Response to cooling of pony stallion semen selected by glass wool filtration

The aim of this study was to compare the sperm separation technique using filtration through glass wool compared with just diluted cooled semen. Eighteen ejaculates were collected from 6 pony stallions of the Brazilian pony breed. Evaluations were done on pH, osmolarity, total motility, membrane functionality (HOST), membrane integrity (CFDA/PI), morphology and mitochondrial viability (MTT) in fresh, 24 and 48 h of cooled semen at 5°C. After dilution, the half of the extended semen was cooled (control group). The other half was cooled after filtration trough glass wool (filtered group). Retained semen was considered the portion of cells that did not transpose glass wool barrier. Total motility from the control, filtered and retained groups after 24 h of cooling was 35.5%, 43.3% and 10% (p < .0001) respectively. Sperm membrane integrity percentage at the CFDA/PI test was 37.9%, 44.8% and 14.8% (p < .0001), on the control, filtered and retained groups respectively. The results confirmed that the passage of spermatozoa through glass wool increased the selection of spermatozoa from pony stallions with higher motility, mitochondrial viability and membrane integrity for cooling in milk extender up to 24 h. Moreover, it was not obtained higher sperm parameters to control after cooling 48 h under the conditions that the study was conducted.     

Addition of caffeine to equine thawed sperm increases motility and decreases nitrite concentration

The aim of this study was to improve the quality of frozen-thawed equine sperm by the addition of caffeine to it. Semen from nine stallions was frozen and different concentrations of caffeine (3, 5 and 7.5 mM) were added to frozen-thawed semen. The sperm kinetic parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite and hydroperoxide concentrations of frozen-thawed semen were measured using spectrophotometry. Sperm fertility was evaluated by artificial insemination (AI) of 16 mares with thawed ejaculates (control and 5 mM caffeine-treated groups). Compared to that in the control, the addition of 5 mM caffeine induced an increase in sperm motility (38.9 ± 2.8 versus 32.6 ± 3.4%), and a decrease in nitrite concentration (11.4 ± 2.1 versus 12.8 ± 2.9 µM/µg protein, p < .05). Moreover, the pregnancy rate from AI in the caffeine group was significantly higher (62.5%) than that in the control group (12.5%). These data suggest that caffeine reduced the nitrite concentration and enhanced sperm motility in thawed equine sperm, thus increasing the fertility rate in mares inseminated with caffeine-treated equine semen.     

Milk Fistula: Diagnosis,Prevention, and Treatment

Milk fistula is an uncommon condition which occurs when there is an abnormal connection that forms between the skin surface and the duct in the breast of a lactating woman, resulting in spontaneous and often constant drainage of milk from this path of least resistance. A milk fistula is usually a complication that results from a needle biopsy or surgical intervention in a lactating patient. Here, the authors present an unusual case of a spontaneous milk fistula which developed from an abscess in the breast of a lactating woman. The patient initially presented to the office with a large open wound on her breast, formed from skin breakdown, within which milk was pooling. She was treated with local wound care and cessation of breastfeeding, with appropriate healing of the wound and closure of the fistula with 6 weeks. Diagnosis, prevention, and treatment of milk fistula were reviewed.     

Effect of storage in short--and long-term commercial semen extenders on the motility, plasma membrane and chromatin integrity of boar spermatozoa

For artificial insemination (AI) in pigs, preservation of liquid boar semen at 16-20 degrees C is still common practice as sperm cryopreservation remains suboptimal in this species. To meet the different needs of the swine industry, several extenders have been developed to preserve semen in liquid form for short--and long-term storage. In the present study, three different commercial extenders devised for short-term (BTS+) or long-term preservation (MR-A and X-Cell), were used to test whether storage of semen from four mature, fertile boars at 17 degrees C for 96 h would affect sperm characteristics relevant for fertility, such as motility, membrane integrity and chromatin stability. Computer-assisted sperm analysis, and stainings with the acylated membrane dye SYBR-14/propidium iodide, and acridine orange in connection with flow cytometry were used to evaluate these variables. Percentages of total motile spermatozoa decreased slightly, but significantly, after 72-96 h. While membrane integrity values varied during the period of study, no significant changes in either membrane integrity or chromatin stability were, however, registered. This suggests a customary 96-day storage at 17 degrees C in these extenders was too short an interval to cause losses of integrity in nuclear DNA in the boar population studied.     

