr of capacitation, we observed reduced percentage of hyperactivated spermatozoa in Sirt1+/− males. Interestingly, prior to capacitation, Sirt1+/− spermatozoa showed higher mitochondrial superoxide levels, which could render mitochondrial injury and thereby motility defects. Accordingly, the fertilization rate of Sirt1+/− males after mating was decreased. We elucidated that SIRT1 male insufficiency underlies posterior sperm defects to hyperactivate during capacitation and propose Sirt1+/− males as a model for the study of the age-related infertility.

Anaplasma phagocytophilum Major surface protein 4 (MSP4) plays a role during infection and multiplication in host neutrophils and tick vector cells. Recently, vaccination trials with the A. phagocytophilum antigen MSP4 in sheep showed only partial protection against pathogen infection. However, in rabbits immunized with MSP4, this recombinant antigen was protective. Differences between rabbit and sheep antibody responses are probably associated with the recognition of non-protective epitopes by IgG of immunized lambs. To address this question, we applied quantum vaccinomics to identify and characterize MSP4 protective epitopes by a microarray epitope mapping using sera from vaccinated rabbits and sheep. The identified candidate protective epitopes or immunological quantum were used for the design and production of a chimeric protective antigen. Inhibition assays of A. phagocytophilum infection in human HL60 and Ixodes scapularis tick ISE6 cells evidenced protection by IgG from sheep and rabbits immunized with the chimeric antigen. These results supported that the design of new chimeric candidate protective antigens using quantum vaccinomics to improve the protective capacity of antigens in multiple hosts.

Incubation with estrous sheep serum (ESS) is required to induce in vitro capacitation of spermatozoa during in vitro fertilization of small ruminants. However, the effect of adding different serum concentrations in the fertilization media on the quality of resulting blastocysts has not yet been studied. Here, 298 sheep oocytes were co-incubated with capacitated spermatozoa with either 10% or 2% ESS. There were no differences between treatments in cleavage (10% ESS: 63.81 ± 5.87% and 2% ESS: 45.31 ± 5.87%) and blastocyst rates (10% ESS: 20.83 ± 2.12% and 2% ESS: 15.93 ± 2.12%). Nonetheless, in vitro-produced blastocysts from the 10% ESS treatment showed a higher transcript abundance of mRNAs involved in apoptosis (ITM2B and BCL2), antioxidant defence (GPX1) and growth-related imprinting (IGF2R). Our data suggest that ESS supplementation during in vitro fertilization can influence the quality of sheep embryos at later stages of development by increasing the transcription of developmentally important genes.

The aim of this study was to assess two models of sperm collection on the quality and fertility of thawed spermatozoa from Black Manchega rams, a threatened breed. Sperm samples were collected by electroejaculation and postmortem from each male. Samples were diluted with Biladyl and frozen. Motility (subjective and objective by means of computer-assisted semen analysis), membrane integrity, and acrosomal status (microscopy) were assessed on fresh and thawed semen; plasmalemma integrity, mitochondrial membrane potential, DNA integrity, and acrosomal status were evaluated by flow cytometry on thawed semen. Thawed spermatozoa were used in a heterologous in vitro fertilization test. After thawing, the proportion of live spermatozoa with intact membrane (YO-PRO-1−/PI−) was higher for postmortem samples (P < 0.001), although the ratio of YO-PRO-1− spermatozoa within the PI− population was higher for ejaculated samples (P = 0.007). Likewise, the proportion of live spermatozoa having high mitochondrial membrane potential (MitoTracker+) and intact acrosomes (PNA−) was higher for postmortem samples (P < 0.001 and P < 0.001, respectively). Considering only live spermatozoa, the ratio of MitoTracker+/PNA− cells was higher for electroejaculated samples (P = 0.026 and P = 0.003). Both electroejaculated and postmortem samples fertilized oocytes. Nevertheless, electroejaculated samples yielded a higher percentage of hybrid embryos (P = 0.041). In conclusion, although postmortem spermatozoa had better sperm quality after thawing, electroejaculated spermatozoa showed higher ratios for sperm quality when only the live population was considered. Electroejaculated and postmortem samples might be used for germplasm banking of this threatened breed, but the fertility of postmortem spermatozoa might be lower.

