Effect of different media additives on capacitation of frozen-thawed ram spermatozoa as a potential replacement for estrous sheep serum

Capacitation is a key process through which spermatozoa acquire their fertilizing ability. This event is required for the successful application of assisted reproductive technologies such as IVF. The aim of the present study was to investigate the effect of using a synthetic oviductal fluid medium supplemented with either heparin–hypotaurine alone, in combination with progesterone (P4), 17β-estradiol (E2), or BSA, or just β-cyclodextrin, in replacement for estrous sheep serum (ESS) for ram sperm capacitation. After incubation in the corresponding media for 15 (time 0) or 60 minutes, sperm function was evaluated by computerized sperm motility analysis and flow cytometry (plasma membrane status and fluidity). Treatments rendering the best results in regards to sperm function parameters related to capacitation were used for an IVF test. Herein, neither heparin–hypotaurine (alone), or in combination with P4, or E2, nor β-cyclodextrin induced capacitation-related changes in frozen–thawed ram spermatozoa. Only the medium supplemented with heparin–hypotaurine–BSA was able to induce changes compatible with in vitro capacitation relating to sperm motility pattern and plasma membrane fluidity, comparable to those in ESS-containing medium. Both media yielded sperm parameter values that differed (P < 0.05) from those obtained in the rest of the media tested. However, after the IVF trial, BSA was unable to support cleavage rates (21.80%) comparable to those obtained with ESS (52.60%; P < 0.05). We conclude that heparin–hypotaurine, P4, E2, β-cyclodextrin, or BSA is not suitable for replacing ESS in capacitation and fertilization media for ram spermatozoa.M. Ramón was supported by the Research Recruitment Program from the National Institute for Agricultural and Food Research program.Peer Reviewe

Beneficial effects of melatonin in the ovarian transport medium on in vitro embryo production of Iberian red deer (Cervus elaphus hispanicus)

This article belongs to the Special Issue Reproductive Biotechnology in Wildlife.[Simple Summary]: The development of in vitro embryo production (IVP) in wild species, such as Iberian red deer, can become a daunting challenge since prolonged ovary transport times to the laboratory are often unavoidable. This may have detrimental effects on the quality and developmental capacity of oocytes. We evaluated the effect of supplementing the ovary transport medium with the antioxidant melatonin and observed an increased level of oocyte intracellular reduced glutathione content. Moreover, melatonin enhanced cleavage and blastocyst rates and had a positive effect on embryo quality in terms of the expression of essential embryo development-related genes. In conclusion, the addition of melatonin to the ovary storage medium could mitigate the negative impacts that long transport times may have on oocyte developmental competence and quality of the resulting blastocysts in Iberian red deer.A major limiting factor for the development of in vitro embryo production (IVP) in wild species, such as Iberian red deer, compared to livestock animals is the poor availability and limited access to biological material. Thus, the use of post-mortem ovaries from slaughtered animals represent a source of oocytes for the large scale production of embryos needed for research and to improve the efficiency of IVP. However, these oocytes are not as developmentally competent as their in vivo counterparts. Moreover, oocytes are usually obtained from ovaries that have been transported for long distances, which may also affect their quality. In order to overcome the issues associated with prolonged storage times of post-mortem material, in this study we examined the effect of melatonin supplementation to the ovary transport medium on oocyte quality, embryo yield, and blastocyst quality in Iberian red deer. When necessary, sheep was used as an experimental model due to the large number of samples required for analysis of oocyte quality parameters. Oocytes were in vitro matured and assessed for early apoptosis; DNA fragmentation; reactive oxygen species (ROS); reduced glutathione (GSH) content, mitochondrial membrane potential, and distribution; and relative abundance of mRNA transcript levels. After in vitro fertilization, embryo rates and blastocyst quality were also investigated. The results revealed that melatonin treatment significantly increased intracellular level of GSH in sheep oocytes. Moreover, the percentage of cleavage and blastocyst yield in red deer was greater compared to the Control group and there was lower abundance of oxidative stress- and apoptosis-related SHC1, TP53, and AKR1B1 mRNA transcripts in blastocysts for the Melatonin group. In conclusion, the supplementation of melatonin to the ovary storage medium had a positive effect on the developmental competence and quality of resulting blastocysts in Iberian red deer.This research was funded by the Spanish Ministry of Economy and Competitiveness (AGL2017-89017-R) and Regional Government (SBPLY/17/180501/000500). M.I.-C. and A.M.-M. were supported by a Ministry of Economy and Competitiveness scholarship. P.P.-F. and D.-A.M.-C. were supported by a University of Castilla-La Mancha scholarship.Peer reviewe