Pre-decondensed sperm head injections into female pronuclei result in chromosomal mingling, zygotic cleavage, and adequate embryonic and fetal development up to delivery of healthy offspring: a novel method of assisted syngamy

Investigation of the developmental potential post-injection of a pre-decondensed or non-pre-decondensed sperm head into the female pronucleus of a pre-activated oocyte. Rat pre-activated oocytes were treated with intrapronuclear pre-decondensed sperm head injections (IPSHI) (n = 133) or intrapronuclear non-pre-decondensed sperm head injections (INPSHI) (n = 138). All injected oocytes were transferred to pseudopregnant female recipients. Rat IPSHI techniques resulted in the delivery of five healthy offspring. Rat INPSHI techniques did not result in any pregnancies. Rat IPSHI techniques can result in delivery of healthy offspring. Successful performance of human IPSHI techniques might serve as a novel method to manage cases of intracytoplasmic sperm injection failure due to lack of development of male pronucleus or due to failure in pronuclei fusion.     

Relationship between semen characteristics, a-glucosidase and the capacity of spermatozoa to bind to the human zona pellucida

Unfertilized oocytes from an in-vitro fertilization programme were stored in different saline solutions and then utilized in a zona binding assay (ZBA). The four saline solutions tested were identical with regard to the capacity of the zona to bind sperniatozoa provided by healthy donors. Spermatozoa from 150 infertile patients were tested in the ZBA. The number of spermatozoa bound to the zona correlated positively with sperm concentration, the percentage sperm motility and the percentage of morphologically normal spermatozoa. The population was then divided into two groups according to the level of α-glucosidase activity, an epididymal marker. The average number of spermatozoa bound to the zona was diminished in the group with low α-glucosidase activity, even when considering strictly equivalent classes of sperm concentration, motility and morphology, respectively.     

Adapting to the Transition Between Gestation and Lactation: Differences Between Rat,Human and Dairy Cow

Adequate blood calcium concentrations are vital for the normal function of mammals. Mechanisms for maintaining normal blood calcium function adequately most of the time; however, occasionally they fail and calcium homeostasis is compromised. Milk fever or periparturient hypocalcemia in dairy cattle is a well-documented example of a breakdown in the mechanisms of calcium homeostasis. This disease occurs at the time of parturition and is unique to adult dairy animals. The disease results from the inability of animals to cope with the sudden demand for calcium in support of colostrum formation. Animals developing the disease become hypocalcemic and require intravenous calcium to survive. The precise metabolic disorder(s) responsible for the onset of milk fever is still being debated. This report will highlight some of the current concepts related to the causes and prevention of milk fever in dairy cattle, as well as contrasting differences in calcium demands that exist between dairy cattle, humans and rats at the onset of lactation.     

Functional Development of the Mammary Gland: Use of Expression Profiling and Trajectory Clustering to Reveal Changes in Gene Expression During Pregnancy, Lactation, and Involution

To characterize the molecular mechanisms by which progesterone withdrawal initiates milk secretion, we examined global gene expression during pregnancy and lactation in mice, focusing on the period around parturition. Trajectory clustering was used to profile the expression of 1358 genes that changed significantly between pregnancy day 12 and lactation day 9. Predominantly downward trajectories included stromal and proteasomal genes and genes for the enzymes of fatty acid degradation. Milk protein gene expression increased throughout pregnancy, whereas the expression of genes for lipid synthesis increased sharply at the onset of lactation. Examination of regulatory genes with profiles similar or complementary to those of lipid synthesis genes led to a model in which progesterone stimulates synthesis of TGF-beta, Wnt 5b, and IGFBP-5 during pregnancy. These factors are suggested to repress secretion by interfering with PRL and IGF-1 signaling. With progesterone withdrawal, PRL and IGF-1 signaling are activated, in turn activating Akt/PKB and the SREBPs, leading to increased lipid synthesis.     