Currently Colombia does not have enough studies on avocado sensory quality. So, the aim of this work was to evaluate the harvest index and storage duration on avocado sensory quality. Avocado fruit were harvested from 5 farms in Antioquia. Sensory descriptors were evaluated on a scale from 0 to 5, and general quality (GQ) was evaluated from 0 to 3. Fruit with low GQ exhibit hard, wet, and fibrous textures; they taste green, astringent, spicy, sour, umami, and acid, and the odor is sour, green, and acid. Fruits have a low sensory quality at the beginning of the harvest season. As DM content increases, the sensory quality improves. Orchard location and fruit storage duration influenced its sensory descriptors and general quality., Colombia actualmente no tiene estudios sobre calidad sensorial de aguacate. Por tanto, el objetivo de este trabajo fue evaluar el efecto del índice cosecha y almacenamiento sobre la calidad sensorial del aguacate. Los frutos se cosecharon de 5 fincas en Antioquia. Descriptores sensoriales fueron evaluados en un escalda de 0 a 5 y calidad general de 0 a 3. Frutos con baja calidad exhibieron textura húmeda, dura y fibrosa; sabor verde, astringente, picante, amargo, umami, ácido; olor amargo, verde y ácido. Al inicio de la cosecha los frutos tuvieron baja calidad sensorial. A medida que aumenta el contenido de DM, mejora la calidad sensorial. La ubicación del huerto y la duración del almacenamiento de la fruta influyeron en sus descriptores sensoriales y en la calidad general.

Introduction: The inequality gaps between urban and rural areas worldwide and especially in developing countries such as Colombia, are increasing. Objective: Among the main purposes of the Sustainable Development Goals -ODS-, is the reduction of these gaps. In this article, some indicators that show the differences between urban and rural territories in Colombia are analyzed, and the way in which the social and solidarity economy sector, especially the cooperative sector, has contributed to this end. Materials and Methods: The foregoing under a qualitative social approach with a comparative method that seeks from the description and statistical analysis of socioeconomic indicators, the relationship between compliance with the SDGs and the contribution of the cooperative sector in some areas of the country. The Results suggest that in Colombia the greatest deficit of indicators and deficiencies are consistent. Conclusion: the SDGs are found in rural areas due to the existence of structural factors that will make it very difficult for our country to comply with and contribute to the 2030 agenda, therefore that the intervention of other actors is urgently required., Resumo Introdução: As lacunas de desigualdade entre áreas urbanas e rurais em todo o mundo e especialmente em países em desenvolvimento como a Colômbia estão aumentando. Objetivos: Entre os principais objetivos dos Objetivos de Desenvolvimento Sustentável -ODS-, está a redução dessas lacunas. Neste artigo, são analisados alguns indicadores que mostram as diferenças entre os territórios urbano e rural na Colômbia e a forma como o setor da economia social e solidária, especialmente o cooperativista, tem contribuído para esse fim. Materiais e métodos: O anterior sob uma abordagem social qualitativa com um método comparativo que busca a partir da descrição e análise estatística dos indicadores socioeconômicos, a relação entre o cumprimento dos ODS e a contribuição do setor cooperativo em algumas áreas do país. Os Resultados: sugerem que na Colômbia os maiores déficits de indicadores e deficiências congruentes. Conclusão: os ODS encontram-se nas áreas rurais devido à existência de fatores estruturais que dificultarão o cumprimento e a contribuição de nosso país para a agenda 2030, portanto que a intervenção de outros atores é necessária com urgência., Resumen Introducción: Las brechas de desigualdad entre zonas urbanas y rurales a nivel mundial y en especial en países en vía de desarrollo como Colombia, son cada vez mayores. Objetivo: Entre los principales fines de los Objetivos de Desarrollo Sostenible -ODS-, está la disminución de dichas brechas. En este artículo son analizados algunos indicadores que evidencian las diferencias existentes entre los territorios urbanos y rurales en Colombia, y la manera como el sector de la economía social y solidaria, especialmente el sector cooperativo, ha contribuido en dicho fin. Materiales y Métodos: Lo anterior bajo un enfoque social cualitativo con método comparativo que busca a partir de la descripción y análisis estadístico de indicadores socioeconómicos, la relación entre el cumplimiento de los ODS y la contribución del sector cooperativo en algunas zonas del país. Los resultados sugieren que en Colombia el mayor déficit de indicadores y carencias son congruentes. Conclusión: los ODS, se encuentran en las zonas rurales debido a la existencia de factores estructurales que harán muy difícil el cumplimiento y aporte de nuestro país a la agenda del 2030, por lo que se requiere de manera urgente la intervención de otros actores.

