Effect of cryopreservation and semen extender on extracellular vesicles isolated from bull semen.

Introduction: Semen cryopreservation is the most popular practice for semen production for artificial insemination and in vitro fertilization in cattle. The Seminal plasma contains extracellular vesicles (spEVs) which modulate sperm viability and function during oocyte fecundation. The study of spEVs in frozen-thawed semen doses may yield novel indicators for predicting bull fertility, but the presence of the semen extender may hinder molecular profiling of spEVs. The aim of this study was to provide extensive characterization of EVs isolated from seminal plasma before and after the cryopreservation process and the addition of a commercial animal protein-free semen extender to understand the potential influence of EVs originating from the extender in hindering the use of spEVs derived biomarkers for assessment of bull fertility. Methods: EVs were isolated from the seminal plasma (with or without the extender), from the cryopreserved straw devoid of spermatozoa, and from the extender using two different methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), and characterized for their structure and composition. Results: Physical characterization of EVs showed that size and particle numbers were related to the method of isolation. spEVs were larger but less abundant (UC: 168.9 nm, n = 2.68 × 109; SEC: 197.0 nm, n = 6.42 × 109) compared to extender EVs (UC: 129.0 nm, n = 2.68 × 1011; SEC: 161.8 nm, n = 6.47 × 1011). Western blotting analysis (WB) confirmed the presence of typical EV markers in spEVS: the membrane bound CD9 (25 kDa) and the luminal markers Alix (96 kDa) and TSG101 (48 KDa). Although Transmission Electron Microscopy confirmed the presence of a lipid bilayer structure in all preparations, no specific EV markers were detected in the vesicles isolated from extender when the Single Molecule Array (SiMoa) was used. A total of 724 Bos taurus miRNAs were identified in at least one preparation. The percentage of miRNAs identified in EVs from the extender (0.05%-0.49% of the total reads) was lower than in the preparation containing spEVs (10.56%-63.69% of the total reads). Edge-R identified a total of 111 DE-miRNAs between EVs isolated from the extender by two methods. Among them, 11 DE-miRNAs (bta-miR-11980, bta-miR-11987, bta-miR-12057, bta-miR-1246, bta-miR-125b, bta-miR-181b, bta-miR-2340, bta-miR-2358, bta-miR-2478, bta-miR-2898, and bta-miR-345-3p) were also abundant in EVs isolated from seminal plasma preparations with extender. Conclusion: This study clearly demonstrates that the presence of the extender does not prevent the characterization of spEVs in cryopreserved semen. However, the molecular profiling of spEVs can be influenced by the isolation method used and by the presence of some miRNAs from the extender. Therefore, in such studies, it is advisable to characterize both spEVs and the vesicles isolated from the extender.

Effects of antioxidant Bis-carboxyethyl germanium sesquioxide added to bovine semen cryopreservation medium on in vitro assessed morphofunctional sperm parameters.

This study investigated the impact of various Ge132 (Bis-carboxyethyl germanium sesquioxide) concentrations on frozen bovine semen. Ejaculates from three bulls were pooled and divided into six groups, each one with different Ge132 concentrations (0, 500, and 1000 µg/mL) and each group was incubated in different conditions (33°C for 30 min (D: D0, D500, and D1000), and the other was immediately cooled to 4°C (R: R0-control; R500 and R1000)). Thawed semen was evaluated for sperm characteristics by CASA and flow cytometer. Results showed better motility in the immediate cooling group without Ge132 compared with high Ge132 concentrations. Values for total motility dropped after 5 and 60 min in groups with high Ge132 levels and some control groups. Linearity increased with 1000 µg/mL Ge132, while straightness differed between moments in multiple groups. Membrane integrity was higher in a control group and certain Ge132 groups. Lower O2 - generation occurred without Ge132. After oxidative stress induction, lipid peroxidation intensity increased with arachidonic acid, but D1000 had lower peroxidation than R0. Overall, Ge132 appears to have provided protection against PLM when subjected to oxidative stress, since even at high concentrations it maintained sperm metabolism.

Investigating the Effect of Chain Extender on the Phase Separation and Mechanical Properties of Polybutadiene-Based Polyurethane.

