Specific microRNAs in stallion spermatozoa are potential biomarkers of high functionality.

Males of some species, from horses to humans, require medical help for subfertility problems. There is an urgent need for novel molecular assays that reflect spermatozoal function. In the last 25 years, studies examined RNAs in spermatozoa as a window into gene expression during their development and, more recently, for their functions in early embryo development. In clinics, more dense spermatozoa are isolated by density gradient centrifugation before use in artificial insemination to increase pregnancy rates. The objectives of the current study were to discover and quantify the microRNAs in stallion spermatozoa and identify those with differential expression levels in more dense versus less dense spermatozoa. First, spermatozoa from seven stallions were separated into more dense and less dense populations by density gradient centrifugation. Next, small RNAs were sequenced from each of the 14 RNA samples. We identified 287 different mature microRNAs within the 11,824,720 total mature miRNA reads from stallion spermatozoa. The most prevalent was miR-10a/b-5p. The less dense spermatozoa had fewer mature microRNAs and more microRNA precursor sequences than more dense spermatozoa, perhaps indicating that less dense spermatozoa are less mature. Two of the most prevalent microRNAs in more dense stallion spermatozoa were predicted to target mRNAs that encode proteins that accelerate mRNA decay. Nine microRNAs were more highly expressed in more dense spermatozoa. Three of those microRNAs were predicted to target mRNAs that encode proteins involved in protein decay. Both mRNA and protein decay are very active in late spermiogenesis but not in mature spermatozoa. The identified microRNAs may be part of the mechanism to shut down those processes. The microRNAs with greater expression in more dense spermatozoa may be useful biomarkers for spermatozoa with greater functional capabilities.

Proteomic analysis of sperm from fertile stallions and subfertile stallions due to impaired acrosomal exocytosis.

Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mainly overrepresented in the "metabolism" and the "metabolism of lipids" pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays revealed that sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of differential abundance proteins, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction.

Factors affecting the analysis and interpretation of sperm quality in frozen/thawed stallion semen.

In the current study, we examined: 1) the agreement (bias) between fluorescence-based methods (NucleoCounter-SP100 [NC] vs. flow cytometry [FC]) for determining the viability (VIAB) of frozen/thawed stallion sperm; 2) the agreement between post-thaw sperm total motility (TMOT) and VIAB; 3) whether a difference between TMOT and VIAB [VIAB - TMOT] in frozen/thawed stallion sperm could be explained by the level of lipid peroxidation in viable sperm (VLPP); 4) the repeatability of post-thaw analysis of sperm quality; and 5) the effect of final post-thaw semen dilution (10, 30, or 50 x 106 sperm/mL) on sperm motion characteristics. Post-thaw VIAB was similar between NC and FC (P > 0.05), and the agreement between these two methods was high (bias: 1 to -3). The agreement between post-thaw TMOT and VIAB decreased as the pre-freeze percentages of morphologically normal sperm and DNA quality decreased: bias - 4 to - 25. The bias between [VIAB - TMOT] and VLPP ranged from - 5 to 7. Differences in post-thaw sperm quality (TMOT, PMOT, VIAB, and sperm concentration) were not observed when analyzing one or three straws per ejaculate (P > 0.05). There was no effect of post-thaw sperm concentration (i.e., 10 vs. 30 vs. 50 x 106 sperm/mL) on sperm motion characteristics (P > 0.05). This study reports factors other than post-thaw sperm motility that warrant further consideration when analyzing frozen/thawed stallion sperm.

Comparison of two processing techniques on the sperm quality of semen contaminated with urine.

Urospermia in stallions can occur intermittently, consistently, or as an isolated event, and may result in reduced sperm quality which is often assumed to reduce fertility. Although sperm quality declines in urospermic ejaculates, fertility has not been assessed in mares bred with urine contaminated semen. The aims of this study were to compare sperm quality after simple dilution (SD), cushioned centrifugation (CC) alone, or cushioned centrifugation combined with a 40 % silane-coated silica solution (SC) in semen contaminated with 0, 20, or 40 % (v/v) urine. Sperm quality values tended to decrease as the percent urine increased within all treatments (SD, CC, SC) after 24 h of cooled storage. However, SC treated groups had higher sperm quality compared to SD and CC when exposed to 20 or 40 % (v/v) urine. Differences in pregnancy rates among treatment groups (SD with 0 or 40 % (v/v) urine, or 40 % (v/v) urine followed by SC) were unable to be detected.

