ProAKAP4 as Indicator of Long-Lasting Motility Marker in Post-Thaw Conditions in Stallions.

ProAKAP4, a precursor of AKAP4 (A-kinase anchor protein) found in the flagellum of mammalian and non-mammalian spermatozoa, serves as a structural protein with established correlations to motility parameters across diverse species. This study aimed to determine the proAKAP4 level evolution in thawed stallion semen over a 3 h period, examining its correlation with motility descriptors and mitochondrial membrane potential. Utilizing sixteen ejaculates from four French warmblood stallions, this study involved maintaining thawed samples at 37 °C for 3 h, conducting proAKAP4 enzyme-linked immunosorbent assays (ELISA), computer-assisted sperm analysis (CASA), and mitochondrial membrane potential by JC-1 probe and flow cytometry at 0, 1, and 3 h post-thawing. The findings indicate significant positive correlations (p ≤ 0.05) between proAKAP4 levels and sperm total or progressive motility at all time points analyzed. Spermatozoa velocity descriptors (VAP, VCL, VSL) and spermatozoa lateral head displacement (ALH) display positive correlations (p ≤ 0.05) with ProAKAP4 at the 0 h post-thawing. ProAKAP4 concentration exhibits no discernible difference between batches with or without a cryoprotectant. Notably, proAKAP4 consumption remains insignificant within the initial hour after thawing but becomes significant (p ≤ 0.05) between 1 and 3 h post-thawing. In summary, proAKAP4 demonstrates positive correlations with total and progressive motility in stallion semen for up to 3 h after thawing, albeit showing a noticeable decrease starting from the first hour post-thawing, indicating a progressive consumption as a result of spermatozoa motile activity.

Impact of Seminal Plasma Antioxidants on DNA Fragmentation and Lipid Peroxidation of Frozen-Thawed Horse Sperm.

Cryopreservation is a stressful process for sperm, as it is associated with an increased production of reactive oxygen species (ROS). Elevated ROS levels, which create an imbalance with antioxidant capacity, may result in membrane lipid peroxidation (LPO), protein damage and DNA fragmentation. This study aimed to determine whether the membrane LPO and DNA fragmentation of frozen-thawed horse sperm relies upon antioxidant activity, including enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and paraoxonase type 1 (PON1)); non-enzymatic antioxidant capacity (Trolox-equivalent antioxidant capacity (TEAC), plasma ferric reducing antioxidant capacity (FRAP) and cupric reducing antioxidant capacity (CUPRAC)); and the oxidative stress index (OSI) of their seminal plasma (SP). Based on total motility and plasma membrane integrity (SYBR14+/PI-) after thawing, ejaculates were hierarchically (p < 0.001) clustered into two groups of good- (GFEs) and poor-(PFEs) freezability ejaculates. LPO and DNA fragmentation (global DNA breaks) were higher (p < 0.05) in the PFE group than in the GFE group, with LPO and DNA fragmentation (global DNA breaks) after thawing showing a positive relationship (p < 0.05) with SP OSI levels and ROS production. In addition, sperm motility and membrane integrity after thawing were negatively (p < 0.05) correlated with the activity levels of SP antioxidants (PON1 and TEAC). The present results indicate that LPO and DNA fragmentation in frozen-thawed horse sperm vary between ejaculates. These differences could result from variations in the activity of antioxidants (PON1 and TEAC) and the balance between the oxidant and antioxidant components present in the SP.

ProAKAP4 as a motility long-lasting marker in Catalan donkey spermatozoa.

ProAKAP4 is identified within the flagellum of spermatozoa in various mammalian species, serving as a structural protein associated with motility parameters. This investigation focuses on the presence of proAKAP4 in donkey sperm, elucidating its localization, molecular characteristics, and its correlation with motility descriptors and mitochondrial membrane potential. Twelve ejaculates from Catalan donkeys were analyzed in this study. The initial steps involved proAKAP4 sequencing and detection through Western blotting and immunofluorescence. Post-thaw assessments were conducted at 0, 1, and 3 h, encompassing proAKAP4 levels, sperm motility analyzed via Computer-Assisted Sperm Analysis (CASA), and mitochondrial membrane potential determined by flow cytometry using the JC-1 stain. The findings reveal that proAKAP4 in donkeys exhibits a characteristic localization at the principal piece of the flagellum, consistent with observations in other mammals. The molecular weight of proAKAP4 is determined to be 100 kDa. Significantly, a positive correlation (p ≤ 0.05) is established between proAKAP4 concentration and both total and progressive motility. The presence of cryoprotectant is associated with a lower proAKAP4 concentration. Notably, proAKAP4 experiences a substantial decrease (p ≤ 0.05) during the initial hour post-thawing. In conclusion, proAKAP4 is identified in donkey sperm, akin to its presence in other mammals. It exhibits a positive correlation with total and progressive motility, its concentration is notably affected by the presence of cryoprotectant with significant consumption observed during the initial hour following thawing. These findings contribute to our understanding of proAKAP4 dynamics in donkey sperm, providing insights that may have implications for semen preservation and reproductive technologies in equids.