Oxidative stress has become a major concern in the field of spermatology, and one of the possible solutions to this acute problem would be the use of antioxidant protection; however, more studies are required in this field, as highly contradictory results regarding the addition of antioxidants have been obtained. Vitamin E is a powerful biological antioxidant, but its low stability and high hydrophobicity limit its application in spermatology, making the use of organic solvents necessary, which renders spermatozoa practically motionless. Keeping this in mind, we propose the use of hydrogels (HVEs) and nanoemulsions (NVEs), alone or in combination, as carriers for the controlled release of vitamin E, thus, improving its solubility and stability and preventing oxidative stress in sperm cells. Cryopreserved sperm from six stags was thawed and extended to 30 × 106 sperm/mL in Bovine Gamete Medium (BGM). Once aliquoted, the samples were incubated as follows: control, free vitamin E (1 mM), NVEs (9 mM), HVEs (1 mM), and the combination of HVEs and NVEs (H + N), with or without induced oxidative stress (100 µM Fe2+/ascorbate). The different treatments were analyzed after 0, 2, 5, and 24 h of incubation at 37 °C. Motility (CASA®), viability (YO-PRO-1/IP), mitochondrial membrane potential (Mitotracker Deep Red 633), lipid peroxidation (C11 BODIPY 581/591), intracellular reactive oxygen species production (CM-H2DCFDA), and DNA status (SCSA®) were assessed. Our results show that the deleterious effects of exogenous oxidative stress were prevented by the vitamin E-loaded carriers proposed, while the kinematic sperm parameters (p ˂ 0.05) and sperm viability were always preserved. Moreover, the vitamin E formulations maintained and preserved mitochondrial activity, prevented sperm lipid peroxidation, and decreased reactive oxygen species (ROS) production (p ˂ 0.05) under oxidative stress conditions. Vitamin E formulations were significantly different as regards the free vitamin E

The heterogeneous nature of ejaculates highlights the relevance of studying the behavior of different sperm subpopulations. Changes in sperm motility and the increase in tyrosine phosphorylation are key events that usually occur during capacitation and can be modified by the cryopreservation process. However, the relationship between both events remains poorly defined throughout capacitation in the different sperm subpopulations. Fresh and frozen-thawed spermatozoa were incubated in capacitating (CAP) and non-capacitating (NC) media up to 240 min. Sperm kinematics, tyrosine phosphorylation and mitochondrial activity were measured by the CASA system and imaging flow cytometry. Four motile sperm subpopulations (SP) were identified in fresh and frozen-thawed ram semen after the cluster analysis. Incubation under CAP conditions over time led to greater changes in the percentage of spermatozoa included in each subpopulation compared to NC conditions, being different between fresh and frozen-thawed spermatozoa. The SP1, characterized by slow spermatozoa, progressively increased after 15 min in frozen-thawed samples incubated in both media but not in fresh ones. The SP4, characterized by fast and non-linear spermatozoa, showed a marked increase during CAP, but not under NC conditions, occurring more rapidly in frozen-thawed spermatozoa. This subpopulation (SP4) was also the only one positively and strongly correlated with mitochondrial activity and all phosphorylated sperm regions during capacitation, either in fresh or frozen-thawed samples. Our results indicated that in vitro capacitation induced significant changes in the distribution of motile sperm subpopulations, being affected by cryopreservation. Notwithstanding, the subpopulation which probably represents hyperactivated-like spermatozoa (SP4) also increased in frozen-thawed samples, occurring faster and simultaneously to the increment of mitochondrial activity and tyrosine phosphorylation of different sperm region

