Surgical coaching applied to laparoscopic TME for continuous professional development in rectal surgery: proof of concept.

BACKGROUND: Coaching is maturing as a strategy for surgeons' continuous professional development in different types of surgery. Laparoscopic total mesorectal excision (LAP TME) is one of the recognized difficult procedures in colorectal surgery. Aim of this trial is to introduce the surgical coaching as a tool for a continuous technical education of LAP TME for cancer in a consultant surgeon carrier. METHODS: Twelve Italian colorectal surgeons were enrolled as trainees in the AIMS Academy rectal cancer surgical coaching project and attended a face-to-face 90-min surgical coaching on a pre-edited 45-min-long video of a laparoscopic proctectomy according to pre-determined guidelines. At the end of the coaching, all mentors were asked to fill a questionnaire evaluating the trainee's skills. All trainees had to fill a post-coaching questionnaire addressing the appropriateness of the coaching with respect to their actual level. RESULTS: Trainees were more confident in performing the extra-pelvic part of the surgical procedures compared to the intra-pelvic dissection. The most challenging steps according to the trainees were the seminal vesicles identification and the pelvic floor dissection. Mentors found the trainees quite confident in the approach to the vascular structures, lymphadenectomy, stapler utilization and bleeding control. The sharpness and the efficacy of the dissection, the dissection of the surgical planes and the anastomosis fashioning were reported at a lower level of confidence. The higher grade of satisfaction reported by the trainee came from the attention that the mentors demonstrated towards them, from the availability of the mentors to take into consideration the surgical issues raised and from the willingness to apply the suggestions received during their next proctectomies. CONCLUSIONS: The surgical coaching applied to LAP TME should be considered as an innovative tool for continuous professional development.

The inhibition of spermatic cystine/glutamate antiporter xCT (SLC7A11) influences the ability of cryopreserved stallion sperm to bind to heterologous zonae pellucidae.

Sperm are redox-regulated cells, and deregulation of their redox status is considered to affect male fertility and to reduce their fertilizing ability following biotechnological procedures, such as cryopreservation. Cystine (CysS), after incorporation in sperm via SLC7A11 antiporter, has been demonstrated to increase intracellular GSH content, the most important non enzymatic antioxidant. This study was aimed at investigating the role of SLC7A11 antiporter on frozen-thawed stallion sperm ability to respond to in vitro capacitating environment after post-thaw incubation with CysS and/or Sulfasalazine (SS), a specific inhibitor of SLC7A11 antiporter. Viability, motility, immunolocalization of tyrosine phosphorylated proteins and the ability to bind to heterologous zonae pellucidae were evaluated. Thawed sperm from seven stallions (2 ejaculates/stallion) was washed and resuspended in Tyrodes media; each thawed ejaculate was divided in Control (CTR) and 3 samples supplemented with: 0.5 mM Cystine (CysS), 500 µM Sulfasalazine (SS) and 0.5 mM CysS + 500 µM SS (CysS + SS). After 1 h of incubation at 37 °C, samples were washed twice, resuspended in capacitating BWW medium and incubated at 38 °C under 5% CO2. After 30 and 60 min, sperm motility, viability and tyrosine phosphorylated protein immunolocalization, used as capacitation status index, were evaluated. After 30 min of capacitation, 4 × 105 sperm were co-incubated with denuded pig oocytes in capacitation medium for 30 min for the heterologous binding assay. None of the sperm parameters studied (motility, viability and tyrosine phosphorylation) showed any difference respective to control. The number of sperm bound per oocyte (mean ± SEM) tended to increase in CysS group (44.0 ± 12.3) respect CTR (40.8 ± 10.8) while decreased in SS group (32.4 ± 7.8) (p < 0.01). Moreover, CysS + SS group showed a lower binding rate (32.0 ± 10.0) compared to CysS (p < 0.001). Our results suggest that CysS supplementation of thawed stallion sperm can influence their ability to bind to heterologous zona pellucidae as the inhibition of CysS incorporation by SLC7A11 reduced the number of sperm bound per oocyte. This effect does not seem to be ascribed to a modification of sperm motility, membrane integrity and tyrosine phosphorylation.

Finding the path of least resistance: Locally adapting the MITIGATE toolkit in emergency departments and urgent care centers.