Comparison of the metabolite profile of donkey and horse seminal plasma and its relationship with sperm viability and motility.

Previous research revealed that several seminal plasma (SP) metabolites are related to sperm functionality, fertility, and preservation. While it is understood that variations between species exist, whether the SP metabolome differs between donkeys and horses has not been previously investigated. The aim of this work, therefore, was to characterize and compare donkey and horse SP metabolites using nuclear magnetic resonance (NMR) spectroscopy, and relate them to sperm viability and motility. For this purpose, ejaculates from 18 different donkeys and 18 different horses were collected and separated into two aliquots: one for harvesting the SP by centrifugation and obtaining the metabolic profile through NMR, and the other for evaluating sperm viability and motility. Based on total motility and sperm viability, samples were classified as with good (GQ) or poor (PQ) quality. The metabolomic profile of donkey and horse SP revealed the presence of 28 metabolites, which coincided in the two species. Yet, differences between horses and donkeys were observed in the concentration of 18 of these 28 metabolites, as well as between ejaculates classified as GQ or PQ and in the relationship of metabolites with sperm motility and viability. These findings suggest that sperm from donkeys and horses differ in their metabolism and energetic requirements, and that the concentration of specific SP metabolites may be related to sperm functionality. Further research should shed light on the metabolic needs of donkey and horse sperm, and evaluate how the knowledge collected from the contribution of these metabolites can help improve semen preservation in the two species.

Anterior knee pain and sit-up tests predicts patients' satisfaction and improvement in quality of life after anterior stabilized total knee replacement without patellar resurfacing.

PURPOSE: The purpose of this study was to assess patient satisfaction and identify risk factors for dissatisfaction after anterior stabilised conventional total knee arthroplasty (TKA) without patellar resurfacing, using the Goodman score. METHODS: We conducted a cross-sectional study using data from our institutional database from 1 January 2018 to 1 March 2021. Patients who underwent TKA with the Vanguard® Cruciate Retaining Anterior Stabilized Knee System (Zimmer Biomet, Warsaw, Indiana, USA) without patellar replacement were included. Patients with other bearing surfaces (posterior stabilised or medial congruent) or diagnosed with infection or instability were excluded. Patients' reported outcomes, body mass index (BMI), passive range of motion, the timed up-and-go test, sit-up test, and algometry were assessed. Patients were also asked if they had anterior knee pain. Satisfaction was assessed using the Goodman scale, and logistic multivariate regression was used to identify variables associated with dissatisfaction and perceived improvement in quality of life. RESULTS: A total of 131 TKA patients were included in the study. The median satisfaction score was 100 (interquartile range [IQR], 87.5 to 100), with the 75-point threshold at the 90th percentile according to Section A of Goodman. Section B of Goodman showed that 113 TKA patients (86.26%) reported "great improvement" or "more than I ever dreamed." Multivariate logistic regression revealed that anterior knee pain (OR 5.16, 95% CI 1.24 to 21.39), the sit-up test (OR 0.63, 95% CI 0.49 to 0.81), and BMI (OR 0.84, 95% CI 0.70 to 0.99) were significantly associated with patient dissatisfaction and a worse perceived improvement in quality of life. The receiver operating characteristics curve for the models had areas under the curve of 0.83 (95% CI 0.69 to 0.97) and 0.82 (95% CI 0.70 to 0.94), respectively. CONCLUSION: Anterior stabilised TKA without patellar resurfacing can achieve 90% satisfaction and 86% improvement in quality of life. To improve these results, it is essential to prevent and treat anterior knee pain and enhance quadriceps strength. LEVEL OF EVIDENCE: Level III (retrospective cohort study).

Gonyautoxins 2/3 Local Periarticular Injection for Pain Management after Total Knee Arthroplasty: A Double-Blind, Randomized Study.