The aim of this study was to assess the effect of melatonin implants on the reproductive performance of yearling Iberian red deer (Cervus elaphus hispanicus) hinds. It also explored exogenous melatonin administration as a tool to minimize the negative effect of a low yearling hind’s liveweight on their reproductive efficiency. In addition, the effect of melatonin-treated yearling hinds on non-treated hinds was studied in order to provide a practical and economical protocol to improve farms’ productivity. A total of 4520 Iberian red deer hinds belonging to the same farm were included in this study. Melatonin (108 mg/hind) implants were administered three-fold every 30 days before the breeding season. Fertility rates, calves’ weights and calving dates were registered for each hind. The results showed that exogenous melatonin increased significantly (p < 0.05) the calves’ weight (32.39 ± 1.07 kg vs. 27.65 ± 1.11 kg for Weight 1calf (July) and 46.59 ± 1.50 kg vs. 41.79 ± 1.54 kg for Weight 2calf (August, at weaning)) and advanced the calving date by 15 days in yearling hinds compared to the non-treated group. In addition, the administration of melatonin implants before the breeding season was able to minimize the negative effect of low yearling hinds’ liveweight (Weight 1hind) on their future reproductive outcomes, as the fertility rates increased by 46% and the calves’ weight increased by 7 kg after the melatonin treatment, regardless of the yearlings’ weight. Finally, when both experimental groups (melatonin and non-treated) were kept separate, higher fertility rates (76.73 ± 7.18% vs. 66.94 ± 7.41%) were observed for the melatonin-treated hinds compared to the non-treated hinds. However, when both groups of yearling hinds were maintained together, no significant differences were observed in their fertility outcomes (78.13 ± 21.26% vs. 78.12 ± 23.32%). Therefore, melatonin implants may be used in yearling Iberian red deer hinds as a management tool to improve their re, [Simple Summary]: Increasing the reproductive performance of hinds is considered to be a key factor of overall farm deer productivity. In the case of yearling hinds, this aspect becomes more important, as a delay in the pubertal onset will compromise the reproductive performance of the entire herd (decreased fertility), and these yearling hinds will carry this ‘late’ condition throughout their reproductive life. The aim of this study was to explore the use of melatonin implants on yearling Iberian red deer (Cervus elaphus hispanicus) hinds to improve their fertility outcomes, advance the calving date and the calves’ weight, and to prevent the negative impact of yearling hinds’ low liveweight on their reproductive outcomes. Melatonin implants (18 mg), administered three-fold (two implants each time) every 30 days before the breeding season, rendered significantly higher fertility rates (regardless of the yearling hind’s weight) and heavier calves, and advanced the calving date in the yearling hinds by 15 days compared to non-treated hinds. In addition, halving the number of yearling hinds that received melatonin provided a similar benefit to a large-scale treatment of the whole herd, which indicates female-to-female stimulation of the ovarian activity. Taken together, this protocol for melatonin treatment simplifies its administration, reduces its costs, and assures the enhancement of the reproductive productivity of the entire farm.

Sperm cryopreservation is a complex process that needs to be adapted to wild and domestic avian species to ensure proper efficiency. Because of its accessibility, the peregrine falcon may be used as a good model for studying other raptor species. To find the most optimal cryopreservation protocol for peregrine falcon ejaculates, sperm parameters such as motility, viability, DNA fragmentation, acrosome integrity, and mitochondrial activity were analyzed under different conditions by varying the freezing method (slow freezing in straws vs. ultrarapid freezing in pellets), thawing conditions (37 °C for 30 s vs. 5 °C for 1 min), type of cryoprotectant (DMA vs. DMSO), and concentration of DMSO (4% vs. 8%). Results show that slow cryopreservation in straws yielded greater percentages (p < 0.05) of motile spermatozoa (22.5% ± 4.4% vs. 0.0% ± 4.1%), viable spermatozoa with intact acrosomes (84.6% ± 4.3% vs. 77.4% ± 4.3%), and spermatozoa with active mitochondria (41.0% ± 6.7% vs.12.8% ± 6.7%), compared with those obtained by the ultrarapid freezing in pellets. However, no differences were found between different thawing conditions. Moreover, all sperm motility parameters were greater (p < 0.05) when DMSO was used during freezing compared with DMA, although the use of 3% and 8% DMSO produced similar results. In conclusion, these results represent important progress in the study of falcon semen cryopreservation protocol, highlighting the crucial steps of the process and the most suitable conditions.