Efecto de diferentes concentraciones de los antioxidantes melatonina, crocina y trolox sobre la calidad seminal a la descongelación en Ciervo Ibérico

Trabajo Fin de Máster: Master en investigación Básica y Aplicada en Recursos Cinegéticos. Línea de Investigación: Bancos de recursos genéticos.El objetivo del trabajo que a continuación se expone fue estudiar el efecto de la adición en el diluyente de congelación de tres antioxidantes (crocina, melatonina y trolox) a distintas concentraciones, sobre la calidad seminal de espermatozoides de ciervo Ibérico (Cervus elaphus hispanicus) descongelados. El material espermático con el que se trabajó procedió de 26 machos de ciervo ibérico abatidos durante la época de celo (septiembre-octubre) y fue recogido postmortem de la cola del epidídimo. El diluyente de congelación utilizado fue un TRIS-citrato-fructosa adicionado en 2 fracciones. La fracción A se formuló con un 20% de yema de huevo y la fracción B fue realizada igual que la fracción A más un 6% de glicerol. Al diluyente de congelación se le adicionaron los siguientes antioxidantes a las concentraciones finales indicadas obteniéndose 7 tratamientos: crocina (0,5 mM, 0,75 mM, y 1 mM), Melatonina (1 mM, 2,5 mM y 5 mM) y Trolox 1 mM. Además, también se utilizó el diluyente sin antioxidantes como un control. La calidad seminal fue estudiada tras la descongelación y después de dos horas de incubación a 37º C mediante evaluación con microscopía de fases y con citometría de flujo valorándose los siguientes parámetros: motilidad individual, calidad de movimiento y porcentaje de espermatozoides con el acrosoma intacto mediante microscopía de contraste de fases, parámetros descriptores del movimiento evaluados mediante sistemas de imágenes computerizados (CASA), y viabilidad, potencial de la membrana mitocondrial e integridad del ADN mediante citometría de flujo. A la descongelación no se encontraron diferencias significativas para los parámetros de movilidad y de integridad acrosomal. Sin embargo, cuando se evaluó la calidad seminal mediante citometría de flujo se observó que la melatonina añadida a concentraciones de 2,5 y 5 mM al diluyente de congelación produce una disminución en la viabilidad y actividad mitocondrial en comparación con el control y con el antioxidante crocina suplementado en el diluyente de congelación a una concentración 0,75 mM. Además, la utilización de crocina en todas sus concentraciones en el diluyente de congelación permitió disminuir el porcentaje de espermatozoides apoptóticos en relación al control. Por otra parte, cuando se realizó la evaluación seminal tras 2 horas de incubación de las muestras espermáticas a 37 °C se observó, que la velocidad curvilínea (VCL) y el desplazamiento lateral de la cabeza (ALH) fueron menores para las muestras espermáticas congeladas en un diluyente con 5 mM de melatonina. De nuevo, el antioxidante crocina fue el mejor para disminuir el porcentaje de espermatozoides apoptóticos. En conclusión la crocina es un buen antioxidante para mejorar la calidad seminal a la descongelación de espermatozoides epididimarios de ciervo Ibérico.Peer reviewe