The MITIGATE toolkit was developed to assist urgent care and emergency departments in the development of antimicrobial stewardship programs. At the University of Washington, we adopted the MITIGATE toolkit in 10 urgent care centers, 9 primary care clinics, and 1 emergency department. We encountered and overcame challenges: a complex data build, choosing feasible outcomes to measure, issues with accurate coding, and maintaining positive stewardship relationships. Herein, we discuss solutions to challenges we encountered to provide guidance for those considering using this toolkit.

Calving time identified by the automatic detection of tail movements and rumination time, and observation of cow behavioural changes.

The use of electronic devices to improve animal health, welfare and farm efficiency in precision livestock farming is a developing area of great scientific and commercial interest. In particular, the use of on-site dairy farm instruments to detect calving is a tool in reproduction livestock farming. The primary aim of this study was to validate the ability of the Moocall device (MD) to detect calving cows. In addition, behavioural changes in parturient dairy cows were evaluated using video-based observations. The MD was applied approximately 9 days before cow delivery. Observational sessions were conducted three times a day for each cow from the day before MD application to calving time. The sensitivity (Se) and specificity (Sp) at 3 and 24 h before calving were measured to test the effectiveness of the MD. In addition, behavioural changes were investigated before and after the MD application as well as before and during calving time. The 3 h Se and the 3 h Sp obtained were 95.2 and 71.4%, respectively. No false negatives were observed in the 24 h before delivery (24 h Se=100%) while the 3 h Se was 95.2%. The MD was well tolerated by the dairy cows since no change in behaviours was observed in this study among the cows with or without the MD, except for an increase in eating behaviour in the animals with the MD. As regards, the behavioural pattern during calving time (8 h before calving) in comparison with the previous phases, a significant increase in tail contraction frequency and raised tail position, and a decrease in eating behaviour and rumination time were observed. The first principal component (PC) was primarily explained by these variables, and calving cows best contributed to this PC. According to the results of the present study, the use of the MD can be a useful tool in detecting the moment of calving.

Vaccines for COVID-19: perspectives from nucleic acid vaccines to BCG as delivery vector system.

This article discusses standard and new disruptive strategies in the race to develop an anti-COVID-19 vaccine. We also included new bioinformatic data from our group mapping immunodominant epitopes and structural analysis of the spike protein. Another innovative approach reviewed here is the use of BCG vaccine as priming strategy and/or delivery system expressing SARS-CoV-2 antigens.

Antimicrobial resistance of staphylococci and streptococci isolated from dogs.

A study was conducted for the examination of bacterial species isolated in dogs from Animal Clinics of Nanjing Agricultural University, China. Forty nasal swabs were taken from dogs having respiratory signs. Staphylococcus pseudintermedius was the most frequently isolated pathogen (37.50 %) followed by Staphylococcus aureus (18.75%), Streptococcus pluranimalium (10.93%), Streptococcus canis (9.37%), Staphylococcus schleiferi (9.37%), Staphylococcus intermedius (6.25%), Staphylococcus cohnii (4.71%) and Staphylococcus hominis (3.12%). S. pseudintermedius and S. pluranimalium were subjected to commonly used antibiotics for determination of resistant drugs. Antimicrobial resistance in S. pseudintermedius was common in gentamicin (70.83%) and tetracycline (50%) while in S. pluranimalium was common in enrofloxacin (71.42%) and gentamicin (57.14%).

Examining determinants of health service utilisation among Filipino older people: A cross-sectional study.

OBJECTIVE: This study was done to study, using Andersen's Behavioral Model, factors affecting health service utilisation among older Filipinos. METHODS: A total of 237 older people from Marikina City, Philippines, were randomly selected and surveyed face-to-face on factors affecting health service utilisation, specifically age, civil status, educational level, employment status, sex, health insurance coverage, household income, chronic disease status and perceived health status. RESULTS: Seventy-nine per cent had used health services in the past year (mean: 5.7 visits per year). Female sex and having a chronic disease were significantly associated with health service utilisation. The odds of using health services was 3.1 times higher for those who had a chronic disease and 2.2 times higher for female sex, assuming all other variables are held constant. CONCLUSION: Findings of this study support targeting men in health promotion campaigns and implementing population-based, chronic disease screening programs to improve the reach of health services among Filipino older people.

Parturition in baboons (PAPIO SPP.).

The Old World non-human primates (NHP) - baboons (Papio spp.) share similarities with humans regarding fetal and placental development and some pregnancy-related complications. Information about the mechanism of birth and complications arising during parturition in these species is relatively sparse. In this manuscript, we add information from a series of pathological and observational cases to highlight insights and selected complications of birth in Papio spp, based on video-recording of the delivery process, X-ray, MRI, and ultrasound evaluations in pregnant baboons. Additionally, we abstracted pathology records obtained from perinatal loss in a large baboon colony during a 17 year period. The presented cases provide important information for the management of pregnancy and delivery in Papio spp.