Lactate as the sole energy substrate induces spontaneous acrosome reaction in viable stallion spermatozoa.

BACKGROUND: Equine spermatozoa appear to differ from spermatozoa of other species in using oxidative phosphorylation preferentially over glycolysis. However, there is little information regarding effects of different energy sources on measured parameters in equine spermatozoa. OBJECTIVE: To determine the effect of three individual energy substrates, glucose, pyruvate, and lactate, on motion characteristics, membrane integrity, and acrosomal status of stallion spermatozoa. MATERIALS AND METHODS: Freshly ejaculated stallion spermatozoa were incubated with combinations of glucose (5 mm), pyruvate (10 mm), and lactate (10 mm) for 0.5 to 4 h. Response to calcium ionophore A23187 (5 µm) was used to evaluate capacitation status. Motility was evaluated using computer-assisted sperm analysis, and plasma membrane and acrosomal integrity were evaluated by flow cytometry. RESULTS: Incubation with lactate alone for 2 h increased acrosomal sensitivity to A23187. Notably, incubation with lactate alone for 4 h induced a significant spontaneous increase in acrosome-reacted, membrane-intact (viable) spermatozoa, to approximately 50% of the live population, whereas no increase was seen with incubation in glucose or pyruvate alone. This acrosomal effect was observed in spermatozoa incubated at physiological pH as well as under alkaline conditions (medium pH approximately 8.5). Sperm motility declined concomitantly with the increase in acrosome-reacted spermatozoa. Sperm motility was significantly higher in pyruvate-only medium than in glucose or lactate. The addition of pyruvate to lactate-containing medium increased sperm motility but reduced the proportion of live acrosome-reacted spermatozoa in a dose-dependent fashion. DISCUSSION: This is the first study to demonstrate that incubation with a specific energy substrate, lactate, is associated with spontaneous acrosome reaction in spermatozoa. The proportion of live, acrosome-reacted spermatozoa obtained is among the highest reported for equine spermatozoa. CONCLUSION: These findings highlight the delicate control of key sperm functions, and may serve as a basis to increase our understanding of stallion sperm physiology.

Effects of Bacillus subtilis PB6 supplementation on production, metabolism, inflammatory biomarkers, and gastrointestinal tract permeability in transition dairy cows.

Objectives were to evaluate the effects of Bacillus subtilis PB6 (BSP) on gastrointestinal tract permeability, metabolism, inflammation, and production parameters in periparturient Holstein cows. Multiparous cows (n = 48) were stratified by previous 305-d mature equivalent milk yield and parity and assigned to 1 of 2 top-dressed dietary treatments 21 d before expected calving through 63 DIM: (1) control (CON; 13 g/d calcium carbonate; n = 24) or (2) BSP (13 g/d BSP; CLOSTAT, Kemin Industries, Des Moines, IA; n = 24). Gastrointestinal tract permeability was evaluated in vivo using the oral paracellular marker chromium (Cr)-EDTA. Effects of treatment, time, and treatment × time were assessed using PROC MIXED of SAS version 9.4 (SAS Institute Inc.). Prepartum dry matter intake (DMI) was unaffected by treatment; however, BSP supplementation decreased postpartum DMI relative to CON (0.7 kg). Milk yield, energy-corrected milk (ECM), fat-corrected milk (FCM), and solids-corrected milk (SCM) increased in BSP cows compared with CON (1.6, 1.8, 1.6, and 1.5 kg, respectively). Decreased DMI and increased production collectively improved feed efficiency of milk yield, ECM, FCM, and SCM for BSP cows (6, 5, 5, and 5%, respectively). No treatment differences were observed for concentrations of milk fat, protein, total solids, somatic cell count, somatic cell score, body weight, or body condition score. Milk urea nitrogen concentrations decreased (5%), whereas milk protein and lactose yield increased (5 and 2%, respectively) with BSP supplementation. Prepartum fecal pH did not differ among treatments; conversely, postpartum fecal pH was increased with BSP supplementation (0.09 pH units). Prepartum fecal dry matter percentage, starch, acetic acid, propionic acid, butyric acid, and ethanol did not differ among treatments. Postpartum concentrations of the aforementioned fecal parameters were also unaffected by treatment, but fecal propionic acid concentration was decreased (24%) in BSP cows relative to CON. Circulating glucose, nonesterified fatty acids, l-lactate, and insulin were similar between treatments both pre- and postpartum. Prepartum ß-hydroxybutyrate (BHB) did not differ between treatments, but postpartum BSP supplementation decreased (21%) circulating BHB relative to CON. Regardless of treatment, inflammatory markers (serum amyloid A and haptoglobin) peaked immediately following parturition and progressively decreased with time, but this pattern was not influenced by treatment. Postpartum lipopolysaccharide binding protein tended to be decreased on d 3 in BSP relative to CON cows (19%). Neither treatment nor time affected Cr-EDTA area under the curve. In summary, supplementing BSP had no detectable effects prepartum, but increased key postpartum production parameters. Bacillus subtilis PB6 consistently increased postpartum fecal pH and decreased fecal propionate concentrations but did not appear to have an effect on gastrointestinal tract permeability.

