Vitrification of feline ovarian tissue: Comparison of protocols based on equilibration time and temperature.

Global contraction of biodiversity pushed most members of Felidae into threatened or endangered list except the domestic cat (Felis catus) thence preferred as the best model for conservation studies. One of the emerging conservation strategies is vitrification of ovarian tissue which is field-friendly but not yet standardized. Thus, our main goal was to establish a suitable vitrification protocol for feline ovarian tissue in field condition. Feline ovarian tissue fragments were punched with biopsy punch (1.5 mm diameter) and divided into 4 groups. Group 1 was fresh control (Fr), while the other three were exposed to 3 vitrification protocols (VIT_CT, VIT_RT1 and VIT_RT2). VIT_CT involved two step equilibrations in solutions containing dimethyl sulfoxide (DMSO) and ethylene glycol (EG) for 10 min each at 4 °C. VIT_RT1 involved three step equilibration in solutions containing DMSO, EG, polyvinylpyrrolidone and sucrose for 14 min in total at room temperature, while in VIT_RT2 all conditions remained the same as in VIT_RT1 except equilibration timing which was reduced by half. After vitrification and warming, fragments were morphologically evaluated and then cultured for six days. Subsequently, follicular morphology, cellular proliferation (expression of Ki-67, MCM-7) and apoptosis (expression of caspase-3) were evaluated, and data obtained were analysed using generalised linear mixed model and chi square tests. Proportions of intact follicles were higher in Fr (P = 0.0001) and VIT_RT2 (P = 0.0383) in comparison to the other protocols both post warming and after the six-day culture. Generally, most follicles remained at primordial state which was confirmed by the low expression of Ki-67, MCM-7 markers. In conclusion, VIT_RT2 protocol, which has lower equilibration time at room temperature has proven superior thus recommended for vitrification of feline ovarian tissue.

Melatonin reduces oxidative stress and improves follicular morphology in feline (Felis catus) vitrified ovarian tissue.

Ovarian tissue vitrification is associated with multiple events that promote accumulation of ROS (reactive oxygen species) which culminate in follicular apoptosis. Thus, this study was aimed at evaluating the role of melatonin in vitrification and culture of feline (Felis catus) ovarian tissue. In phase 1, domestic cat ovaries were fragmented into equal circular pieces of 1.5 mm diameter by 1 mm thickness and divided into four groups (fresh control and 3 treatments). The treatments were exposed to vitrification solutions supplemented with melatonin at 0 M, 10-9 M, and 10-7 M, then vitrified-warmed, histologically evaluated and assayed for ROS. Consequently, phase 2 experiment was designed wherein ovarian fragments were divided into two groups. One group was exposed to vitrification solution without melatonin and the other with 10-7 M melatonin supplementation, then vitrified-warmed and cultured for ten days with fresh ovarian fragments as control prior to assessment for histology, immunohistochemistry (Ki-67, MCM-7 and caspase-3) and ROS. Concentration of ROS was lower (p = 0.0009) in 10-7 M supplemented group in addition to higher proportion of grade 1 follicles. After culture, proportions of intact and activated follicles were higher (p < 0.05) in melatonin supplemented group evidenced by higher expression of Ki-67 and MCM-7. Follicular apoptosis was lower in melatonin supplemented group. In conclusion, melatonin at 10-7 M concentration preserved follicular morphological integrity while reducing ROS concentration in vitrified-warmed feline ovarian tissue. It has also promoted the follicular viability and activation with reduced apoptosis during in vitro culture of vitrified-warmed feline ovarian tissue.

Mini laparotomy for candy cane syndrome at the jejunojejunostomy after a second Roux Y Gastric bypass with multiple surgical history: a case report.

Background: Candy cane syndrome (CCS) is a rare and underreported complication, seldom occurring after bariatric surgeries, especially, the Roux-en-Y gastric bypass (RYGB) type. It refers to an excessively long-blind end of the alimentary limb, usually at the gastrojejunal (GJ) junction, and to a lesser extent, can occur at the jejunojejunal (JJ) junction, that may cause symptoms including abdominal pain, regurgitation, nausea, vomiting and reflux. However, its diagnosis can be challenging and misleading. Case Description: A 34-year-old woman with a multiple past surgical history presented with small bowel obstruction (SBO) symptoms following a second gastric bypass surgery. An esophagogastroduodenoscopy (EGD) was inconclusive, then a computed tomography (CT) scan was done, which reported intussusception. The patient underwent laparoscopy, which revealed an anastomosis with an extra 14 cm of single-loop bowel near the JJ junction rather than intussusception, leading to a diagnostic laparoscopy followed by a mini-laparotomy procedure. Adhesiolysis followed by a resection of the elongated blind end was done, hence, the diagnosis of CCS was established. The patient tolerated the surgery with a complete resolution of her symptoms; no subsequent complications were reported. Conclusions: The frequency of RYGB surgery and the number of past surgeries a patient might have undergone might correlate independently with the risk of developing CCS.