The purpose of this study was to compare the efficacy of periarticular infiltration of gonyautoxin 2/3 (GTX 2/3) and a mixture of levobupivacaine, ketorolac, and epinephrine for pain management after total knee arthroplasty (TKA). Forty-eight patients were randomly allocated to receive periarticular infiltration of 40 µg GTX 2/3 (n = 24) diluted in 30 mL of sodium chloride 0.9% (study group) or a combination of 300 mg of levobupivacaine, 1 mg of epinephrine, and 60 mg ketorolac (n = 24) diluted in 150 mL of sodium chloride 0.9% (control group). Intraoperative anesthetic and surgical techniques were identical for both groups. Postoperatively, all patients received patient-controlled analgesia (morphine bolus of 1 mg; lockout interval of 8 minutes), acetaminophen, and ketoprofen for 72 hours. A blinded investigator recorded morphine consumption, which was the primary outcome. Also, the range of motion (ROM) and static and dynamic pain were assessed at 6, 12, 36, and 60 hours after surgery. The incidence of adverse events, time to readiness for discharge, and length of hospital stay were also recorded. The median of total cumulative morphine consumption was 16 mg (range, 0-62 mg) in the GTX 2/3 group and 9 mg (range, 0-54 mg) in control group, which did not reach statistical difference (median test, p = 0.40). Furthermore, static and dynamic pain scores were similar at all time intervals. GTX 2/3 was inferior in range of motion at 6 and 12 hours; nevertheless, we noted no difference after 36 hours. No differences between groups were found in terms of complications, side effects, or length of hospital stay. No significant differences were found between groups in terms of breakthrough morphine requirement. However, local anesthetic use resulted in an increased ROM in the first 12 hours. This prospective randomized clinical trial shows that GTX 2/3 is a safe and efficient drug for pain control after TKA; nevertheless, more studies using GTX 2/3 with larger populations are needed to confirm the safety profile and efficiency. This is level 1 therapeutic study, randomized, double-blind clinical trial.

Time-lapse imaging and developmental competence of donkey eggs after ICSI: Effect of preovulatory follicular fluid during oocyte in vitro maturation.

Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study, we tested the hypothesis that supplementation with jenny preovulatory follicular fluid (PFF) during IVM could improve oocyte developmental competence in the donkey. For this, in vitro nuclear maturation rates, cumulus cell expansion, and embryo development after ICSI of donkey COCs matured in culture media supplemented with fetal bovine serum (FBS) or donkey PFF, with a known metabolomic profile, were assessed. Time-lapse imagining was performed after ICSI of horse and donkey oocytes. Eight OPU sessions were done in five jennies with an average recovery rate of 69.2% (n = 45 COCs). Although lower cumulus cells expansion was observed in oocytes of PFF group (P = 0.0010), no significant differences were described in nuclear maturation rates and preimplantation embryo development between groups. Donkey ICSI embryos showed similar morphokinetics to horse ICSI embryos. Our study shows that supplementing IVM media with FBS or donkey PFF supports nuclear maturation and early preimplantation embryo development after ICSI in donkeys. To our knowledge, the present study is the first report of ICSI, time-lapse imaging and in vitro blastocyst production in donkey.

ProAKAP4 Concentration Is Related to Sperm Motility and Motile Sperm Subpopulations in Frozen-Thawed Horse Semen.

ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyzed. Spermatic motility descriptors were obtained by the CASA system, and four motile subpopulations (SP) with specific motility patterns were statistically identified. ProAKAP4 concentrations were evaluated by ELISA. The relationship between motility descriptors of sperm subpopulations and proAKAP4 concentrations was evaluated. Following a hierarchical cluster statistical analysis, ejaculates were divided into two groups according to their proAKAP4 concentrations, either having low proAKAP4 concentrations (5.06−35.61 ng/10M spz; n = 23) or high (39.92−82.23 ng/10M spz; n = 25) proAKAP4 concentrations (p < 0.001). ProAKAP4 concentrations were positively correlated (p < 0.05) with total and progressive motility, as well as with parameters of velocity. ProAKAP4 amount also showed a negative correlation (p < 0.05) with sperm motile subpopulation number 3, which was the subpopulation with the lowest velocity parameters. In conclusion, proAKAP4 concentration in stallion semen positively reflects sperm progressive motility with the functional velocity kinematic descriptors. Concentrations of proAKAP4 higher than 37.77 ng/10M spz were correlated with a very good quality frozen/thawed stallion semen.

Metabolic profiling of preovulatory follicular fluid in jennies.