Differences in Left Ventricular Enlargement Secondary to Chronic Volume Loading Between English Springer Spaniels and Two Similar Sporting Breeds.

INTRODUCTION: English springer spaniels have larger, rounder ventricles than most other breeds. How this geometry impacts responses to volume overload remains unknown. We compared left ventricular size between English springer spaniels and two similarly sized sporting breeds (Border collies and Labrador retrievers) in naturally occurring chronic left ventricular volume loading conditions (mitral regurgitation and patent ductus arteriosus [PDA]) to examine whether differences in remodelling responses exist between these breeds. ANIMALS, MATERIALS AND METHODS: We searched records for cases of mitral regurgitation and PDA in three breeds. We recorded age, sex, presence of congestive heart failure (CHF), body weight and specific echocardiographic variables. We compared normalised measures of left ventricular size between breeds. Cases with CHF were further examined as a separate group. RESULTS: One-hundred-and-ninety-one dogs were included: 110 with degenerative mitral valve disease, 42 with mitral dysplasia and 39 with PDA. One third of all cases had CHF. All measures of left ventricular size were larger in English springer spaniels in mitral regurgitation cases (P<0.001), whereas PDA cases did not differ. English springer spaniels with PDA resulting in CHF had larger systolic dimensions and volumes than similarly affected non-English Springer Spaniel dogs (P=0.003). CONCLUSIONS: English springer spaniels have greater left ventricular dimensions when exposed to chronic mitral regurgitation, compared with Border collies and Labrador retrievers, but not when exposed to volume overload from a PDA. English springer spaniels differ in their left ventricular morphology from two other sporting breeds, supporting previous studies that they have a unique cardiac morphotype.

Lactate-induced spontaneous acrosomal exocytosis as a method to study acrosome function in stallion sperm.

Evaluation of acrosome function in stallion sperm is mostly based on the use of inducers of acrosomal exocytosis (AE), such as the calcium ionophore A23187 or progesterone. Recently, it has been reported that incubation of stallion sperm under presumed capacitating conditions (i.e., medium formulated with calcium, bicarbonate, and bovine serum albumin) using a lactate-only containing medium (Lac-MW) results in a high rate of spontaneous AE in viable sperm (AE/Viable). In the current study, we developed an alternative assay of acrosome function for stallion sperm following the incubation of sperm in a medium formulated only with lactate as an energy substrate (Lac-MW). In Experiment 1, freshly ejaculated stallion sperm was incubated with 10 µM A23187, Lac-MW, or Control, for up to 6 h under capacitating conditions. The percentages of motile sperm, viable sperm, total AE (Total AE), and AE in viable sperm (AE/Viable) were compared among treatment groups. Incubation in Lac-MW, but not with Control or A23187, resulted in a time-dependent increase in the percentage of AE/Viable, as determined by flow cytometry, particularly at 4 and 6 h of incubation (P < 0.05). In Experiment 2, freshly ejaculated sperm was incubated in Lac-MW for up to 6 h, and the occurrence of protein tyrosine phosphorylation and AE/Viable were determined. At 4h and 6h of incubation in Lac-MW, ∼40% of the sperm displayed a protein tyrosine phosphorylation immunofluorescence pattern that coincides with that recently associated with stallion sperm capacitation (i.e., immunofluorescence signal at the acrosome and midpiece). In Experiment 3, the rate of AE/Viable sperm was compared among freshly ejaculated, cool-stored, and frozen/thawed stallion sperm. Except at 2h incubation in Lac-MW, differences in mean AE/Viable among fresh, cool-stored, and frozen/thawed sperm were not observed (P > 0.05). In Experiment 4, the relationship between Total AE (A23187), or AE/Viable (Lac-MW), and in vivo fertility of 5 stallions was determined. A linear relationship was observed between mean AE/Viable and the per-cycle (r = 0.93; P < 0.05) and seasonal (r = 0.66; P < 0.05) pregnancy rates of five stallions used for artificial insemination with cool-stored semen. In Experiment 5, frozen/thawed sperm from subfertile Thoroughbred (TB) stallions, known to carry the susceptibility genotype for Impaired Acrosomal Exocytosis (IAE; FKBP6 A/A-A/A) was evaluated following incubation in Lac-MW. Sperm from subfertile TB stallions with IAE had lower mean AE/Viable, at both 4h and 6h incubation in Lac-MW, when compared to that of fertile control stallions (P < 0.05). Overall, the Lac-MW model validated in the current study may be a useful complementary assay to evaluate the ability of stallion sperm to physiologically undergo AE and to study stallion fertility potential. This acrosome function assay can be used to evaluate fresh, cool-stored, or frozen/thawed stallion sperm, and describes a strong linear relationship with in vivo-fertility of stallions used in artificial insemination programs.