The thermodynamic incompatibility between the soft and hard segments of thermoplastic polyurethane (TPU) results in a microphase-separated behavior and excellent mechanical properties. However, the effect of the chain extender on the degree of microphase separation (DMS) and the resultant mechanical properties of TPU have not been well studied because of the complex interactions between the soft and hard segments. Herein, hydroxyl-terminated polybutadiene-based TPUs(HTPB-TPUs) without hydrogen bonding between the soft and hard segments are synthesized using hydroxyl-terminated polybutadiene, toluene diisocyanate, and four different chain extenders, and the effect of the chain extender structure on DMS is analyzed experimentally using a combination of analytical techniques. Furthermore, the solubility parameters of the soft and hard segments, glass transition temperatures, and hydrogen-bond density of the HTPB-TPUs, are computed using all-atom molecular dynamics simulations. The results clearly reveal that the chain extender significantly affects the DMS and thus the mechanical properties of HTPB-TPUs. This study paves the way for studying the relationship between the structure and properties of TPU.

Investigating the safety of antibiotics added to collared peccary (Pecari tajacu) semen extender through a multiparametric thermoresistance test.

The effects of antibiotics on sperm longevity in collared peccary (Pecari tajacu) fresh diluted semen was evaluated. Semen samples from six adult males were collected by electroejaculation and diluted in Tris-citrate-fructose alone (control) and plus streptomycin-penicillin (2 mg/ml-2000 IU/ml) or gentamicin (70 µg/ml). Membrane integrity and functionality, mitochondrial activity and sperm morphology were assessed subjectively. Sperm motility and other kinetic parameters were objectively assessed using CASA (computer-assisted semen analysis). The semen diluted according to the treatments were submitted to the thermoresistance test, incubated at 37 ° C, and the sperm parameters analyzed at 0, 30, 60, 120 and 180 min. The average values of the treatments were compared with each other and between the times. There were no differences (P > 0.05) between treatments until the end of the test. Control and streptomycin-penicillin samples maintained sperm function for up to 180 min (with total motility of 24.3 ± 7.1% and 28 ± 8.7%, respectively). Gentamicin aliquots retained most parameters until the end of the incubation, except for membrane integrity and mitochondrial activity that declined (P < 0.05) at 180 min (53.1 ± 7.1% and 50.7 ± 6.2%, respectively) compared to 0 min (80.5 ± 4.7% and 86.3 ± 3.4%, respectively). In conclusion, a multiparametric thermoresistance test proved that Tris-based extenders used for collared peccary semen can be effectively supplemented by streptomycin-penicillin (2 mg/ml-2000 IU/ml) or gentamicin (70 µg/ml), especially during 180-min incubation at 37 °C.

Evaluation of Ethanolic Extract of Prosopis Farcta and Trehalose on Cryopreserved Goat Epididymal Spermatozoa.

The purpose of this study, carried out in two experiments, was to investigate the antioxidant effect of Prosopis farcta in Experiment 1, and trehalose in Experiment 2 in the freezing extender, on the quality of frozen-thawed goat epididymal spermatozoa. Sperm samples were added to based egg-yolk Tris-extender containing experimental treatments. The first experimental treatments included the following: an extender of the control group without additive and extender containing 50, 100, or 150 µg/mL of Prosopis farcta ethanolic extract (PEE1, PEE2, and PEE3, respectively). Treatments of the second experiment include an extender of the control group without additive, an extender containing 100 mM of trehalose (Tr), an extender containing 100 µg/mL PEE2, and 100 µg/mL PEE2 + 100 mM Tr. The results of the first experiment showed that PEE2 compared with the control group led to a significant decrease (p < 0.05) in malondialdehyde (MDA) concentration. Also, PEE1 and PEE2 treatments resulted in a significant increase (p < 0.05) in motility parameters by computer-assisted sperm analysis, and MDA concentrations decreased significantly (p < 0.05) in all treatments compared with the control group. In general, the results of the present experiment showed that Prosopis farcta ethanolic extract at the level of 100 µg/mL was effective in improving the quality of frozen-thawed goat epididymal spermatozoa. Also, a combination of Prosopis farcta ethanolic extract and trehalose can be successful in freezing goat epididymal spermatozoa.

Effect of Antimicrobial Peptide BiF2_5K7K on Contaminated Bacteria Isolated from Boar Semen and Semen Qualities during Preservation and Subsequent Fertility Test on Pig Farm.