Follicular fluid is formed from the transudation of theca and granulosa cells in the growing follicular antrum. Its main function is to provide an optimal intrafollicular microenvironment to modulate oocyte maturation. The aim of this study was to determine the metabolomic profile of preovulatory follicular fluid (PFF) in jennies. For this purpose, PFF was collected from 10 follicles of five jennies in heat. Then, PFF samples were analysed by nuclear magnetic resonance (NMR) and heteronuclear single quantum correlation (2D 1H/13C HSQC). Our study revealed the presence of at least 27 metabolites in the PFF of jennies (including common amino acids, carboxylic acids, amino acid derivatives, alcohols, saccharides, fatty acids, and lactams): 3-hydroxybutyrate, acetate, alanine, betaine, citrate, creatine, creatine phosphate, creatinine, ethanol, formate, glucose, glutamine, glycerol, glycine, hippurate, isoleucine, lactate, leucine, lysine, methanol, phenylalanine, proline, pyruvate, threonine, tyrosine, valine, and τ-methylhistidine. The metabolites found here have an important role in the oocyte development and maturation, since the PFF surrounds the follicle and provides it with the needed nutrients. Our results indicate a unique metabolic profile of the jennies PFF, as it differs from those previously observed in the PFF of the mare, a phylogenetically close species that is taken as a reference for establishing reproductive biotechnology techniques in donkeys. The metabolites found here also differ from those described in the TCM-199 medium enriched with fetal bovine serum (FBS), which is the most used medium for in vitro oocyte maturation in equids. These differences would suggest that the established conditions for in vitro maturation used so far may not be suitable for donkeys. By providing the metabolic composition of jenny PFF, this study could help understand the physiology of oocyte maturation as a first step to establish in vitro reproductive techniques in this species.

Seminal Plasma Antioxidants Are Related to Sperm Cryotolerance in the Horse.

The objective of this study was to determine the relationship of enzymatic (superoxide dismutase, SOD; glutathione peroxidase, GPX; catalase, CAT; and paraoxonase type 1, PON1) and non-enzymatic antioxidants (measured in terms of: Trolox equivalent antioxidant capacity, TEAC; cupric-reducing antioxidant capacity, CUPRAC; and ferric-reducing ability of plasma, FRAP), as well as the oxidative stress index (OSI) in seminal plasma (SP) with the resilience of horse sperm to freeze-thawing. Twenty-one ejaculates (one per individual) were collected and split into two aliquots: the first was used to harvest the SP and assess the activity levels of antioxidants and the OSI, and the second one was cryopreserved. The following post-thaw sperm quality parameters were evaluated: sperm motility, plasma membrane and acrosome integrity, mitochondrial membrane potential, intracellular levels of reactive oxygen species (ROS), and plasma membrane lipid disorder. Based on post-thaw total motility (TM) and plasma membrane integrity (SYBR14+/PI−), ejaculates were hierarchically (p < 0.001) clustered into two groups of good (GFE) and poor (PFE) freezability. The SP activity levels of PON1, SOD, and TEAC were higher (p < 0.05) in GFE than in PFE, showing a positive relationship (p < 0.05) with some sperm motility parameters and with plasma membrane (PON1 and TEAC) and acrosome (SOD and TEAC) integrity. In contrast, OSI was higher (p < 0.05) in the SP of PFE than in that of GFE, and was negatively correlated (p < 0.05) to some sperm motility parameters and to plasma membrane and acrosome integrity, and positively (p < 0.05) to the percentage of viable sperm with high plasma membrane lipid disorder. In conclusion, enzymatic (PON1 and SOD) and non-enzymatic (TEAC) antioxidants of SP are related to horse sperm cryotolerance. In addition, our results suggest that PON1 could be one of the main antioxidant enzymes involved in the control of ROS in this species. Further investigation is needed to confirm the potential use of these SP-antioxidants and OSI to predict sperm cryotolerance in horses.

ProAKAP4 Semen Concentrations as a Valuable Marker Protein of Post-Thawed Semen Quality and Bull Fertility: A Retrospective Study.

Functional sperm quality markers to predict bull fertility have been actively investigated. Among them, proAKAP4, which is the precursor of AKAP4, the main structural protein in the fibrous sheath of spermatozoa; appears to be promising, especially since spermatozoa lacking AKAP4 expression were shown to be immotile, abnormal, and infertile. In this study, the objective was to evaluate proAKAP4 concentration values with the classic sperm motility descriptors and fertility outcomes (NRR at 90 days) in post-thawed conditions of 10 bulls' semen. ProAKAP4 expression was confirmed by Western blotting and proAKAP4 concentrations were determined by ELISA. Variations in proAKAP4 concentrations were observed independently of the motility sperm descriptors measured using computer-assisted semen analysis (CASA). A ProAKAP4 concentration of 38.67 ± 8.55 ng/10 million spermatozoa was obtained as a statistical mean of all samples. Threshold values of proAKAP4 were then determined between 19.96 to 96.95 ng/10 million spermatozoa. ProAKAP4 concentrations were positively correlated with progressive motility and the linearity coefficient. The sperm showing the lowest progressive motility were the samples exhibiting proAKAP4 concentrations below 20 ng/10 million spermatozoa. Furthermore, proAKAP4 concentrations were significantly higher in bulls with a higher NRR in the field. Our results demonstrate a correlation between the semen concentration of proAKAP4 and NRR-90d (p = 0.05) in post-thawed bull semen, highlighting the potential of proAKAP4 as a predictive marker of bull fertility.