A matter of agreement: The effect of the technique and evaluator on the analysis of morphologic defects in stallion sperm.

Analysis of sperm morphology is an important part of the stallion breeding soundness evaluation since it provides an objective measure of a stallion's sperm quality and is one of many factors that estimate a stallion's fertility potential. To describe the effect of sperm quality level on the technique (Differential Interference Contrast - DIC; Phase-contrast - PH; Dip-Quick staining - DQ; and eosin-nigrosin staining - EN; semen samples fixed in buffered-formal saline) and evaluator (three evaluators; using only DIC), stallions were categorized based on sperm quality into three categories: High: >57% normal sperm, Moderate: 23-56% normal sperm, or Low: <23% normal sperm (four stallions per category). The data were analyzed using three different statistical methods: Analysis of Variance (ANOVA), correlative analysis, and Bland-Altman method (agreement). A higher level of agreement among techniques was observed between DIC and PH for morphologically normal sperm. The agreement between the alternative methods (EN, DQ, or PH) and the standard method (DIC) varied, depending on the sperm quality level (High, Moderate, or Low). Some morphological defects (e.g., AH, AMP) were constantly underestimated with the staining methods (DQ, EN) compared to DIC and PH, particularly in ejaculates with low sperm morphology. Underestimation of some abnormalities, due to the technique or the evaluator, has the potential to alter the clinical interpretation of stallion fertility.

Effects of egg yolk level, penetrating cryoprotectant, and pre-freeze cooling rate, on the post-thaw quality of stallion sperm.

The current study determined the effect of the egg-yolk (phospholipid source) level (egg yolk [20% EY] vs. skim-milk + egg yolk [SM + 2% EY]), cryoprotectant (glycerol [Gly] vs. glycerol + methylformamide [Gly + MF]), and pre-freeze cooling rate (-0.1 vs. -1 vs. -5 °C/min) on post-thaw stallion sperm quality. In Experiment 1, ejaculates (n = 27) from 9 stallions (3 ejaculates each) with varied sperm quality (High, Average, or Low) were frozen in EY-Gly, SMEY-Gly, EY-Gly + MF, or SMEY-Gly + MF extenders. Sperm in each group were cooled from 22° to 5°C using either -0.1 °C/min or -1 °C/min linear cooling rates prior to freezing. In Experiment 2, ejaculates (n = 24) from 12 stallions (2 ejaculates each) with High or Average sperm quality were frozen in EY-Gly, EY-Gly + MF, or in BotuCrio (BC) extenders. Sperm in each group were cooled from 22° to 5°C using either -1 or -5 °C/min linear cooling rates prior to freezing. In Experiment 1, for stallions with High or Average sperm quality, either cooling rate generally resulted in lower sperm quality for the SMEY-based extenders than for the EY-based extenders (P < 0.05). Stallions with Low sperm quality were unaffected by any experimental treatment (P > 0.05). In Experiment 2, a -5 °C/min cooling rate yielded lower sperm quality in BC than in EY-Gly or EY-Gly + MF groups (P < 0.05); however, a -1 °C/min cooling rate yielded similar sperm quality among these treatments (P > 0.05). In summary, the phospholipid level in the freezing extender and the pre-freeze cooling rate, but not the penetrating cryoprotectant, affected the post-thaw quality of stallion sperm.

The stallion sperm acrosome: Considerations from a research and clinical perspective.

