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Prolonging the shelf-life of liquid-preserved semen without compromising its fertilizing capacity may increase the efficiency of artificial insemination in pigs. Many fertilization-relevant processes are adenosine triphosphate dependent. The impact of semen storage and rewarming to body temperature on the energy status of spermatozoa is as yet unknown.To investigate the energy status of boar spermatozoa during storage and subsequent rewarming and to reveal the potential role of mitochondrial function for reactivation and maintenance of sperm motility.Extended semen samples (n = 7 boars) were used. Spermatozoa were challenged by storage at 17°C for 7 days and incubation at 38°C for 180 min. The adenosine triphosphate concentration and energy charge in semen samples and lactate concentration in the extracellular medium were assessed. Viability and mitochondrial activity were determined by flow cytometry, and clustered single-cell analysis of motility parameters was performed.The energy status was not affected by semen storage (p 0.05). Rewarming resulted in a net reduction in adenosine triphosphate concentration, which increased with storage time (maximum Day 5: -24.2 ± 10.3%) but was not accompanied by a loss in viability, motility, or mitochondrial activity. Blocking glycolysis with 2-deoxy-d-glucose prevented the re-establishment of motility and mitochondrial activity after rewarming. Mitochondrial activity gradually subsided in virtually all spermatozoa during incubation at 38°C, while adenosine triphosphate and energy charge remained high. Concomitantly, extracellular lactate levels rose, and sperm populations with lower velocity, increased linearity, and low lateral head displacement grew larger. Size changes for major sperm subpopulations correlated with the percentage of viable spermatozoa with high mitochondrial activity (r = 0.44-0.70 for individual subpopulations, p 0.01).Storage of boar spermatozoa increases the demand of adenosine triphosphate for reactivation of spermatozoa towards fast, non-linear, and hyperactivation-like motility patterns upon rewarming. Maintenance of glycolysis seems to be decisive for sperm function after long-term storage in vitro.

Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca

Hypothermic storage of boar semen may allow antibiotic-free semen preservation but is limited due to chilling sensitivity of boar spermatozoa. Progress in this area requires sensitive tools to detect chilling injury. Therefore, multiparameter flow cytometry panels were evaluated to ascertain whether they are useful tools for identifying sublethal damage of sperm function at a single cell level, thus considering the high intrinsic sperm heterogeneity in a sample. The first fluorochrome panel consisted of Hoechst 33342 to identify DNA-containing events, Yo-Pro 1 to detect viability, merocyanine 540 to describe membrane fluidity, and PNA-Alexa Fluor™ 647 to identify acrosomic integrity. The second fluorochrome panel consisted of SiR700-DNA to identify DNA-containing events, JC-1 to characterize the mitochondrial transmembrane potential (MMP), and Calbryte 630 to assess the intracellular calcium level. Extended boar semen was stored either at 17°C (control) or 5°C (chilled). It is shown that chilling increased membrane fluidity in the viable (Yo-Pro 1 negative) sperm population at 24 h (p < 0.05). At 144 h, the viable, acrosomic intact sperm population with low membrane fluidity was similar for both storage temperatures. Moreover, chilling reduced the main sperm population with high MMP, medium fluorescence for JC-1 monomer and low intracellular calcium level (p < 0.05). However, after in vitro sperm capacitation, this population did not differ between the two storage temperatures. Exemplary computational data visualization in t-distributed stochastic neighbor embedding (t-SNE) maps and moving radar plots revealed similar subpopulations as identified by three-dimensional stacked bar charts. In conclusion, sperm surviving an initial chilling injury withstand long-term storage and respond in a similar manner to capacitation conditions as sperm stored conventionally at 17°C. Multicolor flow cytometry is a valuable tool for detecting chilling-induced alterations of cell function in sperm subpopulations.