Cactus flower extracts may prove beneficial in benign prostatic hyperplasia due to inhibition of 5α reductase activity, aromatase activity and lipid peroxidation

The cactus flower is deemed to be helpful in benign prostatic hyperplasia (BPH) therapy, although there is no published information regarding its clinical effect in patients and on the mechanism of its biological activity. The present study evaluated the ability of cactus flower extracts to exert an effect on BPH through possible inhibition of such processes as lipid peroxidation, androgen aromatization and testosterone reduction. Cactus flower extracts indeed inhibited aromatase and 5α reductase activity in cultured foreskin fibroblasts, and also in human placental and prostatic homogenates. The inhibitory activity in both instances was associated with the dichloromethane or ethanol (methanol) extracts, while a marked antioxidative activity was associated with the aqueous extract. The finding that cactus flower extracts interfere concurrently in vitro with aromatase and reductase activity as well as with free radical processes suggests that these substances may prove beneficial in BPH treatment. Received: 15 August 1997 / Accepted: 31 March 1998     

Bilateral thoracoscopy,mediastinoscopy and laparoscopy,in addition to CT,MRI and PET imaging,are essential to correctly stage and treat patients with mesothelioma prior to trimodality therapy*

Background: Trimodality therapy (TMT; extrapleural pneumonectomy (EPP), chemotherapy and radiation therapy) offers the potential of optimal survival in selected patients with Brigham stage I–II epitheliod mesothelioma based on CT, MRI and PET scanning. We hypothesized that these scanning modalities were inadequate to accurately stage these patients. Methods: Patients suitable for TMT, in addition to CT, MRI and PET scanning, prior to EPP, underwent bilateral thoracoscopy, mediastinoscopy and laparoscopy (surgical staging). Follow‐up CT scans were performed, six monthly, quality of life assessments yearly. Results: From 1 June 2004 to 28 February 2007, 34 patients were referred; mean age was 66 years (range: 44–69). Surgical staging was performed in 30 patients; 24 patients were confirmed as Brigham Stage I–II. However, six were upstaged, five as stage IV disease (one contralateral chest, two contralateral chest and abdomen, two abdomen) and one as mediastinal node positive; two further patients were reclassified histologically (one sarcomatoid, one biphasic). These eight patients fared poorly, 50% dying within 1 year from mesothelioma. Following surgical staging, 3 patients declined further surgery; thus, 19 patients proceeded to surgery, 3 were unresectable and 16 received EPP. Follow‐up of all 34 patients is complete. Conclusion: Surgical staging identified 26% of patients who would have received no benefit from TMT.     

Evaluation of genitofemoral nerve block, in addition to ilioinguinal and iliohypogastric nerve block, during inguinal hernia repair in children

Background. Ilioinguinal and iliohypogastric (IG-IH) nerve blockhas been widely used in children undergoing inguinal herniarepair. This technique may provide insufficient analgesia forintraoperative management as the inguinal region may receivesensory innervation from genitofemoral nerve. We proposed thataddition of a genitofemoral nerve block might improve the qualityof analgesia. Methods. Ninety-eight children undergoing inguinal hernia repairwere assigned randomly to receive either IG-IH nerve block (GroupI) or IG-IH and genitofemoral nerve blocks (Group II). Systolicarterial pressure (SAP) and heart rate (HR) were recorded beforesurgery (control), after skin incision, at sac traction andat the end of surgery. Postoperative analgesic requirementsand incidence of complications were recorded until discharge. Results. At sac traction, SAP and HR were significantly higherin Group I (P<0.05), and the incidence of episodes of increasedHR was also significantly higher in Group II (29 vs 12%, respectively,P<0.05). There were no significant differences in SAP andHR at other time points, postoperative analgesic requirementsor incidence of complications between the groups. Conclusions. The benefit of the additional genitofemoral nerveblock to IG-IH nerve block was limited only to the time of sactraction without any postoperative effect. This suggests thereis little clinical benefit in the addition of a genitofemoralnerve block.     

Dietary trans and saturated fatty acids effects on semen quality,hormonal levels and expression of genes related to steroid metabolism in mouse adipose tissue

Our objectives were to assess sperm alteration and adipose tissue (AT) genes expression related to steroid metabolism subsequent to fatty acids consumption. Twenty‐nine mature male mice were divided into: fat diet (FD; n = 15) and the control group (n = 14). FD group was fed with low level of trans and saturated fatty acids source for 60 days. Sperm parameters, levels of hormones and the mRNA abundance of the target genes in AT were assessed. The sperm concentration, total and progressive motilities were lower in FD group compared to that of control (p < 0.01). Blood estradiol levels increased in FD (p < 0.001), whereas no significant difference was observed in testosterone. The mRNA levels of StAR, CYP11A1, CYP17A1, 17βHSD7 and 17βHSD12 in AT of FD were higher than those of the control (p < 0.05). In contrast, mRNA level of Cyp19a1 in FD was significantly (p < 0.05) lower than that of control. 17βHSD12 and 17βHSD7 (as oestrogenic genes) increased, while 17βHSD5 and 17βHSD3 (as androgenic genes) remained unchanged, indicating that dietary trans/saturated fatty acids affect AT genes expression. Probably, sperm parameters were altered by increment of expression level of genes involved in oestrogenic metabolism rather than those engaged in androgenic metabolism after fatty acids consumption.     