Estudio y optimización de la capacitación in vitro de espermatozoides de ciervo ibérico (Cervus elaphus hispanicus)

En los últimos años, la cría en cautividad de ciervos se ha convertido en un tipo de ganadería alternativa de gran importancia en Europa. Las diferentes biotecnologías reproductivas, como la producción in vitro de embriones (PIVE), representan una herramienta de gran utilidad para la expansión de este sector, así como el aumento de su competitividad y rentabilidad. Sin embargo, el escaso desarrollo de estas técnicas en ciervo no permite, por el momento, proporcionar a los ganaderos una infraestructura comercial de producción, venta y transferencia de embriones. Uno de los principales motivos que impide el desarrollo de la PIVE en esta especie es la falta de conocimiento sobre la fisiología de sus espermatozoides. En este contexto, la información disponible acerca de la fase de capacitación espermática, mediante la cual los espermatozoides adquieren la capacidad de fecundar los ovocitos, es contradictoria, debido sustancialmente a los pocos trabajos que hay realizados en ciervo. Por este motivo, es común el uso de sustancias no definidas como el suero de oveja en celo (ESS) como agente capacitante en las rutinas de fecundación in vitro (FIV) dificultando su estandarización y un uso más amplio de esta tecnología. Los trabajos llevados a cabo en la presente Tesis Doctoral tienen como objetivo mejorar los protocolos de capacitación espermática implementados en la PIVE de ciervo mediante el estudio de la fisiología del espermatozoide durante esta fase, así como el desarrollo de un medio de capacitación definido para producir in vitro embriones de esta especie. En primer lugar, en el Capítulo I evaluamos y comparamos el efecto del ESS obtenido de diferentes hembras sobre la funcionalidad espermática, así como sobre los resultados de capacitación y embriones producidos in vitro utilizando como modelo la especie ovina. Nuestros resultados mostraron que distintos ESSs tuvieron un efecto variable sobre el desarrollo de la fosforilación en los residuos de tirosina asociada al proceso de capacitación, evidenciando la necesidad de eliminar el ESS de los medios de capacitación in vitro. Sin embargo, no observamos ningún efecto de los diferentes ESSs empleados sobre las tasas de embriones producidos ni sobre la actividad transcripcional de los mismos. En el Capítulo II, caracterizamos por primera vez los cambios que los espermatozoides descongelados de ciervo experimentan durante la incubación en condiciones de capacitación in vitro. Para ello, monitorizamos durante diferentes tiempos de incubación (hasta 24 h) diversos parámetros de funcionalidad espermática, marcadores de capacitación y estado de la cromatina. Así, observamos que los espermatozoides de ciervo mostraron parámetros relacionados con la capacitación y un incremento progresivo de la descondensación de la cromatina (% HDS) tras un corto periodo de incubación (t<30min). Además, observamos que períodos de tiempo superiores a los 30 min provocaron una disminución de la funcionalidad de los espermatozoides. En la misma línea, los resultados de FIV corroboraron que las tasas de división de ovocitos fueron superiores durante los 30 primeros min de incubación en condiciones de capacitación, reduciéndose a partir de los 60 min. Finalmente, observamos una correlación positiva entre la fosforilación en los residuos de tirosina, la proporción de espermatozoides que muestran la fosforilación sólo en la región ecuatorial y acrosomal y el %HDS con la tasa de división a las 48 h.p.i., respectivamente, confirmando la importancia de estos parámetros durante la capacitación para incrementar la capacidad fecundante de los espermatozoides de ciervo. En el Capítulo III evaluamos el efecto de diferentes concentraciones de albúmina sérica bovina (BSA) como posible alternativa al uso de ESS en el medio de capacitación de los espermatozoides descongelados de ciervo. Nuestros resultados mostraron que, aunque no existieron diferencias en parámetros relacionados con la capacitación, como la fosforilación en tirosina, para las diferentes concentraciones de BSA evaluadas en relación a los medios control, el uso de BSA resultó adecuado para neutralizar las ROS y reducir el daño acrosomal. Sin embargo, concentraciones de BSA a partir de 3 mg mL-1 disminuyeron la viabilidad y la actividad mitocondrial de los espermatozoides de ciervo durante la capacitación. Por último, en el Capítulo IV evaluamos el efecto de distintas concentraciones de Ca2+ y bicarbonato, así como de diferentes pH sobre la funcionalidad y capacitación espermática con el objetivo de mejorar el medio de capacitación sin ESS definido en el Capítulo anterior. Con esta serie de experimentos observamos que un fluido oviductal sintético formulado con 3 mg mL-1 de BSA, 3 mM de Ca2+, 30 mM de NaHCO3 y con un pH 7 desencadenó cambios compatibles con la capacitación espermática por encima de los observados en los espermatozoides incubados con ESS. No obstante, este medio para la capacitación in vitro (CIV) de espermatozoides de ciervo requiere de mejoras pues su uso durante la técnica de FIV no permitió alcanzar las mismas tasas de fecundación que las obtenidas con el ESS. Estos resultados podrían indicar que los ovocitos de esta especie también necesitan la influencia de determinadas concentraciones de Ca2+ y bicarbonato en el medio, que deberán estudiarse en futuros estudios, para su adecuada fecundación