Evaluation of sperm motility is well-established in farm animals for quickly selecting ejaculates for semen processing into insemination doses and for evaluating the quality of preserved semen. Likewise, sperm motility is a fundamental parameter used by spermatologists in basic and applied science. Motility is commonly assessed using computer-assisted semen analysis (CASA). Recent increases in computational power, as well as utilization of mobile CASA systems and open-source CASA programs, broaden the possibilities for motility evaluation. Despite this technological progress, the potential of computer-generated motility data to assess male fertility remains challenging and may be limited. Relevance for fertility assessment could be improved if measurement conditions would more closely mimic the in vivo situation. Hence, this review is focused on the current trends of automated semen assessment in livestock and explores perspectives for future use with respect to the physiological and physical conditions encountered by sperm in the female reproductive tract. Validation of current CASA systems with more complex, microfluidic-based devices mimicking the female reproductive tract environment could improve the value of sperm kinematic data for assessing the fertilizing capacity of semen samples, not only for application in livestock but also for use in conducting assisted reproduction techniques in other species.

We describe the development of two methods for obtaining confluent monolayers of polarized, differentiated equine oviduct epithelial cells (EOEC) in Transwell inserts and microfluidic chips. EOECs from the ampulla were isolated post-mortem and seeded either (1) directly onto a microporous membrane as differentiated EOECs (direct seeding protocol) or (2) first cultured to a confluent de-differentiated monolayer in conventional wells, then trypsinized and seeded onto a microporous membrane (re-differentiation protocol). Maintenance or induction of EOEC differentiation in these systems was achieved by air-liquid interface introduction. Monolayers cultured via both protocols were characterized by columnar, cytokeratin 19-positive EOECs in Transwell inserts. However, only the re-differentiation protocol could be transferred successfully to the microfluidic chips. Integrity of the monolayers was confirmed by transepithelial resistance measurements, tracer flux, and the demonstration of an intimate network of tight junctions. Using the direct protocol, 28% of EOECs showed secondary cilia at the apical surface in a diffuse pattern. In contrast, re-differentiated polarized EOECs rarely showed secondary cilia in either culture system (>90% of the monolayers showed

1 Mitochondria-cytoskeleton interactions modulate cellular 2 physiology by regulating mitochondrial transport, position-3 ing, and immobilization. However, there is very little struc-4 tural information defining mitochondria-cytoskeleton inter-5 faces in any cell type. Here, we use cryo-focused ion beam 6 milling-enabled cryo-electron tomography to image mam-7 malian sperm, where mitochondria wrap around the ciliary 8 cytoskeleton. We find that mitochondria are tethered to their 9 neighbors through inter-mitochondrial linkers and are an-10 chored to the cytoskeleton through ordered arrays on the 11 outer mitochondrial membrane. We use subtomogram aver-12 aging to resolve in-cell structures of these arrays from three 13 mammalian species, revealing they are conserved across 14 species despite variations in mitochondrial dimensions and 15 cristae organization. We find that the arrays consist of 16 boat-shaped particles anchored on a network of membrane 17 pores whose arrangement and dimensions are consistent 18 with voltage dependent anion channels. Proteomics and in-19 cell cross-linking mass spectrometry suggest that the con-20 served arrays are composed of glycerol kinase-like proteins. 21 Ordered supramolecular assemblies may serve to stabilize 22 similar contact sites in other cell types where mitochondria 23 need to be immobilized in specific subcellular environments, 24 such as in muscles and neurons. 25 sperm | mitochondria-cytoskeleton contact | cryo-electron tomography | cryo-26 FIB milling | cross-linking mass spectrometry | subtomogram averaging 27

