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.

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.

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.

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.

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 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.

Abruptio placentae in cynomolgus macaques (Macaca fascicularis): male bias.

BACKGROUND: Abruptio placentae is a serious problem with a high rate of maternal and fetal mortality and documented sexual dimorphism in reoccurrence. Macaca fascicularis is a well-described reproductive model; however, there are no data available regarding sexual dimorphism in abruptio placentae in these species. METHODS: A retrospective study of pathology and medical records in a large colony of M. fascicularis was performed. Placental specimens were analyzed. RESULTS: The incidence of placenta abruptio in the colony was 15.7/1000 births. In the abruptio placentae group, male fetuses had lower placental disk length and increased femur length compared with female fetuses. The feto-pacental ratio and fetal weight were lower in the male fetuses in the abruption group compared with those in the stillbirth group without abruption placentae. CONCLUSION: This is the first documentation of male bias in placental and fetal development in abruptio placentae in non-human primates.

Pregnancies following artificial insemination with spermatozoa from problem stallion ejaculates processed by single layer centrifugation with Androcoll-E.

Some stallions produce ejaculates of low quality and/or low fertility when used for artificial insemination (AI). The purpose of these five case studies was to use Single Layer Centrifugation (SLC) to select the best spermatozoa from 'problem' ejaculates for subsequent use in AI. Sperm quality, in terms of motility, morphology and chromatin integrity, was improved in the SLC-selected samples compared to the corresponding uncentrifuged samples, with the exception of one stallion thought to have ampullary stasis. In this stallion, neither the incidence of spermatozoa with detached heads nor the proportion of damaged chromatin was decreased by SLC, in contrast to previous results. Pregnancies were obtained after using SLC-selected spermatozoa from the five stallions for AI, indicating that the spermatozoa were functional after SLC. Overall, the results suggest that SLC may be useful when preparing AI doses from some 'problem' ejaculates.

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.

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.

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.

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.

Comparative immunolocalization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa.

Spermatozoa, as other eukaryotic cells, need hexoses to produce energy to maintain membrane homeostasis, to move along the female genital tract and to carry the male genome to the female gamete. GLUTs are a family of proteins that permit and improve the passive transport of hexoses inside cells. This study was aimed at investigating the presence and localization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa by both immunofluorescence and western blotting. GLUTs exhibited a peculiar distribution along the sperm cell depending on the isoforms considered, the hexose they transport and the different species. The localization of GLUTs after capacitation and acrosome reaction highlighted the possible changes in their distribution because of the different functional moment. Only in dog spermatozoa changes in GLUTs distribution were demonstrated; these changes could be related to the different metabolic needs and modifications occurring in the sperm cell.

Quality and fertilizing ability in vivo of sex-sorted stallion spermatozoa.

Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex-sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14-propidium iodide), mitochondrial function (JC-1) and acrosomal status (fluorescein isothiocyanate Pisum sativum agglutinin conjugated). A fertility trial was carried out on four mares (seven oestrous cycles) by hysteroscopic insemination, depositing 5 x 10(6) X-bearing spermatozoa. Sex sorting resulted in a significant decrease (p < 0.001) in all motility characteristics. Sperm viability and percentage of spermatozoa with functional mitochondria were not affected by the sorting process, while the percentage of reacted spermatozoa was higher (p < 0.01) for non-sorted than sorted spermatozoa. Pregnancy rate was 28.6% (2/7) after low dose hysteroscopic insemination. Only one pregnancy was carried to term with the birth of a healthy filly. In conclusion, despite the reduction in sperm motility, sex sorting did not impair stallion sperm viability and mitochondrial activity immediately post-thaw; moreover, the sexed spermatozoa retained the ability to fertilize in vivo.

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.

Administration of sulpiride or domperidone for advancing the first ovulation in deep anestrous mares.

Since results with using sulpiride and domperidone are conflicting and since both have not been tested at the same time, the aim of this study was to compare the efficacy of these substances for the induction of ovulation in deep anestrous mares in the same experimental conditions and to determine their fertility after artificial insemination (AI) at the induced estrus. Twenty-six non-pregnant, non-lactating standardbred anestrous mares were randomly assigned to three groups and treated daily for 25 days (from February 3rd to February 28th) with either sulpiride (1mg/kg of body weight im SID, n=10), or domperidone (1mg/kg po SID, n=10); 6 animals were used as control. The beginning of the transition period and the first ovulation were hastened in sulpiride (16.4+/-0.8 days) but not in domperidone (46.0+/-3.3 days) treated mares (P<0.05). The diameter of the largest follicle was affected by treatment, time and interaction of treatment-by-day (P<0.05) and significantly increased in the sulpiride group (P<0.05). Although a main effect of treatment on plasma LH concentration was not observed (P=0.06), time and interaction of treatment-by-day were statistically significant (P<0.05). The interval from the beginning of treatment to first ovulation was shorter (P<0.05) in the sulpiride group (36.9+/-2.5 days) than in the domperidone (74.7+/-3.3 days) and control (81.4+/-3.1) groups. The establishment of pregnancy was significantly (P<0.05) hastened in sulpiride (61.0+/-35.2 days) but not in domperidone (83.0+/-44.0 days) treated mares. Treated mares not pregnant after the first AI, showed normal estrous cycles with regular interovulatory intervals (P>0.05). It was concluded that sulpiride is effective in advancing the beginning of transition period and the first ovulation whereas domperidone is successful only in some mares.

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.