Cohorte histórica de artroplastia unicompartimental de rodilla en un hospital universitario chileno / Historical cohort of unicompartmental knee arthroplasty in a Chilean university hospital

OBJETIVO Describir el resultado funcional informado por el paciente de una cohorte de pacientes sometidos a artroplastia unicompartimental de rodilla (AUR) en un hospital universitario chileno. MÉTODOS Se diseñó un estudio de cohorte histórica. Se incluyeron todos los pacientes que se sometieron a AUR de platillo fijo entre 2003 y 2019. Un evaluador independiente se puso en contacto con los pacientes en junio de 2020. Se utilizó el índice de artritis de las universidades de Western Ontario y McMaster (WOMAC) para comparar los procedimientos de AUR (medial o lateral), la edad (mayor o menor de 70 años), y el seguimiento (más o menos de 5 años). RESULTADOS Se incluyeron 78 pacientes, en un total de 94 AURs. La mediana de edad fue de 64 años (rango: 43 a 85 años). Hubo 72 (76,6%) casos de AUR medial. Un paciente necesitó revisión para artroplastia total de rodilla (ATR). Un total de 60 pacientes (76,9%), correspondientes a 72 AURs (76,7%), fueron contactados con éxito por teléfono para el seguimiento final. La mediana del puntaje en los dominios del WOMAC fue: dolor ­ 1 (rango: 0 a 12); rigidez ­ 0 (rango: 0 a 4); y función física ­ 2 (rango: 0 a 29). La mediana del puntaje total en el WOMAC fue de 4 (rango: 0 a 44). Los pacientes sometidos a AUR lateral lograron mejores puntuaciones funcionales (p » 0,0432), y el puntaje total en el WOMAC fue similar en pacientes mayores o menores de 70 años (p » 0,3706). CONCLUSIONES La AUR es un tratamiento eficaz y reproducible para pacientes con artrosis de rodilla unicompartimental. La edad parece no afectar los resultados funcionales, y la AUR es un tratamiento eficaz en pacientes mayores de 70 años. Estos resultados deberían animar a los cirujanos de rodilla a aprender esta técnica y a los responsables de las políticas de salud pública a considerar la AUR para la osteoartritis de rodilla.

PURPOSE To describe the patient-reported functional outcome of a cohort of patients undergoing unicompartmental knee arthroplasty (UKA) in a Chilean university hospital. METHODS A historical cohort study was designed. All patients who underwent fixedbearing UKA between 2003 and 2019 were included. An independent evaluator contacted the patients in June 2020. The Western Ontario and McMaster Universities Arthritis Index (WOMAC) was used to compare UKA procedures (medial or lateral), age (over or under 70 years), and follow up (longer or shorter than 5 years). RESULTS A total of 78 patients, corresponding to 94 UKAs, were included. The median age was 64 years (range: 43 to 85 years). There were 72 (76.6%) cases of medial UKA. One patient needed revision to total knee arthoplasty (TKA). A total of 60 patients (76.9%), corresponding to 72 UKAs, were successfully contacted by phone for the final follow-up. The median scores on the WOMAC domains were: pain ­ 1 (range: 0 to 12); stiffness ­0 (range: 0 to 4); and physical function ­ 2 (range: 0 to 29). The median total score on the WOMAC was 4 (range: 0 to 44). Patients submitted to lateral UKA had better functional scores (p » 0.0432), and the total WOMAC score was similar among patients older or younger than 70 years of age (p » 0.3706). CONCLUSIONS For patients with unicompartmental knee osteoarthritis, UKA is an effective and reproducible treatment. Age does not seem to affect the functional results, and UKA is an effective treatment in patients over 70 years old. These results should encourage knee surgeons to learn this technique and those responsible for public health policies to consider UKA for knee osteoarthritis

Impact of Seminal Plasma Antioxidants on Donkey Sperm Cryotolerance.