The purpose of this study was to determine the impact of an antimicrobial peptide, BiF2_5K7K, on semen quality and bacterial contamination in boar semen doses used for artificial insemination. A key factor affecting semen quality and farm production is bacterial contamination in semen doses. Using antibiotics in a semen extender seems to be the best solution for minimizing bacterial growth during semen preservation. However, concern regarding antibiotic-resistant microorganisms has grown globally. As a result, antimicrobial peptides have emerged as interesting alternative antimicrobial agents to replace the current antibiotics used in semen extenders. BiF2_5K7K is an antimicrobial peptide that can inhibit Gram-negative and Gram-positive bacteria isolated from boar semen and sow vaginal discharge. In this study, ten fresh boar semen samples were collected and diluted with one of two types of semen extender: with (positive control) or without (negative control) an antibiotic (i.e., gentamicin). The semen extender without an antibiotic contained antimicrobial peptide BiF2_5K7K at different concentrations (15.625, 31.25, 62.5, and 125 µg/mL). The samples were stored at 18 °C until use. Semen quality parameters were assessed on days 0, 1, 3, and 5, and the total bacterial count was also evaluated at 0, 24, 36, 48, and 72 h after storage. A fertility test on a pig farm was also performed via sow insemination with a commercial extender plus BiF2_5K7K at a concentration of 31.25 µg/mL. No significant difference was found in terms of semen quality on days 0 or 1. On days 3 and 5, the total motility, progressive motility, and viability remained normal in the 15.625 and 31.25 µg/mL groups. However, the sperm parameters decreased starting on day 3 for the 125 µg/mL group and on day 5 for the 62.5 µg/mL group. For total bacterial count at 0, 24, 36, 48, and 72 h, the lowest bacterial count was found in the positive control group, and the highest bacterial count was found in the negative control group compared with the other groups. Comparing antimicrobial peptide groups from 0 to 48 h, the lowest bacterial count was found in the 125 µg/mL group, and the highest bacterial count was found in the 15.625 µg/mL group. For the fertility test, artificial insemination was conducted by using a commercial extender plus BiF2_5K7K at a concentration of 31.25 µg/mL. The results showed a superior pregnancy rate, farrowing rate, and total number of piglets born compared with artificial insemination conducted using a commercial extender plus antibiotic. In conclusion, BiF2_5K7K can inhibit bacterial growth in extended boar semen for 24 h, and thereafter, the bacterial count slightly increases. However, the increase in the number of bacterial counts from days 0 to 3 had no negative effect on sperm quality in the positive control, 15.625, or 31.25 µg/mL groups. This indicates that BiF2_5K7K might be an antimicrobial peptide candidate with potential for use as an alternative antimicrobial agent to replace the conventional antibiotic used in boar semen extenders.

Effect of novel lyophilized extenders on stallion post-thaw sperm motility.

The aim of this study was to assess the effect of lyophilized freezing extenders, which can be stored at room temperature, on stallion post-thaw sperm total motility (TM). Ejaculates of 28 stallions were frozen with four different extenders: two commercial freezing extenders offered worldwide and two novel lyophilized extenders (STAR and MX3), and two different cryopreservation protocols (CP1 with an equilibration period of 20 min. and CP2 with an equilibration period of 60 min.). The TM was assessed after thaw. Mean TM did not show significant differences between cryopreservation protocols within each extender. Mean TM was greater in samples diluted with STAR than in samples diluted with Botucrio (P ˂ 0.05), but no significant differences were observed for this variable between the other studied extenders. From all evaluated samples, twenty ejaculates showed the greatest TM when using the lyophilized extenders and the CP1. Thus, lyophilized extenders are a promising option for stallion sperm cryopreservation and have the advantage of storage and distribution at room temperature for at least one year.

The Effects of Supplementation of the Freezing Extender with Silymarin on the Quality Parameters of Frozen-Thawed Arabian Stallion Sperm: A Preliminary Evaluation.

This study evaluated the effects of supplementation of the freezing extender with different concentrations of silymarin on the quality of frozen-thawed Arabian stallion spermatozoa. Semen samples from three stallions (1, 2, and 3) were suspended in the freezing extender without or with silymarin (0, 25 µg/mL, 50 µg/mL, 75 µg/mL, and 100 µg/mL) and cryopreserved in 0.5 mL straws. After 1 month of storage, the frozen semen samples in straws were thawed and evaluated in terms of viability, mitochondrial membrane potential, kinematic parameters, total and progressive motility, plasma membrane integrity, lipid peroxidation, and DNA fragmentation. The findings indicated that 25-100 µg/mL of silymarin significantly improved viability and mitochondrial membrane potential while reducing the stallion sperm lipid peroxidation, DNA fragmentation, and apoptosis compared with the control group (p < 0.05). Silymarin concentrations of 75 µg/mL and 100 µg/mL significantly increased progressive motility and plasma membrane integrity (p < 0.05). Based on our findings, it can be inferred that silymarin exhibited a dose-dependent enhancement in the frozen-thawed Arabian stallion sperm quality. The most favorable outcomes were observed when 100 µg/mL silymarin was used.