Cryopreservation of donkey sperm using non-permeable cryoprotectants.

The aim of this study was to evaluate the effect of different concentrations of sucrose combined with bovine serum albumin (BSA), as non-permeable cryoprotectants, on donkey sperm parameters after cryopreservation, in comparison to a control extender containing glycerol. Semen from five Andalusian donkeys (n = 12) were centrifuged and resuspended with a commercial extender for equine sperm (Gent A, Minitube) adding 1% BSA and different concentrations (M, mol/l) of water-diluted sucrose: 0.05, 0.1, 0.25, 0.35 and 0.45. Thereafter, semen (n = 24) were diluted in the same base extender containing 0.25 M sucrose (S25) or glycerol (GLY, Gent B). Sperm were slowly cooled, filled in 0.5 ml straws and frozen in nitrogen vapours. Post-thaw samples were assessed for sperm motility, plasma membrane and DNA integrity and results were compared by ANOVA. In Experiment 1, sperm motility was significantly higher (P < 0.001) for S25 than the remaining treatments, and no differences were found for plasma membrane or DNA integrity. In Experiment 2, no differences were found between S25 or GLY for sperm motility and DNA integrity but plasma membrane integrity was significantly higher (P < 0.05) for S25. In conclusion, the extender with sucrose 0.25 M combined with BSA can be considered as an alternative to conventional extenders with glycerol for donkey sperm cryopreservation.

Exercise in vivo marks human myotubes in vitro: Training-induced increase in lipid metabolism.

BACKGROUND AND AIMS: Physical activity has preventive as well as therapeutic benefits for overweight subjects. In this study we aimed to examine effects of in vivo exercise on in vitro metabolic adaptations by studying energy metabolism in cultured myotubes isolated from biopsies taken before and after 12 weeks of extensive endurance and strength training, from healthy sedentary normal weight and overweight men. METHODS: Healthy sedentary men, aged 40-62 years, with normal weight (body mass index (BMI) < 25 kg/m2) or overweight (BMI ≥ 25 kg/m2) were included. Fatty acid and glucose metabolism were studied in myotubes using [14C]oleic acid and [14C]glucose, respectively. Gene and protein expressions, as well as DNA methylation were measured for selected genes. RESULTS: The 12-week training intervention improved endurance, strength and insulin sensitivity in vivo, and reduced the participants' body weight. Biopsy-derived cultured human myotubes after exercise showed increased total cellular oleic acid uptake (30%), oxidation (46%) and lipid accumulation (34%), as well as increased fractional glucose oxidation (14%) compared to cultures established prior to exercise. Most of these exercise-induced increases were significant in the overweight group, whereas the normal weight group showed no change in oleic acid or glucose metabolism. CONCLUSIONS: 12 weeks of combined endurance and strength training promoted increased lipid and glucose metabolism in biopsy-derived cultured human myotubes, showing that training in vivo are able to induce changes in human myotubes that are discernible in vitro.

Epigallocatechin-3-gallate (EGCG) and green tea polyphenols do not improve stallion semen parameters during cooling at 4°C.

Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24-72 hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin-3-gallate (EGCG) at 20, 60 and 120 µm and green tea polyphenols, and p at .001, .01 and .1 mg/ml) on some sperm parameters (sperm motility, viability/acrosome integrity and DNA quality) in extended semen immediately after its collection (T0) and after 2, 6, 24 and 48 hr of cool storage. Two ejaculates from three trotter stallions were analysed after 48 hr of storage at 4°C. No beneficial effect on the analysed parameters was observed: the two antioxidants were not able to improve sperm quality after 48 hr of storage. These results are in agreement with previous findings on the effect of different antioxidants reported by other researches, who have demonstrated that stallion semen keeps good antioxidant capacity after dilution for 24 hr. In conclusion, the positive effect exerted by antioxidant molecules in other species is not confirmed in the equine one.

VIABILITY, IN VITRO DIFFERENTIATION AND MOLECULAR CHARACTERIZATION OF EQUINE ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS CRYOPRESERVED IN SERUM AND SERUM-FREE MEDIUM.