Sperm osmotic adaptability to anisosmotic conditions is important for sperm epididymal maturation, motility activation at ejaculation, and female tract colonization, or for conducting technological procedures such as cryopreservation. Several factors affect this adaptability, including the fluid composition that contributes to water flow dynamics, and the temperature at which osmotic stress is initiated. This study was designed to investigate the effect of medium composition (electrolyte- or sugar-based extender) and temperature (25 and 5 °C) on rabbit sperm adaptability to anisosmotic conditions. Rabbit spermatozoa, therefore, were diluted at both temperatures (25 and 5 °C) in electrolyte- or sugar-based media at increasing osmotic conditions (100 to 1,000 mOsm/kg), and values for sperm variables (sperm kinetics, membrane integrity, mitochondrial membrane potential) were estimated as endpoints. Sperm kinetics seemed to be more sensitive to osmotic stress than membrane integrity or mitochondrial function. The effect of moderate hypoosmotic stress did not differ when there was use of sugar- and electrolyte-based extenders at 25 °C (P0.05). In hyper-tonic conditions at 25 °C, the sugar-based extender was more effective in protecting sperm membrane integrity and mitochondrial function (P0.05). The lesser temperature made the differences more relevant because of the detrimental effect of hyperosmotic stress was more evident in the electrolyte-based extender at 5 °C (P0.05). The results from this study indicated rabbit spermatozoa have different adaptability to anisosmotic conditions induced by sugar- and electrolyte-based media and that the temperature at which the osmotic stress is initiated affects the cellular response.

SummaryMotile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures rein-forcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.

In embryos from most animal species a zygotic centrosome is assembled by the centriole derived from the sperm cell and pericentriolar proteins present in the oocyte. This zygotic centrosome acts as a microtubule organizing center (MTOC) to assemble the mitotic spindle in the first and all subsequent cell divisions. As MTOC formation has been studied mainly in adult cells, very little is known about the formation of the first zygotic MTOC. Here we find that zebrafish (Danio rerio) embryos lacking maternal or paternal Cfap53, a centriolar satellite protein, arrest during the first cell cycle due to a failure in proper formation of the mitotic spindle. During the first cell cycle Cfap53 co-localizes with γ-tubulin and other centrosomal and centriolar satellite proteins to the very large MTOC. Furthermore, we find that γ-tubulin localization to the MTOC is impaired in the absence of Cfap53 or when the microtubule network is disrupted. Based on these results we propose a model in which maternal and paternal Cfap53 participates in the organization of the first zygotic MTOC of the embryo. Once the zygotic MTOC is formed, Cfap53 is dispensable for MTOC formation and integrity in subsequent cell divisions.

The developmental competence of male and female gametes is frequently reduced under in vitro conditions, mainly due to oxidative stress during handling. The amino-acid derived hormone melatonin has emerged as a potent non-enzymatic antioxidant in many biological systems. The goal of the present study was to evaluate the effects of melatonin on post-thaw sperm quality, fertilizing ability, and embryo development and competence in vitro after in vitro fertilization. Frozen-thawed bovine spermatozoa were incubated either in the presence of 10−11 M melatonin (MT), or its solvent (ethanol; Sham-Control), or plain Tyrode’s Albumin Lactate Pyruvate medium (TALP, Control). Computer-Assisted Sperm Analysis (CASA) and flow cytometry data after 30 min, 120 min, and 180 min incubation did not reveal any significant effects of melatonin on average motility parameters, sperm subpopulation structure as determined by hierarchical cluster, or on the percentage of viable, acrosome intact sperm, or viable sperm with active mitochondria. Nevertheless, in vitro matured cumulus-oocyte-complexes fertilized with spermatozoa which had been preincubated with 10−11 M melatonin (MT-Sperm) showed higher (P < 0.01) rates of monospermic fertilization, reduced (P < 0.05) polyspermy and enhanced (P < 0.05) embryo development compared to the Control group. Moreover, the relative abundance of MAPK13 in the in vitro-derived blastocysts was greater (P < 0.05) than observed in the Control group. In conclusion, adding melatonin to the sperm-preparation protocol for bovine IVF improved proper fertilization and enhanced embryonic development and competence in vitro.