This study investigated whether the activities of the antioxidant components of donkey seminal plasma (SP)-both enzymatic (superoxide dismutase (SOD), catalase-like (CAT), glutathione peroxidase-like (GPX), and paraoxonase type 1 (PON1)) and non-enzymatic (measured in terms of total thiol, copper-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP), and Trolox equivalent antioxidant capacity (TEAC))-and oxidative stress index (OSI) are related to sperm cryotolerance. For this purpose, 15 ejaculates from jackasses (one per individual) were collected and split into two aliquots. The first one was used for measuring the activities levels of enzymatic and non-enzymatic antioxidants and OSI in SP, whereas the other aliquot was cryopreserved. Before cryopreservation, sperm quality parameters (concentration, motility, and viability) were evaluated. After thawing, sperm motility, plasma membrane integrity, lipid disorder, mitochondrial membrane potential, reactive oxygen species (ROS), and calcium intracellular levels were also determined. Based on the percentages of total motility (TM) and of sperm with an intact plasma membrane (SYBR14+/PI-) after thawing, samples were classified as good-freezability (GFE) or poor-freezability (PFE) ejaculates through cluster analyses. The SP activity levels of enzymatic (SOD and PON1) and non-enzymatic antioxidants (CUPRAC, FRAP, and TEAC) were higher (p < 0.05) in GFE than in PFE, whereas SP-OSI was higher (p < 0.05) in PFE than in GFE. In addition, the activity levels of SOD, PON1, GPX, CUPRAC, FRAP, and TEAC were positively (p < 0.05) related to post-thaw sperm motility and plasma membrane integrity and negatively to intracellular ROS levels. The SP-OSI was negatively correlated (p < 0.05) to post-thaw sperm quality parameters and positively to intracellular ROS levels. It can thus be concluded that donkey SP antioxidants are related to sperm cryotolerance and that measurements of antioxidants PON1, SOD, CUPRAC, FRAP, and TEAC, as well as SP-OSI, could be used as markers of sperm cryotolerance. Further research addressing the relationship of these antioxidants and SP-OSI with sperm cryotolerance and their potential use as freezing markers is warranted.

Advances in sperm cryopreservation in farm animals: Cattle, horse, pig and sheep.

Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. In some farm animals, the use of cryopreserved sperm has so many benefits for which relevance has become more evident in recent decades. Values for post-thaw sperm quality, however, are variable among species and within individuals of the same species. There is no standardized methodology for each of the stages of the cryopreservation procedure (andrological examination, semen collection, dilution, centrifugation, resuspension of the pellet with the freezing medium, packaging, freezing and post-thaw sperm evaluation), which also contributes to differences among studies. Cryotolerance markers of sperm and seminal plasma (SP) have been evaluated for prediction of ejaculate freezability. In addition, in previous research, there has been a focus on supplementing cryopreservation media with different substances, such as enzymatic and non-enzymatic antioxidants. In most studies, inclusion of these substances have led to improved post-thaw sperm quality and fertilizing capacity as a result of minimizing the adverse effects on sperm structure and function. Another approach is the use of different cryoprotectants. The aim with this review article is to provide an update on sperm cryopreservation in farm animals. The main detrimental effects of cryopreservation are described, including the negative repercussion on reproductive performance. Furthermore, the potential use of molecular biomarkers to predict sperm cryotolerance is discussed, as well as the addition of substances that can mitigate the harmful impact of freezing and thawing on sperm.

Seminal plasma, and not sperm, induces time and concentration-dependent neutrophil extracellular trap release in donkeys.