Effect of autologous platelet-rich plasma on the fertility and quality of cryopreserved buffalo bull semen: a comparative study using OptiXcell® and tris egg yolk extenders.

BACKGROUND: Buffalo spermatozoa have a distinct membrane structure that makes them more vulnerable to cryopreservation, resulting in lower-quality post-thawed sperm. This decreases the success rate of artificial insemination in buffaloes. Understanding and addressing these specific vulnerabilities are essential for improving reproductive techniques in buffalo populations. The properties of cryopreserved buffalo bull semen were examined in this study regarding the impact of adding autologous platelet-rich plasma (PRP) to OptiXcell® or Tris egg yolk-based extenders. Ten buffalo bulls were used to collect semen. Each bull's ejaculate was separated into two main equal amounts, each of which was then diluted with either OptiXcell® or Tris egg yolk-based extender, supplemented with various PRP concentrations (5%, 10%, and 15%), and the control (0%), before being cryopreserved according to established protocols. Following equilibration and thawing, the quality and functionality of the sperm were evaluated, along with the antioxidant enzyme activities (GSH and TAC), malondialdehyde (MDA) content, and in vivo fertilization rate of the thawed semen. RESULTS: All PRP concentrations in both extenders, particularly 10% PRP, improved the quality and functionality of the sperm in both equilibrated and frozen-thawed semen. Additionally, the antioxidant enzyme activities in both extenders were higher in the PRP-supplemented groups compared to the control group in thawed semen (P < 0.05). All post-thaw sperm quality, antioxidant enzyme activities, and functionality aside from DNA integrity were higher (P < 0.05) in the PRP-supplemented OptiXcell® than in the PRP-supplemented Tris egg yolk-based extender. The fertility of cryopreserved semen in the extenders supplemented with 10% and 15% PRP increased (P < 0.05) significantly more than that of the control extenders, with 10% PRP being the optimum concentration in OptiXcell® (80%) compared to that of Tris egg yolk-based extender (66.67%) and control of two extenders (53.33% and 46.67%, respectively). CONCLUSIONS: Even though autologous PRP-supplemented extenders have a protective impact on equilibrated and cryopreserved semen, 10% PRP-supplemented OptiXcell® extenders are more effective at preserving post-thaw semen quality, functionality, and antioxidant capacity, which increases the in vivo fertility of buffalo bulls.

The comparison of gasoline powered vehicle and serial hybrid vehicle on emissions.

In this study, a vehicle model was developed to examine the performance of a gasoline-powered vehicle and a vehicle with a serial-hybrid-drive system. An extra-downsized gasoline hybrid engine was used as a range extender in the vehicle model. Utilizing OBD II output to collect data, engine data for a sample vehicle was established with a neural network. Vehicle models were subsequently built using Matlab Simulink to complete the study. A comparison was made between the vehicle equipped with a serial-hybrid-drive system and the one with a gasoline engine-powered system regarding their fuel consumption and CO2 emissions for NEDC (New European Drive Cycle) and WLTC (Worldwide Harmonized Light Vehicles Test Cycle) Class 2 cruise cycles. Based on two driving cycles with different speed-time profiles, the results demonstrate the significant impact that powertrains can have on a vehicle's efficiency and performance, particularly during the transition from NEDC to WLTC which takes into account higher speeds, dynamic driving cycles, and factors such as air conditioning usage. Based on the findings, there was a 3.3% rise in CO2 emissions during an NEDC driving cycle with an additional downsized serial-hybrid-drive system. However, the opposite occurred during the WLTC driving cycle, resulting in a 1.7% decline in the serial-hybrid vehicle's fuel consumption and emissions performance.

Post-cooling semen processing and sperm re-suspension as an alternative method to circumvent poor semen cooling in stallions.