The oviduct was long considered a largely passive conduit for gametes and embryos. However, an increasing number of studies into oviduct physiology have demonstrated that it specifically and significantly influences gamete interaction, fertilization and early embryo development. While oviduct epithelial cell (OEC) function has been examined during maintenance in conventional tissue culture dishes, cells seeded into these two-dimensional (2-D) conditions suffer a rapid loss of differentiated OEC characteristics, such as ciliation and secretory activity. Recently, three-dimensional (3-D) cell culture systems have been developed that make use of cell inserts to create basolateral and apical medium compartments with a confluent epithelial cell layer at the interface. Using such 3-D culture systems, OECs can be triggered to redevelop typical differentiated cell properties and levels of tissue organization can be developed that are not possible in a 2-D culture. 3-D culture systems can be further refined using new micro-engineering techniques (including microfluidics and 3-D printing) which can be used to produce ‘organs-on-chips’, i.e. live 3-D cultures that bio-mimic the oviduct. In this review, concepts for designing bio-mimic 3-D oviduct cultures are presented. The increased possibilities and concomitant challenges when trying to more closely investigate oviduct physiology, gamete activation, fertilization and embryo production are discussed.

Evidence is emerging that the interaction between male seminal fluid and female tissues promotes fertility, pregnancy, and health of offspring. This includes the acceleration of ovulation in a species known as a spontaneous ovulator, the domestic pig. Earlier studies revealed that seminal plasma acts by a local mechanism in the female pig. The aim of the present study was to examine local short-term and mid-term effects of seminal plasma (SP) on mRNA expression of immunoregulatory genes and transcripts associated with follicle- and oocyte maturation. In the porcine animal model, effects on mRNA expression in the female tract and preovulatory follicles were examined. SP suppressed mRNA expression of Prostaglandin-Endoperoxide Synthase 2 (PTGS2) ipsilateral to the infused uterine horn which was associated with a lower presence of immune cells in the uterine epithelium and lower PTGS2 immunoreaction. Depending on the sampling time (2 h vs. 17 h) and hormonal status, SP altered significant correlative relations of mRNA expression between PTGS2 and the transcripts Tumor Necrosis Factor Alpha, Tumor Necrosis Factor Alpha-Induced Protein 6 and Pentraxin 3 in uterus, granulosa and cumulus cells. A modulatory effect of SP on the oocyte gene network comprising eight oocyte transcripts was observed: uterine exposure to SP induced positive correlations (r >0.08, p

Corporate environmental compliance managers are required to perform and supervise a set of complex tasks. It can be expected that the growing density of environmental regulations will contribute to even more challenges for compliance managers in the near future. As a result, companies are increasingly pressured to improve the efficiency of their compliance management processes by the use of automation approaches. For some specific compliance management processes it is expected that a partial process automation can be obtained through the use of a ticket system. However, so far only little is known about the general usage options and benefits of ticket systems for environmental compliance management. Given this gap, the article contains a corresponding demonstration and evaluation study. A reference process model for environmental compliance management and an industry survey of the top software capabilities desired by compliance managers serves as foundation for this study. The process automation options based on a ticket system are exemplified through a reference process for the new regulation management process. The process specification is given in the popular standardized graphical notation called “Business Process Model and Notation” (BPMN).

Environmental temperatures may influence the temperature inside commercial transport boxes during semen shipment and thereby storage conditions of diluted dog semen. To evaluate the temperature changes inside boxes and their influence on sperm quality, split semen samples (n=8) were placed in Neopor boxes(®) exposed for 48h to room temperature (RT) (Box 1), 40°C for 6h and then kept at RT (Box 2) or 40°C (Box 3). A fourth subsample was kept at 4-5°C in a refrigerator (control). Inside Box 1 temperature initially decreased to3°C before it stabilized at 7-8°C, while in Box 2 no decrease occurred and temperature was at 7-8°C for 48 h. Temperature inside Box 3 was at 14-15°C for 24h and, thereafter, increased to 36.1°C. Analysis of sperm motility (CASA) and viability (PI and FITC-PNA) after 24 and 48 h revealed marked sensitivity of dog spermatozoa to temperature fluctuations (Box 1). A constant storage temperature of 7-8°C (Box 2) provided the most desirable semen quality in terms of motility, viability, as well as osmotic resistance when samples were stored for 48 h. Furthermore, results indicate that during 24h preservation a storage temperature of 14-16°C may provide optimum conditions for maintenance of sperm viability and function. An increase of the inside temperature to30°C (Box 3) resulted in an almost complete loss in sperm integrity. In conclusion, results suggest a revision of current recommendations for storage temperature of diluted dog semen. Boxes for semen transport should be prepared depending on the expected environmental temperatures.