BACKGROUND: The effect of freezing-thawing equine adipose tissue-derived mesenchymal stem cells (eATMSCs) have been poorly investigated. OBJECTIVE: This study is to test the influence of cryopreservation solution and temperature when adding the cryoprotectant for freezing eATMSCs, and to investigate the effects of cryopreservation on their stemness features. MATERIALS AND METHODS: Four freezing protocols were evaluated. Viability and proliferation ability of cryopreserved cells were investigated by MTT assay. Fresh and frozen thawed eATMSCs were compared for morphology, phenotypic characteristics (flow cytometry), and differentiation potential. RESULTS: A higher value of viable cells for samples frozen in FBS and a positive effect of CPA equilibration at low temperature in samples frozen in medium were observed. Morphology was similar for fresh and cryopreserved cells, such as CD expression and differentiation potential. CONCLUSION: eATMSCs can be safely stored for clinical use. FBS is superior to medium for freezing, but CPA equilibration at low temperature is beneficial when freezing in serum- free medium.

Beta-mercaptoethanol supplementation of in vitro maturation medium does not influence nuclear and cytoplasmic maturation of equine oocytes.

In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low-molecular thiol compounds can be added to culture media. Beta-mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1 mM) and high (0.7 mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recovered from slaughterhouse ovaries were used. Meiotic configuration after in vitro maturation (IVM) and early embryo production after intracytoplasmic sperm injection (ICSI) were considered as criteria for assessing nuclear and cytoplasmic maturation, respectively. A total of 1,076 oocytes were analysed over two experiments: 848 (control n = 293, BME 0.1 n = 270, BME 0.7 n = 285) were stained with Hoechst 33342 and examined for nuclear stage after 26 hr of IVM, and 228 MII oocytes were fertilized by ICSI (control n = 83, BME 0.1 n = 65, BME 0.7 n = 80). Cleavage rates were determined after 60 hr of culture. Unlike results obtained in other species, the addition of BME did not influence maturation rates (51.9% control vs 55.6% BME 0.1 mM and 55.1% BME 0.7 mM), nor cleavage rates after ICSI (38.6% vs 38.5% and 41.3%, respectively). In conclusion, the addition of BME at 0.1 and 0.7 mM to the maturation medium, in our culture conditions, has no effect on nuclear and cytoplasmic maturation of equine oocytes.

Pregnancy-driven cardiovascular maternal miR-29 plasticity in obesity.

BACKGROUND: Obesity in pregnancy (MO) is a risk factor for maternal and/or fetal cardiovascular system disorders. This study evaluated maternal CVS expression of microRNA-29 family and its target molecules in MO to test the hypotheses: CVS miR-29 concentrations are increased in pregnancy and decreased in MO. METHODS: Non-pregnant (n=4), pregnant obese (POb, n=4), and pregnant non-obese (PnOb, n=4) baboons (Papio spp.) were studied. Maternal left ventricle (LV), left atrium (LA), and aortic arch (AA) were collected at the end of gestation. Expression of MiR-29 and elastin (ELN) mRNA were quantified. RESULTS: LA miR-29 (a, c) expression was highest in PnOb. In the LV, miR-29b expression trended lower (P=.059) for PnOb animals. ELN mRNA expression correlated positively with miR-29b expression in AA (r=.76, P=.03). CONCLUSION: Maternal obesity diminishes miR-29 adaptation to pregnancy. Pharmacologic, tissue-specific targeting of miRNA-29 may represent a strategy for prevention and treatment of MO complications.

Epigallocatechin-3-Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm-Zona Pellucida Heterologous Binding.

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin-3-gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2-h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm, 500 nm and 5 µm) and EGCG (10, 20 and 60 µm), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 µm (but not of 60 µm) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm, the presence of EGCG at all the concentrations tested (10, 20 and 60 µm) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone.

Effect of cushioned or single layer semen centrifugation before sex sorting on frozen stallion semen quality.

The aim of this study was to compare the effect of presorting centrifugation (cushioned [CC] or single-layer colloid [SLC]), with simple dilution (SD), on the quality of sex-sorted stallion semen before and after sorting and after freezing and thawing. Four ejaculates from each of two fertile stallions were collected 1 week apart and evaluated for percent total sperm motility (TM), percent viable acrosome-intact sperm (VAI), and DNA quality (percentage of DNA fragmentation index). Freezing caused, independently from CC and SLC treatments, a significant decrease of TM (P < 0.05) and VAI (P < 0.05) in both unsorted and sorted semen. On the other hand, sorting did not impair TM and VAI and, interestingly, improved DNA quality in all treatments only before freezing (28 vs 13, 28 vs 10, 22 vs 7 in SD, CC, and SLC for unsorted vs sorted groups, respectively; P < 0.05); this positive effect was lost in the same samples after freezing and thawing, suggesting that the freezing process reduces the DNA quality of sex-sorted sperm. Our results suggest that CC and SLC are not able to select those spermatozoa that possess a better ability to withstand sperm processing associated with sperm sorting and freezing.