BACKGROUND: Artificial insemination with cooled-shipped semen is the primary method used in the equine breeding industry; yet, sperm quality and fertility can be suboptimal for some stallions when standard techniques are used. Therefore, there is a critical need to develop alternative approaches for these stallions. OBJECTIVE: To assess sperm quality parameters and fertility of cooled-stored stallion semen processed by SpermFilter® or centrifugation and resuspended in three extenders. STUDY DESIGN: Controlled and field study. METHODS: In Experiment 1, semen was collected from 21 stallions classified as having good ('Good-coolers', n = 8) or poor ('Bad-coolers', n = 13) semen cooling. The semen was extended at 30 million spermatozoa/mL in a skimmed milk-based (SM) diluent, and refrigerated for 24 h. Then, the cooled-stored semen was processed through SpermFilter® or centrifugation, and the resulting sperm pellets were resuspended in SM, SM containing pentoxifylline (SM-P), or an egg yolk-based (EY) extender. Unprocessed cooled-stored semen served as control. Sperm motility parameters, plasma membrane integrity (PMI), and mitochondrial membrane potential (HMMP) were assessed in cooled-semen pre- and post-processing. Experiment 2, cooled semen from 9 stallions classified as Bad-coolers was used to inseminate 18 embryo donor mares at 66 cycles (Unprocessed, n = 22; SpermFilter®/SM-P, n = 16; or SpermFilter®/EY, n = 28). Data were analysed with a mixed model and Tukey's as posthoc, and logistic regression. RESULTS: Processed semen resuspended in EY had superior sperm motility compared to unprocessed, SM and SM-P (p < 0.0001). Semen processed by SpermFilter® resuspended in SM-P was similar to EY (p > 0.05). Pellet resuspension with EY and SM-P improved the HMMP of Bad-cooler stallions (p = 0.0010). Semen processed by SpermFilter® had superior PMI to centrifuged semen (p < 0.0001). Mares inseminated with SpermFilter®/SM-P (50%, 8/16) or SpermFilter®/-EY (68%, 9/28) had higher pregnancy rates than mares bred with unprocessed semen (14%, 3/22) (p < 0.001). MAIN LIMITATIONS: Low number of mares in the fertility trial. CONCLUSION: Sperm quality and fertility of Bad-cooler stallions can be enhanced by SpermFilter® and pellet resuspension with either EY or SM-P.

Zinc oxide nanoparticles preserve the quality and fertility potential of rooster sperm during the cryopreservation process.

Sperm cryopreservation is one of the main methods for preserving rooster sperm for artificial insemination (AI) in commercial flocks. Yet, rooster sperm is extremely susceptible to reactive oxygen species (ROS) produced during the freezing process. Oxidative stress could be prevented by using nanoparticles containing antioxidants. The present study was conducted to investigate the effect of zinc oxide nanoparticles (ZnONP) in rooster semen freezing extender on quality parameters and fertility potential. For this aim, semen samples were collected and diluted in Lake extenders as follows: control: Lake without ZnONP, ZnO100: Lake with 100-µg zinc oxide (ZnO), ZnONP50: Lake with 50-µg ZnONP, ZnONP100: Lake with 100-µg ZnONP and ZnONP200: Lake with 200-µg ZnONP. After freezing and thawing, sperm motility, viability, membrane integrity, morphology, mitochondrial activity, acrosome integrity, DNA fragmentation, lipid peroxidation and ROS, as well as fertility and hatchability were assessed. According to the current results, higher rates of motility, membrane integrity, mitochondrial activity, acrosome integrity and live cells were detected in the ZnO100, ZnONP50 and ZnONP100 groups compared to other groups (p ≤ .05). Yet, the percentage of dead cells, DNA fragmentation, lipid peroxidation and ROS levels were lower in the mentioned groups (p ≤ .05). Furthermore, a higher percentage of fertility was observed in the ZnO100 and ZnONP100 groups than in the control group (p ≤ .05). In conclusion, the use of 100-µg ZnO and 50- to 100-µg ZnONP represents a valuable and safe additive material that could be used to improve the quality and fertility potential of rooster sperm under cryopreservation conditions.

Exploring the Impact of the Synthesis Variables Involved in the Polyurethane Aerogels-like Materials Design.

This research presents a novel approach to synthesising polyurethane (PUR)-based aerogels at the pilot scale, optimizing synthesis variables such as the gelation solvent, solids content, chain extender/isocyanate ratio, and dispersion mode. The solids content (2-11 wt.%) is the parameter with the most influence on the density of the aerogels, with a clear decrease in this property as the solids content decreases. On the other hand, it was demonstrated that minimizing the excess of ethylenediamine (used as chain extender) in relation to the isocyanate is a valuable consideration to improve the thermal conductivity of the aerogel. Related to the chain extender/isocyanate ratio, a compromise situation where the initial isocyanate reacts almost completely is crucial. Fourier-transform infrared spectroscopy was used to conduct such monitoring during the reaction. Once the conditions were optimised, the aerogel showing improved properties was synthesised using ethyl acetate as the gelling solvent, a 3.7 wt.% solids content, an ethylenediamine/isocyanate ratio of 0.20, and sonication as the dispersion mode, attaining a thermal conductivity of 0.030 W m-1 K-1 and a density of 0.046 g cm-3. Therefore, the synthesized aerogel emerges as a promising candidate for use in the construction and automotive industries.