During the fertilization process, the interaction between the sperm and the oocyte is mediated by a process known as acrosomal exocytosis (AE). Although the role of the sperm acrosome on fertilization has been studied extensively over the last 70 years, little is known about the molecular mechanisms that govern acrosomal function, particularly in species other than mice or humans. Even though subfertility due to acrosomal dysfunction is less common in large animals than in humans, the evaluation of sperm acrosomal function should be considered not only as a complementary but a routine test when individuals are selected for breeding potential. This certainly holds true for stallions, which might display lower levels of fertility in the face of "acceptable" sperm quality parameters determined by conventional sperm assays. Nowadays, the use of high throughput technologies such as flow cytometry or mass spectrometry-based proteomic analysis is commonplace in the research arena. Such techniques can also be implemented in clinical scenarios of males with "idiopathic" subfertility. The current review focuses on the sperm acrosome, with particular emphasis on the stallion. We aim to describe the physiological events that lead to the acrosome formation within the testis, the role of very specific acrosomal proteins during AE, the methods to study the occurrence of AE under in vitro conditions, and the potential use of molecular biology techniques to discover new markers of acrosomal function and subfertility associated with acrosomal dysfunction in stallions.

Sperm factors associated with the production of equine blastocysts by intracytoplasmic sperm injection (ICSI) using frozen/thawed semen.

Intracytoplasmic Sperm Injection (ICSI) using frozen/thawed sperm is a common procedure to obtain embryos from fertile or subfertile mares and stallions. Stallion-associated factors that impact the efficiency of ICSI have been studied less than those associated with the mare. Three experiments were conducted: Experiment 1: the effect of freezing extender composition and cryoprotectant; Experiment 2: the effect of sperm exposure to seminal plasma prior to freezing (ejaculated vs. epididymal sperm; two-freeze/thaw cycles each); and Experiment 3: the effect of sperm morphologic feature used for fertilization (normal vs. cytoplasmic droplet vs. bent tail); on the blastocyst rate after ICSI. In Experiment 1, stallion sperm was cryopreserved using commercially available extenders containing: a) 2% egg-yolk + milk + 4% glycerol (MFR5); b) 2% egg-yolk + milk + 2% glycerol + 3% methyl formamide (CMMFR5); c) 20% egg-yolk + 4.75% glycerol (LE); or d) 20% egg-yolk + 2% glycerol + 3% methyl formamide (CMLE). Sperm from each of the treatment groups were used for Piezo-driven ICSI on in vitro-matured equine oocytes (n = 321). Extender CMLE resulted in a lower cleavage rate (35%) than the other treatment groups (MFR5: 74%, CMMFR5: 62%, LE: 68%; P < 0.05). Extender MFR5 yielded a higher blastocyst rate per injected oocyte (21/82 [26%]) than the Groups LE (8/77 [10%]), CMLE (4/80 [5%]) or CMMFR5 (4/82 [5%]; P < 0.05). Extender MFR5 also yielded a higher blastocyst rate per cleaved oocyte (34%) than Groups LE, CMLE or CMMFR5 (15%, 14%, 8%; respectively P < 0.05). In Experiment 2, ejaculated (EJ) and epididymal (EPD) sperm from a fertile stallion which was initially cryopreserved in the CMLE extender, was thawed and re-cryopreserved in MFR5 extender for use in ICSI. Sperm from both groups (EJ vs. EPD) were used for ICSI on in vitro matured oocytes (n = 127). Differences were not detected for cleavage rate (EJ: 36/63 [57%] vs. EPD: 49/64 [77%]), blastocyst rate per injected oocyte (EJ: 11/63 [17%] vs. EPD: 11/64 [17%]), or blastocyst rate per cleaved oocyte (EJ: 31% vs. EPD: 22%) between treatment groups (P > 0.05). In Experiment 3, morphologically normal sperm (N), or sperm with proximal droplets (PD) or bent tails (BT), were obtained from a single fertile stallion and were used for ICSI on in vitro matured oocytes (n = 75). No differences were detected among treatment groups for cleavage rate (N: 19/25 [77%] vs. PD: 20/25 [88%] vs. BT: 18/25 [72%]), blastocyst rate per injected oocyte (N: 6/25 [24%] vs. PD: 5/25 [20%] vs. BT: 2/25 [8%]), and blastocyst rate per cleaved oocyte (N: 32% vs. PD: 23% vs. BT: 11%; P > 0.05). In conclusion, the current study indicates that freezing extender composition used for stallion sperm cryopreservation has an impact on the developmental competence of in vitro-matured equine oocytes after ICSI and in vitro culture. Furthermore, we were unable to detect differences on cleavage and blastocyst rates when performing ICSI when using: 1) ejaculated or epididymal sperm; or 2) sperm with different morphologic features. The results from the current study provide additional insight regarding stallion-related factors that should be considered when performing ICSI in horses.