Freshly collected boar spermatozoa are sensitive to a fast reduction in temperature because of lipid phase transition and phase separation processes. Temperature management during semen processing may determine the quality of stored samples. The aim of this study was to evaluate the influence of isothermic and hypothermic semen processing protocols on boar sperm quality under laboratory and field conditions. In the laboratory study, ejaculates (n = 12) were first diluted (1:1) with Beltsville Thawing Solution (BTS) at 32 °C, then processed either with isothermic (32 °C) or hypothermic (21 °C) BTS, stored at 17 °C, and assessed on days 1, 3, and 6. Temperature curves showed that 150 minutes after the first dilution, semen doses of both groups reached the same temperature. Two-step hypothermic processing resulted in lower sperm motility on days 1 and 6 (P < 0.05). Concomitantly, hypothermally processed samples contained less membrane intact sperm on days 3 and 6 (P < 0.05). Using AndroStar Plus extender instead of BTS reduced the negative effect of hypothermic processing. In the field study, 15 semen samples from each of 23 European artificial insemination studs were evaluated as part of an external quality control program. Semen quality based on motility, membrane integrity, mitochondrial activity, and a thermoresistance test was higher for stations using one-step isothermic dilutions (n = 7) compared with artificial insemination centers using two-step hypothermic protocols (n = 16). Both studies show that chilling injury associated with hypothermic dilution results in lower quality of stored boar semen compared with isothermic dilution and that the type of semen extender affects the outcomes.

Spermatozoa, especially those of the porcine species, are highly susceptible to in vitro chilling and ageing. Extenders are continuously developed to protect boar spermatozoa from chilling injury. New semen extenders and other modified preservation strategies require sensitive testing for essential sperm functions. The key process on the pathway of fertilization is capacitation. The aim of the present study was to examine whether the specific response to capacitating stimuli is sensitive enough to indicate different preservation capacities of extenders during hypothermic storage of boar spermatozoa. Semen was diluted in Beltsville Thawing Solution (BTS) and Androstar Plus and kept for 3 h at 22°C or stored at 17°C, 10°C, and 5°C. Semen was analyzed at 24 and 96 h of storage. Motility and membrane integrity remained at high levels, except for lower values when stored in BTS at 5°C. Washed subsamples were incubated in capacitating medium (Tyrode) and control medium and were assessed for intracellular calcium concentration and integrity of plasma membranes using a flow cytometer. On the basis of the loss of low-calcium live cells in a kinetic approach, the specific response to capacitation stimuli was determined. There was a higher loss of response in semen stored hypothermically in the standard extender BTS compared to Androstar Plus. Assessment of the extent of phospholipid disorder under capacitating and control conditions by use of merocyanine staining did not reveal any significant extender-related differences. A field insemination trial with 778 sows was performed to relate in vitro results to fertility. Fertility parameters did not differ in semen stored up to 48 h at 10°C in Androstar Plus compared to controls stored at 17°C in BTS. In conclusion, assessment of specific reactivity to capacitating stimuli appears to be a sensitive tool for detection of extender-dependent alterations in functionality of chilled boar spermatozoa.