BACKGROUND: In several mammalian species, acute endometritis driven by the recruitment of polymorphonuclear cells (PMN) occurs in response to semen. These PMNs release DNA to form neutrophil extracellular traps (NETs) in cattle, horse and human, leading to sperm entrapment. While there is no evidence of this phenomenon occurring in donkeys, artificial insemination (AI) with frozen-thawed semen, which results in very poor pregnancy rates, leads to a large PMN recruitment to the uterus. OBJECTIVES: To investigate whether donkey semen can trigger NET release (NETosis) and if excessive NETosis occurs in response to frozen-thawed semen. STUDY DESIGN: In vitro experiments. METHODS: Jenny PMNs were exposed to jackass fresh or frozen-thawed semen, isolated sperm or seminal plasma (SP), over the course of three experiments. NET formation in response to different treatments was assessed through manual quantification of stained slides. A one-way analysis of variance (ANOVA), followed by a post hoc Sidak test, was carried out to determine statistical significance. RESULTS: NET release occurred in a semen concentration- and incubation-time-dependent manner. Surprisingly, frozen-thawed donkey sperm did not increase NETosis rate in comparison with the control (23 ± 2.5% vs. 31 ± 3.7%; P > .05), whereas fresh semen exposure did (78 ± 5.7% vs. 26 ± 3.2%, P < .01). NETosis increased in the presence of SP, regardless of the presence or absence of sperm, in comparison with the control in both fresh (84 ± 5.2% and 77 ± 5.0% vs. 12 ± 2.7%, respectively; P < .01) and frozen (95 ± 2.2% and 94 ± 2.9% vs. 14 ± 3.8%, respectively; P < .01) samples. Moreover, exposure of PMN to viable and motile sperm, in the absence of SP, did not increase NETosis rates (P > .05). CONCLUSIONS: Donkey SP, and not sperm-intrinsic factors, is able to trigger NETosis in both time- and semen concentration-dependent manner. The physiological relevance of such response against semen in the donkey remains to be elucidated.

Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm.

In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and motile sperm subpopulations were significantly (p < 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O2-) were not affected (p > 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM or 10 mM GSH reduced (p < 0.05) the percentages of viable sperm with high overall ROS levels compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity suggests that ROS could be important during post-AI and that the impact of using exogenous antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species.

Simple Tube Centrifugation Method for Platelet-Rich Plasma (PRP) Preparation in Catalonian Donkeys as a Treatment of Endometritis-Endometrosis.

The aim of this study was to standardize a simple, manual platelet-rich plasma (PRP) protocol in Catalonian donkeys using single-spin tube centrifugation as a treatment for jenny endometritis. The objective was to obtain a blood product with a moderate concentration of platelets (2 or 3 times baseline physiologic values) and a low WBC (White Blood Cells) concentration. Blood was drawn from six Catalonian donkeys using acid citrate dextrose (ACD) as an anticoagulant, and then processed by single centrifugation at 133× g for two different centrifugation times (10 and 15 min). The PRP samples were evaluated by flow cytometry, and TGF-ß1 (Transforming Growth Factor-Beta1) concentrations were determined by enzyme-linked immunosorbent assay (ELISA). The 10 min centrifugation protocol resulted in a slightly greater release of TGF-ß1 (6044.79 ng/mL), a 2.06-fold increase in platelet concentration, and a 15-fold reduction in leukocyte concentration when compared to the initial values. The 15 min centrifugation time resulted in a 2.44-fold increase in baseline platelet concentration, a reduction in WBC count by a factor of 20, and slightly lower TGF levels (5206 ng/mL). We conclude that both protocols are adequate for the obtention of PRP, and both may have an acceptable therapeutic potential for use in this species, although this needs to be further validated.

Extracellular Reactive Oxygen Species (ROS) Production in Fresh Donkey Sperm Exposed to Reductive Stress, Oxidative Stress and NETosis.

Jenny shows a large endometrial reaction after semen influx to the uterus with a large amount of polymorphonuclear neutrophils (PMN) migrating into the uterine lumen. PMN act as a sperm selection mechanism through phagocytosis and NETosis (DNA extrudes and, together with proteins, trap spermatozoa). While a reduced percentage of spermatozoa are phagocytosed by PMN, most are found to be attached to neutrophil extracellular traps (NETs). This selection process together with sperm metabolism produces a large amount of reactive oxygen species (ROS) that influence the reproductive success. The present study aimed to determine the extracellular ROS production in both sperm and PMN. With this purpose, (1) donkey sperm were exposed to reductive and oxidative stresses, through adding different concentrations of reduced glutathione (GSH) and hydrogen peroxide (H2O2), respectively; and (2) PMN were subjected to NETosis in the presence of the whole semen, sperm, seminal plasma (SP) or other activators such as formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular ROS production (measured as H2O2 levels) was determined with the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. Donkey sperm showed more resilience to oxidative stress than to the reductive one, and GSH treatments led to greater H2O2 extracellular production. Moreover, not only did SP appear to be the main inducer of NETosis in PMN, but it was also able to maintain the extracellular H2O2 levels produced by sperm and NETosis.

The TUNEL assay underestimates the incidence of DNA damage in pig sperm due to chromatin condensation.