Evaluation of a training intervention to improve cancer care in Zimbabwe: Strategies to Improve Kaposi Sarcoma Outcomes (SIKO), a prospective community-based stepped-wedge cluster randomized trial.

INTRODUCTION: Most Zimbabweans access medical care through tiered health systems. In 2013, HIV care was decentralized to primary care clinics; while oncology care remained centralized. Most persons in Zimbabwe with Kaposi sarcoma (KS) are diagnosed late in their disease, and the prognosis is poor. Little is known about whether educational interventions could improve KS outcomes in these settings. METHODS: Interventions to improve KS detection and management were evaluated at eight Zimbabwe primary care sites (four rural/four urban) that provided HIV care. Interventions included a standardized KS clinical evaluation tool, palliative care integration, standardized treatment and improved consultative services. Interventions were implemented between February 2013 and January 2016 using a randomized stepped-wedge cluster design. Sites were monitored for KS diagnosis rates and KS outcomes, including early diagnosis (T0 vs. T1 tumour stage), participant retention and mortality. Analyses controlled for within-clinic correlations. RESULTS: A total of 1102 persons with suspected KS (96% HIV positive) were enrolled: 47% incident (new diagnosis), 20% prevalent (previous diagnosis) and 33% determined as not KS. Early (T0) diagnosis increased post-intervention, though not significant statistically (adjusted odds ratio [aOR] = 1.48 [95% confidence interval (95% CI): 0.66-3.79], p = 0.37). New KS diagnosis rates increased 103% (95% CI: 11-273%), p = 0.02) post-intervention; although paired with an increased odds of incorrectly diagnosing KS (aOR = 2.08 [95% CI: 0.33-3.24], p = 0.001). Post-intervention, non-significant decreases in 90-day return rates (adjusted hazard ratio [aHR] = 0.69 [95% CI: 0.38-1.45], p = 0.21) and survival (aHR = 1.36 [95% CI: 0.85-2.20], p = 0.20) were estimated. CONCLUSIONS: KS training interventions at urban and rural Zimbabwe decentralized primary care clinics significantly increased overall and incorrect KS diagnosis rates, but not early KS diagnosis rates, 90-day return rates or survival.

Outcome clinical audit: analyses of interventional closure of patent ductus arteriosus in dogs.

OBJECTIVES: The objectives of this study were to determine whether conducting a clinical audit was achievable in a group of centres that perform interventional cardiac procedures and to report the success and complications rates in dogs diagnosed with patent ductus arteriosus. METHODS: This was a multicentre, European-wide, prospective study. Patient data were entered into a bespoke database prior to commencing interventional closure of patent ductus arteriosus in all animals undergoing this procedure during the study period. The database was designed to gather clinical audit information, after completion of the procedure, such as discharge outcome, complication rate, and medium-term outcome. RESULTS: A total of 339 cases were included from five participating centres. The process of performing clinical audit was achieved in all centres. Successful discharge outcome was 95.9% with a complication rate of 4.1%. The procedure-related mortality was 0.6%. 149 cases (43.9%) were either lost to follow-up or had not yet had a follow-up within the time period. Of the remaining 169 cases in which follow-up was available, 157 (92.9%) cases had a successful medium-term outcome CONCLUSIONS: This study demonstrates that the process of performing a clinical audit is achievable in veterinary clinical interventions across different centres. These results provide a benchmark for future comparison in our ongoing clinical audit and validate the process of clinical audit for other centres performing cardiac interventions. The use of clinical audit should be considered in other aspects of veterinary medicine.

Assessment of community-based flood early warning system in Malawi.