Summary Boar spermatozoa are sensitive to storage temperatures below 15 °C. Chilling injury causes loss of motility and membrane integrity in a minority of cells, whereas the main population displays sublethal changes compromising fertility. In this study, changes of the response to capacitation conditions in hypothermically stored boar spermatozoa have been examined using a kinetic approach with well-defined test and control media. Ejaculates of seven boars were diluted in Beltsville Thawing Solution kept for 3 h at 22 °C or cooled to 17, 10 and 5 °C and stored for 24 and 96 h. At each time point, the standard sperm parameters motility and membrane integrity were evaluated. Subsequently, washed subsamples were incubated in capacitating and control medium before flow cytometric analysis of intracellular calcium content using the Fluo-3 probe and changes in phospholipid disorder using merocyanine. Kinetic changes of response parameters were monitored in viable (plasma membrane intact) cells. Chilling led to a loss of standard sperm quality traits in a minor subpopulation of cells, whereas storage length had no effect on these parameters. However, responses to incubation as determined by the loss of live cells with low intracellular calcium content showed marked changes in relation to storage conditions. The specific responsiveness to capacitation conditions decreased in close relation to storage temperature and length. In contrast, the merocyanine probe revealed to be limited to detect effects of hypothermic storage. Using Fourier transform infrared spectroscopy, no influence of chilling on membrane phase behaviour was found that might implicate decreased sperm function. In conclusion, assessment of response to capacitating media by monitoring intracellular calcium levels provides a sensitive measure for chilling injury in extended boar semen, and therefore, deserves implementation in hypothermic storage tests.

Contents Fertility of extended boar semen declines within the first 72 h of storage in vitro. Standard semen assessment, such as motility and membrane integrity, allows detection of lethal damage of spermatozoa. However, conventional sperm assessment often lacks standardization and does not allow identification of sub-lethal changes of sperm quality during the initial 72 h of storage. In the present brief review, recent strategies for quality assessment of liquid preserved boar semen are discussed and basic implications for experiments designed to detect storage effects are given.

Short-term storage of equine sperm at 5°C in an extender containing milk and/or egg yolk components is common practice in the equine breeding industry. Sperm motility, viability, DNA integrity and, consequently, fertilizing ability decline over time, partly due to reactive oxygen species (ROS) generation. We investigated whether adding the anti-oxidant d-penicillamine to a commercial milk/egg yolk extender delayed the decrease in semen quality. Semen was recovered on four consecutive days from eight 3-year old Warmblood stallions. On day 5, seven of the stallions were castrated and sperm recovered from the caudae epididymides. Ejaculated samples were split, and one portion was centrifuged and re-suspended to reduce seminal plasma content. All samples were diluted to 50millionsperm/ml and divided into two portions, one of which was supplemented with 0.5mM d-penicillamine. After 48h, 96h, 144h and 192h storage, sperm motility was assessed by computer-assisted semen analysis (CASA), viability by SYBR14/PI staining, and DNA integrity using the sperm chromatin structure assay (SCSA). d-Penicillamine had no effect on motility of ejaculated sperm (P>0.05) but reduced total and progressive motility of epididymal sperm. Sperm chromatin integrity was not influenced by storage time, seminal plasma or d-penicillamine. In short, adding d-penicillamine to a commercial semen extender was neither beneficial nor detrimental to the maintenance of quality in ejaculated semen stored at 5°C. The negative effect on motility of epididymal sperm may reflect differences in (membrane) physiology of spermatozoa that have not been exposed to seminal plasma.

Summary Density gradient centrifugation of semen is commonly used in many assisted reproduction techniques. Although gradients have the potential to isolate and enrich motile and viable spermatozoa, the centrifugation force presents a stress factor to cell organelles and membranes. The objective of the study was to evaluate the impact of density gradient centrifugation stress on sperm capacitation dynamics, cell stability and the ability of spermatozoa to specifically respond to bicarbonate in extended semen undergoing in vitro ageing. Extended boar semen (n = 7) was stored for 12, 24, 72 and 120 h respectively at 17 °C before centrifugation and incubation in variations of an in vitro capacitation medium. The number of viable, acrosome intact sperm and motility parameters as assessed by computer-assisted semen analysis did not change during storage. Kinetic changes in viability (plasma membrane integrity) and intracellular calcium levels (calcium influx) during in vitro capacitation were assessed after preparation of semen samples with both, a Percoll and a sucrose gradient centrifugation, either only Percoll, only sucrose centrifugation or no centrifugation. Changes in the viable sperm population that could be specifically attributed as a response to either bicarbonate or calcium were determined. In in vitro-aged (>12 h stored) spermatozoa, centrifugation reduced the proportion of spermatozoa which specifically responded to the capacitating stimulus bicarbonate. Concomitantly, centrifugation increased the proportion of spermatozoa responding to calcium in absence of bicarbonate, thus indicating an increased sensitivity to incubation per se. Absence of centrifugation steps during semen preparation, revealed a highly conserved ability of in vitro-aged spermatozoa to specifically respond to bicarbonate. In conclusion, density gradient centrifugation alters the physiological property of spermatozoa for controlled capacitation, which may influence the success rates of centrifuged semen in assisted reproductive technologies and confound interpretation of capacitation assays.