Effect of storage and single layer centrifugation before cryopreservation on stallion sperm cryosurvival.

The objectives of this study were to evaluate the effect of a short, cooled storage before cryopreservation on sperm progressive motility (PM) and compare the effect of different centrifugation methods on post-thaw PM of stored samples. Semen was diluted in chilling extender and aliquoted in 6 protocols: i) Standard centrifugation (SC) followed by freezing; ii) Single Layer Centrifugation (SLC) followed by freezing; iii) Storage for 8 h/5 °C before SC; iv) Storage for 8 h/5 °C before SLC; v) Storage for 8 h/15 °C before SC; and vi) Storage for 8 h/15 °C before SLC. PM was assessed before centrifugation, after centrifugation, and post-thawing. Stallions were classified as "good freezers" (GF) or "bad freezers" (BF). The PM in samples immediately frozen was greater than in the stored ones (71.98 ± 14.2, 52.91 ± 17.8, 53.93 ± 18.9 for no storage, 5 ºC storage and 15 ºC storage, respectively) (P˂ 0.0001). There was an effect of storage condition (p ˂ 0.0001), centrifugation method (p ˂ 0.0001), and freezability (P=0.0016), with an interaction between them (P= 0.0004), on PM after centrifugation. Post-thaw PM was greater in samples treated by SLC than in samples processed by SC, for all storage conditions (p ˂ 0.05). All BF stallions 'showed post-thaw PM ˂ 30 % when samples were previously stored. Storage at 5 ºC or 15º C for 8 h maintains an appropriate quality in GF stallions. Applying a sperm selection technique as SLC is suggested to improve post-thaw motility, allowing GF straws to be frozen after storage, although BF semen should be prepared by SLC immediately after collection.

Effect of Trehalose Supplementation in Egg-Yolk-Free Extender on Conventional Parameters and Gene Expression Related to Reactive Oxygen Species, Apoptosis, and Motility of Frozen Dog Spermatozoa.

The present study was conducted to evaluate the effects of trehalose supplementation in egg-yolk (EY)-free tris extender on dog spermatozoa. Pooled spermatozoa were diluted with extender 1 (EY-free tris extender supplemented with 0, 10, 15, 20, or 30 mM trehalose) and cooled (2 × 108 sperm/mL) for 1 hour at 4°C. After that, extender 2 (extender 1 containing 1 M glycerol) was added (v:v) to the diluted sperm, loaded in 0.5-mL straws (1 × 108 sperm/mL), and incubated at 4°C for 30 minutes. The sperm straws were frozen over liquid nitrogen (LN2) vapor for 20 minutes and then plunged directly into LN2. After thawing at 37°C for 25 seconds, sperm progressive motility (CASA), viability (SYBR-14/PI), apoptosis (Annexin V/PI), and reactive oxygen species (ROS; H2DCFDA/PI) were evaluated. Thereafter, the optimal concentrations of trehalose were selected, and the gene expression of BAX, BCL2, NOX5, SMOX, OGG1, and ROMO1 was evaluated after freeze-thawing. Supplementation with 20 and 30 mM trehalose significantly increased sperm progressive motility and viability compared to the control. However, trehalose had no significant effect on sperm ROS or phosphatidylserine translocation index. There were minor numerical increases and decreases in gene expression when the selected optimal concentrations of trehalose (20 and 30 mM) were compared to the control. However, there were no significant differences. We conclude that the addition of trehalose (20 and 30 mM) in EY-free extender could improve sperm motility and viability without significant effects on ROS, apoptosis, or gene expression.

Measuring the impact of scientific publications and publication extenders: examples of novel approaches.