Effect of Fixatives and Fixation Period on Morphology and Immunohistochemistry of Feline Ovarian Tissue.

Fixatives and fixation protocol have a profound effect on both the morphology and epitope sensitivity of ovarian tissue, which hampers accurate ovarian tissue evaluation. We aimed to establish the most suitable fixation protocol for feline (Felis catus) ovarian tissue. Fragments (1.5 mm diameter) were punched from 1 mm-thick feline ovarian tissue, divided into three groups then fixed with three different fixatives (Bouin, neutral buffered formalin [NBF] and form acetic acid [new compound fixative formulation for ovarian tissue composed of 5% acetic acid in NBF]) for five fixation periods. Subsequently, fragments were processed and evaluated for the morphology and intensity of immunohistochemical signals against three antigens (Ki-67, MCM-7 and activated caspase-3). Proportions of grade 1 or morphologically intact follicles were significantly lower in NBF when compared with Bouin and form acetic acid fixatives. However, Bouin fixative had the lowest mean DAB intensity (p < 0.05) in all three antigen targets, while NBF had the highest (p < 0.05) in Ki-67 and caspase-3, but in MCM-7, it was no different from form acetic acid. In conclusion, form acetic acid maintained ovarian tissue architecture with excellent follicular morphology in the same manner as Bouin fixative, and it also maintained reasonable DAB signals similar to NBF, thus providing a better alternative for feline ovarian tissue studies.

Culture of vitrified bovine ovarian tissue on agarose gel inserts maintains follicle integrity.

In brief: Ovarian tissue cryopreservation and culture provide an option for fertility preservation without tissue grafting, but need optimization. This study reveals that vitrified bovine ovarian tissue can be cultured on agarose gel and maintain follicle morphology, low activation, and low apoptosis. Abstract: Ovarian tissue preservation is hitherto a promising fertility insurance option for precious animals. Ovarian tissue vitrification and culture combined approach would eliminate the need of transplanting ovarian tissue to obtain mature oocytes. We aimed at optimizing vitrification and in vitro culture conditions for improved bovine ovarian tissue viability. Ovaries obtained from the slaughterhouse were punched into fragments and divided into three groups. Group 1 (fresh) was divided into two and immediately placed in two-culture systems (culture inserts and agarose inserts). Group 2 was vitrified, warmed, and placed in the two-culture systems, while group 3 was only equilibrated and then placed in the two-culture systems. All cultures were maintained for 6 days and spent media were collected on alternate days for cytokine (interleukin 1ß and interleukin 6) evaluation. Fragments were fixed for morphology assessment and immunohistochemistry. Higher percentages (P < 0.05) of grade 1 (morphologically intact) follicles were observed in fragments on agarose compared to those on culture inserts on days 2 and 4 of the culture. Conversely, we found higher (P < 0.05) shifts of primordial follicles to transitional follicles in fragments on culture inserts vis-à-vis agarose inserts which was consistent with a higher proportion of Ki-67 and MCM-7 and activated caspase-3-positive follicles. In conclusion, in vitro culture of bovine ovarian tissue on agarose inserts maintained follicle morphology, low follicle activation, and low apoptosis compared to culture inserts.

Precision Denavit-Hartenberg Parameter Calibration for Industrial Robots Using a Laser Tracker System and Intelligent Optimization Approaches.

Precision object handling and manipulation require the accurate positioning of industrial robots. A common practice for performing end effector positioning is to read joint angles and use industrial robot forward kinematics (FKs). However, industrial robot FKs rely on the robot Denavit-Hartenberg (DH) parameter values, which include uncertainties. Sources of uncertainty associated with industrial robot FKs include mechanical wear, manufacturing and assembly tolerances, and robot calibration errors. It is therefore necessary to increase the accuracy of DH parameter values to reduce the impact of uncertainties on industrial robot FKs. In this paper, we use differential evolution, particle swarm optimization, an artificial bee colony, and a gravitational search algorithm to calibrate industrial robot DH parameters. A laser tracker system, Leica AT960-MR, is utilized to register accurate positional measurements. The nominal accuracy of this non-contact metrology equipment is less than 3 µm/m. Metaheuristic optimization approaches such as differential evolution, particle swarm optimization, an artificial bee colony and a gravitational search algorithm are used as optimization methods to perform the calibration using laser tracker position data. It is observed that, using the proposed approach with an artificial bee colony optimization algorithm, the accuracy of industrial robot FKs in terms of mean absolute errors of static and near-static motion over all three dimensions for the test data decreases from its measured value of 75.4 µm to 60.1 µm (a 20.3% improvement).