Fatty acids,food groups and semen variables in men referring to an Italian Fertility Clinic: Cross-sectional analysis of a prospective cohort study

Diet has been suggested as a factor affecting sperm quality, both in healthy and infertile men. To study whether specific food groups and fatty acids (FA) intake were associated with sperm parameters of men from couples referring to an Italian Fertility Clinic, we conducted a cross-sectional analysis. A semen sample was collected and analysed before proceeding with assisted reproduction. To evaluate food groups and fatty acids intake, we used a questionnaire of food frequency. We calculated odds ratios (OR) and 95% confidence intervals (CI) for abnormal semen parameters. Among 323 men, 19% had semen volume (SV) <1.5 ml, 31% sperm concentration (SC) <15 mil/mL, 33% total sperm count (TSC) <39 mil and 27% sperm progressive motility <32%. Low SC was more frequent in men with higher saturated FA (SFA) and polyunsaturated FA (PUFA) intake. Better SC and TSC were related to higher monounsaturated FA (MUFA)/SFA ratio. Monounsaturated FA and linoleic/linolenic acid ratio were not related to any considered parameters. Low TSC was associated with low vegetable intake. In conclusion, normal sperm parameters were more frequent in men with low intake of SFA and PUFA, and high vegetable intake. Higher MUFA/SFA ratio was associated with better sperm quality parameters.     

Effects of high dose E. coli lipopolysaccharide on rabbit lung function,and of subsequent addition of chemotactic granulocyte activators to their isolated,blood-perfused lungs

E. coli LPS was infused (1 μg/kg to 5 mg/kg over 30 min) to spontaneously breathing rabbits, and their arterial blood pressure (ABP), blood leukocyte count and blood gases were observed for 2.5–3.5 h. Pulmonary vascular and airway function were subsequently evaluated in vitro by comparing weight changes, fluid filtration rates, pulmonary vascular resistance (PVR) and airway pressures in their isolated, blood-perfused lungs with those in lungs from untreated rabbits. Lung preparations from both groups of animals were then exposed to autologous zymosan-activated plasma (ZAP) or n-formyl-methionyl-leucyl-phenylalanine (FMLP) and perfused for 2 more hours. LPS addition to isolated rabbit leukocytes increased cell aggregation; cell chemiluminescence after activation with FMLP was also enhanced. Infusion of 1–5 mg/kg LPS decreased the count of all types of leukocytes and caused a metabolic acidosis (BE< –8 mmol), but no decrease in ABP. PAo₂-Pao₂ increased by about 2.0 kPa. No vascular permeability increase was detected in the lungs of these animals during subsequent in vitro perfusion. Addition of ZAP or FMLP during perfusion markedly increased the PVR in lungs from LPS animals, but did not induce major microvascular leakage. No significant differences in edema between lungs from LPS-treated and control animals were found by microscopy.     

First‐in‐human clinical trial to assess pharmacokinetics,pharmacodynamics, safety,and tolerability of iscalimab,an anti‐CD40 monoclonal antibody

Iscalimab is a fully human, CD40 pathway blocking, nondepleting monoclonal antibody being developed as an immunosuppressive agent. We describe a first‐in‐human, randomized, double‐blind, placebo‐controlled study investigating the safety, tolerability, pharmacokinetics, and pharmacodynamics of iscalimab in healthy subjects and rheumatoid arthritis patients. Healthy subjects (n = 56) received single doses of intravenous iscalimab (0.03, 0.1, 0.3, 1, or 3 mg/kg), or subcutaneous iscalimab (3 mg/kg), or placebo. Rheumatoid arthritis patients (n = 20) received single doses of intravenous iscalimab (10 or 30 mg/kg) or placebo. Iscalimab exhibited target‐mediated drug disposition resulting in dose‐dependent and nonlinear pharmacokinetics. Complete (≥90%) CD40 receptor occupancy on whole blood B cells was observed at plasma concentrations >0.3‐0.4 µg/mL. In subjects receiving 3 mg/kg iscalimab, antibody responses to keyhole limpet hemocyanin were transiently suppressed. CD40 occupancy by iscalimab prevented ex vivo human rCD154‐induced expression of CD69 on B cells in whole blood. All doses were generally safe and well tolerated, with no clinically relevant changes in any safety parameters, including no evidence of thromboembolic events. Iscalimab appears to be a promising blocker of the CD40‐CD154 costimulatory pathway with potential use in transplantation and other autoimmune diseases.     