Male SIRT1 insufficiency leads to sperm with decreased ability to hyperactivate and fertilize

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.This study was supported by the Charles University Research Fund (Progress Q39) and by the Ministry of Education, Youth and Sports of the Czech Republic, due to project No. CZ.02.1.01/0.0/0.0/16_019/0000787 ‘Fighting Infectious Diseases’, and the grant SVV–2020-2022 No 260 536.Peer reviewe

Recombinant SPINK3 improves ram sperm quality and in vitro fertility after cryopreservation

Capacitation-like changes affect sperm of several species, such as ram, reducing cell survival and fertilizing competence. Proteins from seminal plasma stabilize sperm plasma membranes, being an interesting focus to develop strategies for improving cryopreserved ram semen performance. To date, biotechnologies are focused to reduce damage in frozen-thawed ram spermatozoa through the addition of bioactives. Serine Protease Inhibitor Kazal-type 3 (SPINK3) is a little protein synthesized by mouse seminal vesicle and secreted to seminal plasma. While attached to the sperm, this protein binds to non-capacitated sperm and blocks calcium entry, avoiding a premature physiological capacitation and consequently, acrosome reaction. Due to these characteristics, SPINK3 has been proposed as a decapacitating factor. The aim of this work was to assess whether heterologous SPINK3 is able to protect ram sperm from the well-known cell damages produced by freezing/thawing and to understand the mechanisms by which it is acting. Sperm were supplemented with 13 μM SPINK3 before freezing in an egg yolk-based extender or after thawing and selection. Under both conditions, SPINK3 decreased intracellular calcium content (p < 0.05) and reduced the 25 kDa tyrosine phosphorylated protein demonstrating a decapacitating effect, although the addition of the protein before cryopreservation was not enough to improve other sperm parameters. However, the addition of SPINK3 post thawing was able to significantly ameliorate viability, motility, mitochondrial status and to avoid the increase of lipid peroxidation (p < 0.05). Moreover, sperm treated with SPINK3 was not only still capable to fertilize, but also improved it, as evidenced by an increase in the oocyte cleavage rate (p < 0.05) although, the embryo development and embryo quality were not affected. Our findings would contribute to develop a strategy for improving sperm quality by using decapacitating proteins. In fact, the outcomes of this work demonstrate that SPINK3 is able to reduce sperm cryo-injuries when is added after thawing, improving functionality and thus in vitro fertilization results.This research was supported by the National Agency for Scientific and Technological Promotion (ANPCyT, grant PICT-2015-3682 awarded to A.C), M.I.C was supported by Ministry of Economy and Competitiveness scholarship and the BeCAR program that facilitates the L.Z′ s stage in the UCLM, Spain.Peer reviewe