Microenvironment factors promoting the quality of vitrified cat oocytes.

In oocyte cryopreservation programs, vitrification has overthrown conventional slow freezing both in veterinary and human medicine. In animals, its feasibility in field conditions makes it the preferred technique for the safeguard of genetic resources from zoo or wild animals, including threatened felids, for which the domestic cat is an excellent model. However, many cellular injuries, such as cytoskeleton, mitochondria and meiotic spindle alterations, DNA damage, zona pellucida hardening and cumulus cell loss, might occur following vitrification. After warming, although the exact mechanisms are still unclear, degeneration is a frequent outcome for cat vitrified oocytes. For immature (germinal vesicle) gametes, in vitro maturation after warming is a challenge, and cleavage after fertilization barely reaches 15-30%, while for mature (metaphase II) cryopreserved gametes it can get to 30-50%. Anyway, the progression to late embryos stages is often impaired, and improvements are needed. Standard cryopreservation protocol and the use of conventional in vitro culture systems after warming may not be enough for vitrified oocytes to recover and demonstrate their full developmental potential. Physical or chemical factors applied to oocytes undergoing vitrification, as an enrichment to the vitrification step, or to the culture microenvironment, could create more favorable conditions and promote vitrified oocyte survival and development. From the use of three-dimensional culture systems to the regulation of metabolic activities and cellular pathways, this review aims to explore all the possibilities employed so far, including the studies performed by our own lab, and the future perspectives, to present the most effective strategies for cat oocyte vitrification and the best time for their application (i.e., before, during, or after vitrification-warming).

Spermatozoa Survival in Egg Yolk-Based and Soybean-Based Extenders at Ambient and Chilling Temperature in Domestic Turkeys (Meleagris gallopavo).

Populations of many galliform species have declined mainly due to habitat loss and over-hunting, notably the Congo peacock, which has been classified as a vulnerable species by the International Union for Conservation of Nature (IUCN). The domestic turkey, being a species of least concern, which has been reported to be closely related to peacocks, could serve as a model for the optimization of assisted reproductive technologies for the Congo peacock. This study was aimed at developing a suitable turkey semen extender for artificial insemination in field conditions. Semen was collected using the dorso-abdominal massage technique from seven turkey toms and analyzed. Ejaculates with >70% motility and >80% live spermatozoa were pooled and divided into four aliquots (four treatments). Each of the four treatments was extended in a soybean-based extender or an egg yolk-based extender, with or without L-ascorbic acid. Two liquid preservation protocols (ambient temperature (35 °C) and chilled (4 °C)) were employed, and quality parameters including motility, viability and morphology were evaluated. The results show that the two extenders were similar with regard to semen quality parameters, and L-ascorbic acid supplementation of the turkey semen extenders improved semen quality during liquid storage.

Fighting Like Cats and Dogs: Challenges in Domestic Carnivore Oocyte Development and Promises of Innovative Culture Systems.

In vitro embryo production in cats and dogs still presents some challenges, and it needs to be optimized to transfer efficient protocols to related wild, endangered species. While the chemical composition of culture media has been the focus of several studies, the importance of culture substrates for oocyte and embryo culture has often been neglected. Traditional in vitro systems, i.e., two-dimensional cultures, do not resemble the physiological environments where cells develop, and they may cause morphological and functional alterations to oocytes and embryos. More modern three-dimensional and microfluidic culture system better mimic the structure and the stimuli found in in vivo conditions, and they could better support the development of oocytes and embryos in vitro, as well as the maintenance of more physiological behaviors. This review describes the different culture systems tested for domestic carnivore reproductive cells along the years, and it summarizes their effects on cultured cells with the purpose of analyzing innovative options to improve in vitro embryo production outcomes.

Identification of Differentially Expressed Proteins in the Serum of Colorectal Cancer Patients Using 2D-DIGE Proteomics Analysis.