Laser-induced thermal injury to dermal blood vessels: analysis of wavelength (585 nm vs. 595 nm), cryogen spray cooling, and wound healing effects

BACKGROUND AND OBJECTIVES: Successful laser treatment of cutaneous hyper-vascular lesions requires appropriate laser irradiation parameters for selective photothermolysis of ectatic dermal blood vessels as well as appropriate cooling parameters for epidermal protection based on an individual patient basis. Using the rabbit ear as an in vivo model for dermal vasculature, we investigated the influences of laser wavelength (585 nm vs. 595 nm) and cryogen spray cooling with various spurt durations on the laser-induced thermal injury to dermal blood vessels. Wound healing response was also evaluated in 2 hours and 4 days. STUDY DESIGN/MATERIALS AND METHODS: Flashlamp-pumped pulsed dye laser ScleroPlus (operated at the wavelength of 585 or 595 nm) was used for the comparison between the influences of two wavelengths (585 nm vs. 595 nm). R134-a cryogen spurts with the durations from 50 to 300 milliseconds were sprayed onto the sites to be irradiated and terminated 20 milliseconds before the onset of the laser pulses. In vivo rabbit ear was used as the model for cutaneous hyper-vascular lesions. Totally 10 New Zealand Albino white rabbits were experimented and in each rabbit ear six to seven sites were irradiated. Five animals were sacrificed 2 hours after the irradiation, and the remaining five sacrificed 4 days after the irradiation. Thermal injury to the blood vessel was assessed by hematoxylin and eosin stained histological sections and confirmed by an apoptosis assay. RESULTS: When the radiant exposures were above 10 J/cm2, 595 nm wavelength induced equivalent or more severe thermal injury to dermal blood vessels than 585 nm. Cryogen spray cooling with the spurt durations above 100 milliseconds resulted in increased depth of the most superficial thermal injury to dermal blood vessels than without cooling, indicating that superficial blood vessels were non-specifically cooled by the cryogen spurts applied at these parameters. Laser-induced thermal injury was significantly healed in the rabbit ear vasculature at 4 days post irradiation. CONCLUSIONS: Given sufficient radiant exposure, 595 nm wavelength can induce equivalent or more severe vascular injury compared with 585 nm. Cryogen spray cooling with the spurt durations above 100 ms may impair the photocoagulation of superficial blood vessels. Irreversible thermal injury to blood vessel can be achieved only when the basement membrane of blood vessel wall is irreversibly damaged.     

The effectiveness of a new dried human amnion derived membrane in addition to standard care in treating diabetic foot ulcers: A patient and assessor blind,randomised controlled pilot study

Recent reviews suggest that amniotic membrane products may accelerate healing of diabetic foot ulcers. A new dried human amniotic membrane (dHAM) has been used for ocular ulcers but not for diabetic foot ulcers. This was a multi‐centre, prospective, patient and observer blind, randomised controlled pilot trial, to investigate whether 2 weekly addition of the dHAM to standard care versus standard care alone increased the proportion of healed participants'' index foot ulcers within 12 weeks. Thirty‐one people (mean age 59.8 years, 81% male, 87% type 2 diabetes) were randomised (15 dHAM, 16 usual care). Within 12 weeks, healing occurred in 4 (27%) ulcers in the dHAM group versus 1 (6.3%) usual care group (P = .1). Percentage wound area reduction was higher in the dHAM versus control group. (P = .0057). There was no difference in AEs between the two groups. Six participants allocated to dHAM correctly identified their treatment group, although 5 in usual care incorrectly thought they were in the intervention arm. This pilot trial result is encouraging showing that this dHAM preparation is safe and promising treatment. These results will be used to design a statistically powered, definitive double blind randomised controlled trial.