One of the major natural hazards the world is facing these days are floods. Malawi has not been spared. Floods have affected the countries' socio-economic developmental plans. River gauges have been installed along major rivers to monitor water levels in a bid to warn communities of imminent flooding. In Malawi, ever since the installation of river gauges no study has been done to assess their effectiveness. This study examines the effectiveness of these river gauges as part of community-based early warning system. The research employs both qualitative and quantitative approach. Questionnaires, interviews, group discussions, document analysis were all used in order to understand the behavioural aspect of communities under study. The current community-based early warning system practices were benchmarked against the following elements: risk knowledge, technical monitoring and warning services, dissemination and communication of warnings and response capability. The study revealed that Malawi has two distinct systems in place: at national level (managed by several government departments) and at community level [managed by Civil Protection Committees (CPCs)]. These systems were installed by non-governmental organisations (NGOs) and faith-based organisations. Apparently, no direct link exists between the two. Operational bureaucracy affects the speedy presentation of warning messages at national level. Lack of capacity and necessities affects the operation of the community-based system. Despite the efforts to develop the early warning systems, the failures outweigh the successes. Government needs to provide enough funding for systems sustainability, build capacity of CPCs and install more technologically advanced systems.

The role of impaired acrosomal exocytosis (IAE) in stallion subfertility: A retrospective analysis of the clinical condition, and an update on its diagnosis by high throughput technologies.

Acrosomal dysfunction has been considered as a cause of subfertility in males of different species, including stallions. A subset of subfertile stallions with acrosomal dysfunction is unique because they have normal sperm quality (motility, morphology, viability, and DNA quality). The current work aims to describe the clinical characteristics of subfertile stallions that were diagnosed with Impaired Acrosomal Exocytosis (IAE) by using two high throughput methods: flow cytometry and molecular genetic analysis, and to identify the prevalence of subfertility due to IAE in stallions evaluated at Texas A&M University. Clinical data from 1,128 stallions evaluated during 17 years at a Veterinary Teaching Hospital was retrospectively analyzed. Only stallions with a history of subfertility not explained following a breeding soundness examination and/or conventional semen analysis, were included. For those stallions, the acrosomal exocytosis test (AE test), in which sperm is incubated at 37 °C for up to 2 h in the presence of the calcium ionophore A23187, was used to determine IAE. The difference in AE-Rate (AE-Diff) between each pair of fertile control stallion and subfertile stallion was categorized as: Normal: AE-Diff < 14%; Questionable: AE-Diff 15-29%; Abnormal: AE-Diff > 30%. In selected cases, blood or hair was procured for identification of the susceptibility genotype for IAE, A/A-A/A, in the FKBP6 gene, exon 5. Twenty-one (21) stallions (1.86% total population analyzed) had reduced fertility despite having acceptable sperm quality. Sperm from these stallions were subjected to the AE Test. Of these, 8 stallions had reduced sperm AE-rate, based on the AE Test (8/21; 38.1%). Subsequently, blood or hair samples from these 8 stallions which had either questionable (AE-Diff 15 - 29%; n = 5) or abnormal (AE-Diff > 30%; n = 3) responses to the AE Test were analyzed for the susceptibility genotype for IAE, A/A-A/A (FKBP6 gene, exon 5). Seven out of the eight (7/8) stallions carried this susceptibility genotype. All of these were Thoroughbreds. After 2 h of incubation, the viability in fertile stallion sperm was lower than in A/A-A/A stallions (4% vs. 25%, respectively; P < 0.05), while the AE-rate was higher for fertile than for A/A-A/A stallions (85% vs. 56%, respectively; P < 0.05). The use of two high throughput tests (i.e., flow cytometry and molecular genetic analysis) may complement each other in the diagnosis of IAE in breeding stallions. In this study, 5/7 subfertile stallions diagnosed with the IAE susceptibility genotype would have been diagnosed as normal with the AE Test. This study introduces a subset of stallions with the IAE genotype with fertility higher than has been previously reported (i.e., <15% per-cycle pregnancy rate), suggesting that IAE manifests as a broader range of subfertility.

Incidence, causes and factors associated with torso injury in Cameroon: A community-based study.

BACKGROUND: Morbidity and mortality linked to injury has become an increasingly important public health concern worldwide, especially in developing countries. Despite the potentially severe nature of torso injury, little is known about the population-based epidemiology of torso injury in sub-Saharan Africa. OBJECTIVES: To determine the incidence, identify common mechanisms, and assess the socioeconomic consequences of torso injury in Cameroon. METHODS: We performed a torso injury sub-analysis of a larger descriptive cross-sectional community-based study on injury epidemiology in the preceding 12 months in the Southwest region of Cameroon. Sampling was done using the three-stage cluster sampling technique. The differences between groups were evaluated using χ² and adjusted Wald tests. RESULTS: We identified 39 cases of torso injuries out of 8 065 participants, providing a yearly incidence estimate of 488 (95% confidence interval (CI) 356 - 668) per 100 000 person-years. Road traffic injury was the most common mechanism of torso injury. The median (interquartile range (IQR)) cost of treatment for torso injury was USD58 (10 - 137), over four times the median (IQR) cost for non-torso injury at USD12 (3 - 43) (p=0.0004). About half of affected households (51%) reported being unable to afford necessities such as rent and food after injury v. 33% of households with members with non-torso injuries (p=0.018). CONCLUSION: Torso injuries have an incidence of 488/100 000 person-years, and road traffic injuries account for the majority of the injuries. Road traffic control measures and trauma care strengthening may reduce the impact of torso injuries and injuries in Cameroon.