Effect of post-thaw addition of seminal plasma on motility, viability and chromatin integrity of cryopreserved donkey jack (Equus asinus) spermatozoa.

Pregnancy rates in donkeys after artificial insemination with cryopreserved semen are still low, compared to the horse species. Addition of autologous seminal plasma to frozen-thawed semen appeared to improve pregnancy rates. The aims of this study were to evaluate (1) sperm motility and plasma membrane integrity after thawing (T0) and after one and 2 h (T1 and T2) of post-thaw incubation in either 0% (SP0) or 70% (SP70) autologous seminal plasma and (2) sperm motility, plasma membrane integrity and DNA quality (%COMP-αt) after thawing (T0) and after 2 and 4 h (T2 and T4) of post-thaw incubation in either 0% (SP0), 5% (SP5) or 20% (SP20) homologous seminal plasma. In experiment 1, seminal plasma decreased total and progressive sperm motility and plasma membrane intact spermatozoa immediately after dilution and at all following time points (p < 0.05). In experiment 2, total and progressive motility did not differ between treatments immediately after dilution and between SP0 and SP5 at T2, while they were lower in both SP5 and SP20 than in SP0 at T4. Plasma membrane intact sperm cells did not differ between SP0 and SP5 and were lower in SP20 at all time points. DNA quality was not affected by treatment immediately after dilution and was significantly worse for SP20 after 4 h of incubation (p < 0.05). The post-thaw addition of seminal plasma at the tested concentrations did not improve donkey frozen semen characteristics in vitro over time.

Effects of repeated transvaginal ultrasound-guided aspirations performed in anestrous and cyclic mares on P4 and E2 plasma levels and luteal function.

The aim of the present study was to verify how repeated ovum pick-up (OPU), performed in anestrous and cyclic mares, affect ovarian activity, measured by progesterone (P4) and 17ß-estradiol (E2) plasma levels. Ovum pick-up of all visible follicles was performed every 9 to 12 days, and four sessions were carried out during anestrous (A) and breeding season (BS). The number of aspirated follicles per mare at each session was not significantly different between the two periods (BS: 6.1 ± 2.4; A: 7.5 ± 4.4; P > 0.05), but the mean follicular diameter was significantly higher during BS (16.0 ± 7.1 vs. 10.2 ± 5.1 mm; P < 0.05); during A the number of aspirated follicles less than 15 mm in diameter resulted significantly higher than that registered in BS (5.1 ± 2.7 vs. 3.0 ± 1.8; P < 0.05). The total mean value of P4 was higher in BS than in A (6.3 ± 4.4 vs. 0.3 ± 1.8 ng/mL; P < 0.05), whereas the total mean level of E2 was not different between the two periods (3.8 ± 3.4 vs. 2.5 ± 2.7 pg/mL; P > 0.05). Estradiol plasma values resulted positively correlated, in A and BS, with diameter of follicles detected on the ovaries (R = 0.345 and R = 0.331, respectively), whereas a negative correlation was observed between P4 and follicular diameter in BS (R = -0.162). Both E2 and P4 presented a high individual variability during BS; in particular, in three of seven mares, P4 trend was compatible with a normal estrous cycle, and the interval between two consecutive peaks was 21 days. In two of seven mares, with CL at first OPU, P4 concentrations remained more than 3 ng/mL throughout the entire treatment period. Finally, in two of seven animals, P4 levels initially showed a similar pattern to that of a normal estrous cycle, then, after the second aspiration, they remained consistently higher than 3 ng/mL. When the procedure was carried out in cyclic animals, the influence of this technique on ovarian activity seemed to be related to individual variability although, according to progesterone values, structures observed on the ovaries after aspirations presented luteal function. Furthermore, the resumption of normal ovarian activity, after repeated OPU sessions, occurred in a period not much longer than the duration of a normal estrous cycle (25.4 ± 5.2 days). Data recorded during nonbreeding period showed that repeated OPU in anestrous mares do not affect ovarian activity and do not anticipate the resumption of ovarian cyclicity. However, based on the number of aspirated follicles in anestrous and cyclic mares, both types of subjects could be considered as oocyte donors.