Different stakeholders, such as authors, research institutions, and healthcare professionals (HCPs) may determine the impact of peer-reviewed publications in different ways. Commonly-used measures of research impact, such as the Journal Impact Factor or the H-index, are not designed to evaluate the impact of individual articles. They are heavily dependent on citations, and therefore only measure impact of the overall journal or researcher respectively, taking months or years to accrue. The past decade has seen the development of article-level metrics (ALMs), that measure the online attention received by an individual publication in contexts including social media platforms, news media, citation activity, and policy and patent citations. These new tools can complement traditional bibliometric data and provide a more holistic evaluation of the impact of a publication. This commentary discusses the need for ALMs, and summarizes several examples - PlumX Metrics, Altmetric, the Better Article Metrics score, the EMPIRE Index, and scite. We also discuss how metrics may be used to evaluate the value of "publication extenders" - educational microcontent such as animations, videos and plain-language summaries that are often hosted on HCP education platforms. Publication extenders adapt a publication's key data to audience needs and thereby extend a publication's reach. These new approaches have the potential to address the limitations of traditional metrics, but the diversity of new metrics requires that users have a keen understanding of which forms of impact are relevant to a specific publication and select and monitor ALMs accordingly.

Different readers have different ways of deciding how important scientific articles are. The usual methods used to measure the impact of research, like the Journal Impact Factor or the H-index, are not meant to measure this for individual articles. These methods mainly look at how many times the articles are mentioned by others, and it can take a long time to see the impact.But in the past ten years, new tools called article-level metrics (ALMs) have been created. These tools measure how much attention an article gets online, like on social media, in the news, or when other researchers talk about it. ALMs are better at explaining how important a specific article is. They can work together with the usual methods to measure impact.This paper talks about why ALMs are important and gives examples of these tools, like PlumX Metrics, Altmetric, the Better Article Metrics score, the EMPIRE Index, and scite. It also explains how these tools can help us see the value of animations, videos, or summaries in simple language. These make it easier for more people to understand and learn from the articles.These new ways of measuring impact can help us see how important articles are in a more complete way. But because there are many different ways to measure this, it's important for users to understand which methods are relevant for a specific article and keep track of them.

L-Proline: A Promising Tool for Boosting Cryotolerance and Fertilizing Ability of Cryopreserved Sperm in Animals.

Sperm cryopreservation technology significantly contributes to the safeguarding of genetic resources, particularly for endangered species, and supports the use of artificial insemination in domestic animals. Therefore, cryopreservation can negatively affect sperm health and function leading to reduce the freezing ability and fertility potential. Therefore, it is essential to prioritize the improvement of cryotolerance in cryopreserved sperm to enhance reproductive efficiency and ensure sustainability in livestock herds. The main reason for sperm dysfunction after thawing may be related to the excessive amount of oxidative stress (OS) produced during cryopreservation. Scientists have different ways for counteracting this OS including the use of plant extracts, enzymes, minerals, anti-freezing proteins, and amino acids. Recently, one such amino acid is L-proline (LP), which has multiple roles such as osmotic and OS defense, nitrogen, and carbon metabolism, as well as cell survival and signaling. LP has been found in seminal plasma and has recently been added to the freezing extender to improve the various post-thaw parameters of sperm. This improvement is related to the ability of LP to reduce the OS, sustain the plasma membrane and to act as an osmoregulatory agent. Moreover, LP can suppress cell apoptosis by modulating intracellular redox in sperm. This review addresses the ongoing research on the addition of L-proline as an osmoregulatory agent in freezing extenders to increase the cryotolerance of animal spermatozoa to freeze-thaw.

Vibration emissions affect the quality of liquid-preserved AI doses in stallions.

Artificial insemination (AI) with liquid-preserved stallion semen is a widely used reproductive technology. As the demand for AI doses of high-class stallions is transnational, they are frequently exposed to long-distance transport. Since recent studies in boars indicated that vibration emissions caused by transport negatively affected sperm quality in vitro, this study questioned whether sperm quality in stallions is similarly impaired. Furthermore, we investigated stallion and extender-related differences in the spermatozoa's resistance to transport-related quality loss. Stallion ejaculates (n = 30) were collected at a German AI center, split in half, and subsequently diluted to a final sperm concentration of 50 × 106 sperm/mL using the semen extenders EquiPlus or Gent (both Minitüb GmbH, Germany). Four 12 mL aliquots of each sample were filled in plastic syringes according to a split-sample design and exposed to vibration (Displacement index Di = 3.0 ± 0.1) at 5 °C for 0 h (control), 3 h, 6 h or 9 h. All samples were stored for four days at 5 °C after transport simulation and analyzed for total sperm motility, thermo-resistance, membrane integrity, and mitochondrial activity determined by flow cytometry as well as the pH. After calculating generalized linear mixed models for each sperm quality trait, a negative impact of the duration of transport simulation could be shown on total sperm motility (P = 0.001), thermo-resistance (P = 0.030), and the pH (P = 0.001). Simulated transport for 6 h and 9 h diminished sperm quality (P ≤ 0.01), with 9 h reducing thermo-resistance by 5 ± 2.2% points (PP) for EquiPlus and sperm motility by 2.2 ± 1.7 PP for Gent compared to the control group. In contrast, samples exposed to vibration for 3 h showed no decline in sperm quality (P > 0.05). The individual stallion influenced every semen trait (P < 0.05) and transport-related losses in sperm thermo-resistance of up to 15.9 PP were demonstrated. Furthermore, EquiPlus was superior to Gent in all semen assessments (P < 0.001). We conclude that in vitro sperm quality is impaired by vibration. As the quality loss depends on the transport time, we recommend keeping shipping time as short as possible especially for spermatozoa of stallions that are susceptible to vibration-induced sperm quality loss.