Inconsistencies in the relationship between sperm DNA fragmentation and reproductive outcomes as well as the low incidence in farm animals raise concerns on its actual value as a sperm quality parameter. Previous studies suggested that the different sensitivity of techniques evaluating DNA fragmentation could explain variations in the correlation with reproductive outcomes. While the TUNEL assay is one of the most standardized methods to detect DNA damage and cell death, the steric impediment for the terminal nucleotidyl transferase enzyme to access the highly condensed sperm nucleus may decrease the ability of this test to detect internal DNA breaks. In the present study, we sought to determine whether increasing chromatin decondensation makes the TUNEL assay more sensitive to detect DNA damage in pig sperm. We compared three chromatin decondensation treatments (2 mM DTT for 45 min; 5 mM DTT for 8 min and further 45 min; and 5 mM DTT+ 1 M NaCl for 8 min) through the Chromomycin A3 test (CMA3). While incubation with DTT increased the percentages of sperm with decondensed chromatin, regardless of concentration and time of incubation (P < 0.05), the extent of that decondensation was higher when 5 mM DTT was combined with 1 M NaCl. In addition, the TUNEL assay detected a higher number of DNA breaks in sperm with decondensed chromatin (1.89% ± 1.63% vs 8.74% ± 6.05%; P = 0.003). This study shows, for the first time, that previous chromatin decondensation increases the sensitivity of the TUNEL assay to detect DNA damage in pig sperm. These findings also support that larger chromatin decondensation is needed in order for DNA damage to be evaluated properly in species containing protamine P1 only.

Evolución epidemiológica de la artroplastia de cadera y rodilla durante 17 años en pacientes con artrosis severa en un hospital Chileno / Evolution of hip and knee arthroplasty epidemiology in a single hospital of Chile: a 17 years overview

INTRODUCCIÓN: La artroplastia total de cadera (ATC), es la cirugía del siglo XX por el impacto en la calidad de vida. Por su parte, la artroplastia de rodilla (AR), ha demostrado ser un procedimiento costo-efectivo en gonartrosis. OBJETIVO: Describir la evolución de ATC y AR, en cuanto a número de procedimientos, edad y género en pacientes operados en un hospital general. MÉTODO: Estudio transversal analítico. Fueron incluidos todos los procedimientos realizados en un único centro, entre enero de 2002 y diciembre 2018, que estuvieran registrados por los códigos de prestación Fonasa 2104129 y 2104153. Fueron excluidos todos los procedimientos de revisión de artroplastia, tumores y fracturas. Se realizó un análisis de correlación de Spearman, se estableció una significancia de 0,05. RESULTADOS: Fueron incluidos 3270 procedimientos, 1975 corresponden a ATC (60,4%) y 1295 AR (39,6%). El número de ATC ha ido aumentando, en 2002 fueron realizados 122, mientras que en 2018, fueron 164. Por su parte, las AR también aumentaron, en 2002 fueron realizadas 40, mientras que en 2018 fueron 155. La proporción ATC/AR ha disminuido significativamente. La edad promedio ha disminuido en AR y aumentado en ATC, sin significación estadística. En ATC, se observa un incremento en la proporción de hombres operados. CONCLUSIONES: El perfil epidemiológico de los pacientes operados de artroplastia ha cambiado significativamente. La epidemiología mundial y la de este estudio muestran un significativo incremento de pacientes que requieren AR, por lo cual, parece importante incluir ese procedimiento en las necesidades sanitarias a satisfacer, en el diseño de nuevas políticas públicas.

OBJECTIVE: To describe the evolution of hip and knee arthroplasty in a general hospital, in terms of number of procedures, average age and gender. METHOD: Analytical cross-sectional study. All procedures performed between January 2002 and December 2018 in a single center were included. All procedures performed for revision surgery, tumors or fracture were excluded. The following data were obtained from the registry: age at the time of surgery, gender of the patient and year of the procedure. A Spearman correlation analysis was performed, a significance of 0.05 was established. RESULTS: 3270 procedures were included, 1975 correspond to hip arthroplasty (60.4%) and 1295 to knee arthroplasty (39.6%). The number of hip arthroplasty has increased over time, in 2002 a total of 122 procedures were performed and in 2018 there were 164. On the other hand, knee arthroplasty procedures have also increased, from 40 in 2002 to 155 in 2018. The ratio between hip and knee arthroplasty has been decreasing, this finding being significant. The average age has been decreasing in knee arthroplasty, meanwhile, decreasing in hip arthroplasty during the studied period. On the other hand, the proportion of men that underwent hip arthroplasty has been increasing over time. CONCLUSIONS: The number of knee arthroplasty has increased significantly, decreasing its difference with respect to the number of hip arthroplasty. An explosive increase in knee arthroplasty has been described in the literature, consistent with the outcomes of this study, so it seems important to include this procedure the new health public policies.