The natural polyphenol resveratrol may be beneficial to many aspects of cell function and animal health, although its actions in the male reproductive system vary depending on animal species. This work investigates resveratrol effects on the quality of preserved boar semen during liquid storage at 17oC. We used three approaches: 1) evaluation of conventional parameters of seminal quality, 2) measurement of specific response to capacitating stimuli, and 3) evaluation of mitochondria membrane potential and ATP content. Resveratrol supplementation causes i) a loss in the response of liquid stored boar spermatozoa to capacitating stimuli, ii) a decrease in the sperm ATP content and iii) a reduction in the mitochondrial membrane potential. Moreover, higher concentrations of resveratrol increase plasma membrane phospholipid disorder and reduce the percentage of motile spermatozoa. These results suggest that semen doses supplemented with resveratrol could be considered sub-fertile compared with semen stored hypothermically in standard conditions.

Oviduct epithelial cells (OECs) generate the microenvironment for mammalian fertilization. When cultured in vitro OECs rapidly lose their differentiated cell properties (e.g. secretory activity and cilia), while suspended cells have a limited lifespan. These limitations, likely due to the lack of folded tubular geometry of the oviduct, prompted us to combine transwell cell culture and 3-D printing technologies to mimic the in vivo OEC niche in order to better study the unique role of the oviduct and its microenvironment during the processes of fertilization and early embryonic development. U-shape inserts were 3-D printed using a multi-arm acrylate-based resin (PIC100) on an Envisiontec Perfactory P3 stereolithographer. Post-printing treatments of custom-made tubular transwell inserts were first tested in order to determine any possible negative impact of the plastics on cell growth. Inserts were either untreated, post-cured with 1000 flashes/side (Otoflash, 66 W), post-cured and Soxhlet-extracted overnight in isopropanol, or post-cured and Soxhlet-extracted over the weekend in water at 37°C. The post-cured and Soxhlet-extracted overnight in isopropanol inserts were selected as best pretreatment for culturing OECs. These inserts were mounted with track-etched PET membranes (12 µm thick, 0.4 µm pore diameter) to create a U-shape geometry that allows perfusion. Bovine OECs were obtained by squeezing the whole oviduct collected from slaughterhouse cows (on luteal phase) and cultured as monolayers for 7 days (n = 2 cows). These de-differentiated OECs were seeded on the membranes, grown to confluence (7 days), and cultured (1) at an air-liquid interface for 6 and 14 days (air-liquid culture) or (2) under perfusion (6 mL h–1) for 6 days (perfusion culture). OECs were also cultured on coverslips as monolayers (2-D culture) for 6 and 14 days. After this period, the OECs were fixed and immune labelled to determine their polarized state. Polarization of OECs (laminin and primary cilia detection) was observed on Day 6 for perfusion culture, on Day 14 for air-liquid culture, and was not detected in 2-D culture. The presence of secondary cilia (acetylated α-tubulin) was observed in 6% of the cells cultured under perfusion at Day 6; secondary cilia was not present in air-liquid or 2-D cultures during the period analysed. In conclusion, post-curing and Soxhlet extraction of leachable compounds is crucial to avoid toxic effects on cell growth. The U-shape custom-designed inserts are able to create a tube-like surface in which bovine oviducal cells can be cultured to confluency and thereafter repolarize (presence of primary cilia and detection of laminin); this polarization occurs faster when the U-shape culture is under perfusion. Further studies will examine the ability of the cells to differentiate further (development of secondary cilia and secretory ability) and support in vitro fertilization. To this end, 3-D designs will be tested to determine their use for live cell imaging and for collecting secreted fluids.