Strategies to Reduce the Use of Antibiotics in Fresh and Chilled Equine Semen.

The study assessed the impact of four equine semen processing techniques on sperm quality and microbial load immediately post-processing and after 48 h of refrigeration. The aim was to explore the potential reduction of prophylactic antibiotic usage in semen extenders. Semen from ten adult stallions was collected and processed under a strict hygiene protocol and divided into four aliquots: Simple Centrifugation with antibiotics (SC+), Simple Centrifugation (SC-), Single-Layer Colloidal Centrifugation (CC-), and Filtration (with SpermFilter®) (F-), all in extenders without antibiotics. Sperm motility, viability, and microbial load on three culture media were assessed. No significant differences were observed in the main in the sperm quality parameters among the four protocols post-processing and at 48 h (p < 0.05 or p < 0.1). Microbial loads in Columbia 5% Sheep Blood Agar and Schaedler vitamin K1 5% Sheep Blood Agar mediums were significantly higher (p < 0.10) for raw semen than for CS+, CC-, and F- post-processing. For Sabouraud Dextrose Agar medium, the microbial load was significantly higher (p < 0.10) in raw semen compared to CS+ and F-. No significant differences (p < 0.10) were found in 48 h chilled samples. Regardless of antibiotic presence, the evaluated processing methods, when combined with rigorous hygiene measures, maintained semen quality and reduced microbial load to the same extent as a traditional protocol using antibiotics.

Improving the quality of chilled semen from Thai native chicken using phosphorus and vitamin B12 supplementation in semen extender.

This study aimed to determine phosphorus and vitamin B12 supplementation effect in semen extender on the quality and fertility ability of chilled Thai native rooster semen. Eighty-four ejaculates of semen from 26 Thai native roosters (Burmese × Vietnam crossbreed) were included. Semen was collected by applying dorsal-abdominal massage once a week, pooled, diluted to 500 million sperms per dose, and divided into 6 groups. The semen samples used for control group were diluted with modified Beltsville poultry semen extender (BPSE). For the treatment groups 2 to 6: semen samples were diluted with modified BPSE and enriched with phosphorus and vitamin B12 (Octafos Octa Memorial Co., Ltd., Bangkok, Thailand) at concentrations 0.02, 0.04, 0.06, 0.08, and 0.10%. Semen fertility ability was tested in 6 replications by inseminating layer hens. Thirty-six Thai native hens were randomly assigned to 3 groups (control, 0.04, and 0.08%) of 12 hens and were inseminated with a dose of 0.2 mL on collecting day. Sperm motion characteristics (i.e., sperm motility, sperm progressive motility, and sperm kinetic parameters) were measured using a computer-assisted sperm analysis system (SCA, Proiser S.L., Valencia, Spain). Sperm viability, mitochondrial activity, acrosome integrity, plasma membrane integrity, and malondialdehyde (MDA) concentration were also evaluated. The sperm motion characteristics were the highest in the 0.04% supplementation group on all days of collection, especially the VCL and VAP (P < 0.05). The viability, mitochondrial activity, plasma membrane and acrosome integrity of spermatozoa were greater in the 0.04% supplementation group than in the control groups (P < 0.05). The 0.04% supplementation group had the lowest MDA concentration in all days of collection. The 0.04% supplementation group were higher both fertility (66.59 vs. 48.50%: P < 0.05) and hatching rates (58.80 vs. 43.18%: P < 0.05) than in the control group. In conclusion, 0.04% phosphorus and vitamin B12 concentrations supplementation in semen extender improved rooster semen quality and fertility in chilled rooster semen.