Cryopreservation of ram sperm alters the dynamic changes associated with in vitro capacitation

The aim of this study was to investigate the dynamic changes that ram sperm experience during in vitro capacitation before and after cryopreservation. Using flow cytometry and computer assisted sperm analysis system (CASA), protein tyrosine phosphorylation and several functional parameters were evaluated in fresh and cryopreserved ram sperm incubated under capacitating and non-capacitating conditions at 0, 1, 5, 15, 30, 60, 120, 180 and 240 min. A short incubation period (5–30 min) under capacitating conditions was enough to increase mitochondrial activity and tyrosine phosphorylation in cryopreserved sperm, inducing also changes in the motility pattern, which could be related to hyperactivation. However, fresh sperm required a longer incubation (180–240 min) under capacitating conditions to undergo similar modifications. In both types of samples, tyrosine phosphorylation increased in a sequential manner in the midpiece, principal piece and tail at specific time points during in vitro capacitation. Moreover, the proportion of viable sperm with intact acrosome begun to decrease during capacitation, occurring before in cryopreserved sperm. Our findings suggest that cryopreserved ram sperm become competent for fertilization after a short exposure to capacitating conditions as a result of drastic changes inflicted by the freezing-thawing procedure, while prolonged incubations after cryopreservation severely impair sperm quality.PP-F was supported by University of Castilla-La Mancha (UCLM) fellowships. MV was supported by the Research Plan of University of Castilla- La Mancha (UCLM). MI-C was supported by the Ministry of Economy and Competitiveness fellowship.Peer reviewe

Reduced glutathione addition improves both the kinematics and physiological quality of post-thawed red deer sperm

The potential protective effect of reduced glutathione (GSH) and trolox (TRX), an analogue of vitamin E, supplementation during in vitro culture (2. h, 39. °C) of electroejaculated frozen/thawed red deer sperm was investigated. Cryopreserved sperm were thawed and incubated with no additive (Control) and 1. mM or 5. mM of each antioxidant to find out whether these supplementations can maintain the sperm quality, considering the use of thawed samples for in vitro techniques such as in vitro fertilisation (IVF), sperm sex sorting or refreezing. The effect of GSH on sperm motility was positive compared to TRX which was negative (P<. 0.001). After 2. h of incubation at 39. °C, use of GSH improved motility while TRX supplementation reduced sperm motility compared with Control samples without antioxidant. Use of TRX at both concentrations (1 and 5. mM; TRX1 and TRX5) resulted in lesser percentages of apoptotic sperm (12.4 ± 1.1% and 11.7 ± 0.9%) than GSH1, GSH5 (15.2 ± 1% and 14.6 ± 1.1%) and Control samples (16.9 ± 1.2%) (P<. 0.001). Use of GSH at both concentrations (1 and 5. mM) resulted in greater mitochondrial activity as compared with findings for the Control, TRX1 and TRX5 groups. Results of this study indicate that GSH is a suitable supplement for electroejaculated red deer sperm. It would be necessary to conduct fertility trials (in vivo and in vitro), to assess whether GSH supplementation of thawed red deer sperm could improve fertility rates.This research was supported by Junta de Castilla y La Mancha (PRE123/2010).Peer Reviewe

Selection of red deer spermatozoa with different cryoresistance using density gradients

The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post-thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post-thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll, Puresperm and Bovipure, and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post-thawing values (p >.05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll and Puresperm considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p =.040) and apoptosis (p =.003). Thus, Percoll selected less live and more apoptotic spermatozoa than Puresperm and Bovipure for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.This work was supported by the Spanish Ministry of Science and Innovation (Project AGL2010-21487). O García-Álvarez, A Maroto-Morales, M Iniesta-cuerda were supported by a fellowship of CYTEMA-UCLM, Junta de Castilla-La Mancha and MINECO, respectively.Peer Reviewe