Early detection of colorectal cancer (CRC) is vital for the improvement of disease prognosis. However to date there are no blood-based biomarkers sensitive and specific enough for early diagnosis. We analysed the differences in serum protein expression of early stage CRC (Dukes' A and B) and late stage CRC (Dukes' C and D) against normal controls using 2D Fluorescence Difference Gel Electrophoresis (2D-DIGE). Analysis of the 2D maps showed that 23 proteins were differentially expressed between groups (p≤0.05) and these proteins were identified with LC-MS/MS. Eight proteins were up-regulated and 2 down-regulated in patients with early CRC, whereas 14 proteins were up-regulated and 4 downregulated in those with late CRC compared to normal controls (p≤0.05). Five proteins, namely apolipoprotein A1 (APOA1), apolipoprotein E (APOE), complement factor H (CFH), galectin-7 (GAL7) and synaptojanin-2 (SYNJ2) were validated using ELISA and only APOA1 and GAL-7 showed consistent findings. Further validation using immunohistochemistry showed negative immunoreactivity for GAL-7 in CRC tissues, suggesting that GAL-7 detected in the serum did not originate from the CRC tumour. APOA1 showed positive immunoreactivity but its expression did not correlate with Dukes' staging (p=0.314), tumour grading (p=0.880) and lymph node involvement (p=0.108). Differences in APOA1 isoforms and/or conformation between serum and tissue samples as well as tumour heterogeneity may explain for the discrepancies between DIGE and ELISAwhen compared to immunohistochemistry. Structural and functional studies of APOA1 in future would best describe the role of APOA1 in CRC.

Role of pre-eclamptic toxaemia or eclampsia in hypertensive women attending cardiac clinic of Ahmadu Bello University Teaching Hospital Zaria, Nigeria.

BACKGROUND: Patients with pre-eclampsia and eclampsia constitute a special high risk group for future hypertension. They require a long term follow up to be able to detect and treat emerging hypertension early enough to prevent complications. Unfortunately, this is not so. This study was undertaken to find out the incidence of history of pre-eclamptic toxaemia (PET) in our female hypertensive patients attending cardiac clinic and to also determine the incidence of complications of hypertension in those with previous history of PET. METHODS: Fifty consecutive female hypertensive patients seen in cardiac clinic were recruited. Detailed history including full obstetric and family history was taken. A full clinical examination was done including blood pressure and a search for complications of hypertension. Findings were then analyzed and various frequencies determined. RESULTS: Forty-nine patients were studied. The mean age was 47.29 +/- 11.46 years. The mean SBP, DBP and MAP were 143.18 +/- 25.05, 90.49 +/- 14.19 and 108.12 +/- 16.71 mmHg respectively. Between the last child birth and the time of established hypertension in those who had PET ranged from 3-25 years. Sixteen, (32.7%) of the 49 patients had history of PET and 7(43.75%) of these 16 patients had complications of hypertension. CONCLUSION: The incidence of history of PET in our female hypertensive patients attending cardiac clinic is significant (32.7%). Also the 43.8% incidence of complications of hypertension seen in those patients with history of PET in this study is high. PET patients, therefore, constitute a special risk group for future hypertension. Therefore collaboration between the Obstetricians and the Cardiologists is important for patients with PET and eclampsia.

Diastolic dysfunction in persons with hypertensive heart failure.

Arterial hypertension is the commonest cause of heart failure in the Savannah region of Nigeria. The prevalence of diastolic dysfunction in patients with hypertensive heart failure in this region is unknown. This needs to be determined, since it may influence therapy. Left ventricular function of 95 patients with hypertensive heart failure and 92 age-/sex-matched, healthy normotensive control was determined by echocardiography using ALOKA SSD 1700 two-dimensional echocardiograph/ Doppler and color flow ultrasound machine. Compared to the controls, the hypertensive heart failure patients had increased left ventricular mass index (LVMI), p = 0.0001; cardiothoracic ratio (CTR), p = 0.0001; and electrocardiographic left ventricular hypertrophy (ECG-LVH), p = 0.0001. Isolated diastolic dysfunction, E/A ratio < 1.0, and EF > 50%, was seen in 12 patients (12.6%). Twenty-three patients (24.2%) had combined diastolic and systolic dysfunction (E/A ratio < 1.0 and EF < 50%). Six patients (6.3%) had pseudonormalization (E/A ratio 1.0-2.0 and deceleration time, DT, 150-200 msec), while restrictive pattern of diastolic dysfunction was seen in 25 patients (26.3%). This prevalence of 69.4% of diastolic dysfunction in Nigerians with hypertensive heart failure seen in this study emphasizes the need for Doppler echocardiographic evaluation and diagnosis where these facilities are available. This will aid the physician to prescribe appropriate treatment.