Evaluation of Stallion Testicular Cell Types by Flow Cytometry.

Flow cytometry procedures can be used for evaluation of both spermatogenic efficiency and diagnose disorders of stallion spermatogenesis. Aims of this study were to compare two testicular sample acquisition techniques (needle aspirate-N and tissue wedge-T) and results when using flow cytometry and histology procedures. Testicular cell types were stained with acridine orange, and nine regions (R2 to R10) were identified and enumerated following acquisition by either N or T. Testes were also grouped and analyzed by size and sexual maturity (Small [immature] compared with Large [mature]) and used to determine if flow cytometry procedures could be used to detect differences. For both N and T, percentages of 2n cell types were greater in the Small than Large testes, whereas percentages of 1n cell types in N were greater in the Large than Small testes (P < .05). Testicular cell types in N regions were correlated to similar T regions (r between 0.51 and 0.99; P < .05) in both groups. Flow cytometry and histology scores were correlated in both groups (r between -0.95 and 0.93, P < .05). There were small differences in number of testicular cell types from N and T. With both sample acquisition methods, there was discrimination between the Small and Large testes, therefore, evaluation of testicular cell types using flow cytometry procedures might have clinical applications. Results with comparison of flow cytometry to histology procedures indicate that flow cytometry can be applied clinically to identify changes in testicular cell types of stallions using a needle aspirate.

The effects of activity, body weight, sex and age on echocardiographic values in English setter dogs.

BACKGROUND: Breed-specific reference intervals improve echocardiographic interpretation and thereby reduce misdiagnoses, especially in athletic breeds. OBJECTIVES: The objectives of the study were to examine transthoracic echocardiographic values in healthy adult English setter dogs and determine the effects of activity, body weight, sex and age on these values. ANIMALS, MATERIALS AND METHODS: One hundred and one adult English setter dogs, recruited from local veterinary clinics and from the Norwegian English setter club, underwent routine transthoracic echocardiography. The population was stratified into two groups based on the reported level of activity. The effects of activity level, body weight, sex and age on echocardiographic variables were examined. Results were compared with published data from other breeds and from a pre-existing species-wide allometric model. RESULTS: Of the 100 dogs between 19 months and 10 years of age included in the study, 72 were reported as very active and 28 as less active. Echocardiographic intervals were calculated for body size-independent echocardiographic variables. The upper limits of the intervals for left-atrial-to-aortic ratios and normalised left ventricular volumes exceeded those of various, previously published studies of other breeds. Normalised left ventricular dimensions exceeded published allometric 95th percentile upper reference values in 13% of dogs in diastole and 32% of dogs in systole. More active dogs had larger cardiac dimensions than less active dogs; however, the activity level did not predict echocardiographic variables when included in a multiple regression model. CONCLUSIONS: The study provides breed specific transthoracic echocardiographic values for English setter dogs, thereby contributing to improve diagnostic assessment of cardiac health in this breed.

Lignin Quantification of Papyri by TGA-Not a Good Idea.

Papyri belong to the oldest writing grounds in history. Their conservation is of the highest importance in preserving our cultural heritage, which is best achieved based on an extensive knowledge of the materials' constituents to choose a tailored conservation approach. Thermogravimetric Analysis (TGA) has been widely employed to quantify cellulose and lignin in papyrus sheets, yielding reported lignin contents of 25% to 40%. In this work, the TGA method conventionally used for papyrus samples was repeated and compared to other lignin determination approaches (Klason-lignin and acetyl bromide-soluble lignin). TGA can lead to a large overestimation of the lignin content of commercial papyrus sheets (~27%) compared to the other methods (~5%). A similar overestimation of the lignin content was found for the pith and rind of the native papyrus plant. We concluded that the TGA method should, therefore, not be used for lignin quantification.