bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuroma Prevention and Implantation Effects of NEUROCAP in Rat Sciatic Nerve Model
Authors: Peterson Steven L. , de Vries Harm , Collins Kami , Geraedts Hilde , Wheatley Michael J.
Published: January 2021

Introduction Symptomatic neuroma with neuropathic pain can develop following peripheral nerve injury. Current interventions for symptomatic neuroma have unpredictable results. NEUROCAP (Polyganics, Groningen, The Netherlands) is a bioresorbable nerve capping device intended to protect a peripheral nerve end and separate the nerve from the surrounding environment, to prevent the recurrence of a symptomatic neuroma.

Materials and Methods This study aims to assess the implantation effects of the NEUROCAP device in a rat sciatic nerve model during 12 months (±2 days). Forty-one adult male Sprague-Dawley rats were used in this study. They were randomly divided into a capping or test group, or a noncapping or control group for different time points of survival (12 weeks, 6 months, and 12 months). The objective of this study was evaluated regarding procedural data, adverse events, clinical observations, and histopathology.

Results The overall general health of the animals was adequate throughout the study, with the exception of autotomy during the first 4 months of survival. Eight animals were euthanized early due to autotomy, excluded from the study and seven of them have been replaced. Autotomy was an expected outcome and a known limitation of the animal model, particularly as this was a full sciatic nerve transection model. Neuroma formation was observed in the control group while there was no neuroma formation present in the test group. The control group showed increased nerve outgrowth and more chaotic fascicles in comparison with the test group. The test group also had a higher percentage of myelinated fibers compared to the control group. These results indicate a preventive mode of action of the NEUROCAP with regard to neuroma formation after nerve transection in a rat sciatic nerve model.

Conclusion The results indicate that NEUROCAP is safe and effective in preventing the recurrence of neuroma formation and inhibiting nerve outgrowth.
Simulation of vacuum arc cathode spot movement between transverse magnetic field contacts
Authors: Jing Hao , Yang Junfei , Pang Xianhai , Xiu Shixin
Published: January 2021

Vacuum switches have been extensively used in the field of medium voltage. Carrying out relevant research on vacuum arc is particularly important for improving the performance and reliability of vacuum switches. The vacuum arc cathode spots provide metal vapor and electrons, which determine the breaking capacity of the vacuum switches to some extent. Therefore, based on the simulation model of the cathode spots between the transverse magnetic field (TMF) contacts, the motion phenomenon and distribution of the cathode spot were simulated and the characteristics of the initial diffusion stage of cathode spots between TMF contacts were studied. The simulation results showed that the probability of generating a cathode spot in the direction of retrograde motion increased with the increase in the TMF component and the direction of motion varied with the angle between the magnetic field and the contact. The diffusion process of cathode spots under wan-shaped contacts, cup-shaped contacts, and spiral-shaped contacts was simulated. The ring-shaped distribution of the cathode spots between cup-shaped contacts with a large current in the initial diffusion stage was formed by the self-generated magnetic field of cathode spots, while the axial magnetic field component and the clustered cathode spots destroyed the ring-shaped distribution. Taking the distribution of cathode spots and current from experiments as initial conditions, the simulation results of cathode spot distribution in the initial diffusion stage and the arc root radius were consistent with the experimental results.
The missing role of hydrodynamic stresses on ascending aortic dissection
Authors: Karachaliou Marina G. , Messaris Gerasimos A. T. , Loukopoulos Vassilios C. , Koutsojannis Constantinos M. , Zampakis Petros E. , Karahalios George T.
Published: January 2021

Ascending aortic dissection is a lethal illness characterized mainly by a tear that develops in the aortic wall when the wall stress by the blood pressure exceeds the wall endurance limits. The estimation of the timing for a surgical treatment of a dilated ascending aorta is based, in practice, on the aortic size, and a recommended criterion for preventive surgical repair is an aortic diameter equal to 5 cm–5.5 cm. However, cardiothoracic surgeons refer to the dissection size paradox, which means that they often encounter unsuspected cases of ascending aortic dissection and/or rupture at aortic diameters less than the recommended criterion. The Laplace law, which is employed by the clinicians for the estimation of wall stress, is generally insufficient to predict ascending aorta dissection because it applies to a straight tube under uniform static blood pressure, whereas an ascending aorta is a curved tube under static and hydrodynamic stresses. Here, we take into account the dynamic stresses generated by the curvilinear motion of the blood and produce novel expressions, which explain the dissection size paradox and indicate the location at which a dissection may appear. Our results are in agreement with the increasing clinical evidence that the aortic length is an indispensable complement to the aortic diameter in the risk stratification and in the decision making for an in time surgical intervention. Employing the derived expressions, a preventive health care system tool can be developed, which will unveil “unsuspected” cases that previously were considered of lower risk for surgical intervention.
Low-frequency noise assessment of ferro-electric field-effect transistors with Si-doped HfO2 gate dielectric
Authors: Simoen E. , O’Sullivan B. J. , Ronchi N. , Van den Bosch G. , Linten D. , Van Houdt J.
Published: January 2021

The low-frequency noise of planar transistors with ferroelectric Si-doped HfO2 as a gate dielectric is investigated and compared with that of undoped HfO2 reference devices. Predominantly 1/f-like spectra have been observed, which are governed by carrier number fluctuations or trapping in the gate stack. The corresponding noise power spectral density is about a factor of three higher for the reference devices, indicating that Si-doping reduces in a way similar to the trap density in the HfO2 layer.
Effects of sodium chloride on structure and compressive properties of foamed AZ91
Authors: Kang Li , Shi Yixuan , Luo Xiaoping
Published: January 2021

The effects of additional sodium chloride particles on the structure and quasi-static compressive properties of open cell AZ91 alloy foams were studied using the infiltration method. Optimal variables, such as the particle size, preheating temperature, and pressure during formation, were obtained using experiments. Furthermore, the fragility and the resultant fracturing of the particles during foam formation were studied. The experimental results show that the physical characteristics of semi-fragile sodium chloride have a prominent effect on the pore structure formation and the compressive behavior of foamed AZ91. Thus, this work reveals that sodium chloride particles can be stabilized to effectively control the pore structure of foam alloys and produce improvements for use in various engineering applications.
Charge trapping analysis in sputtered BixSe1-x based accumulation-mode FETs. II. Gate capacitance characteristics
Authors: Sahu Protyush , Chen Jun-Yang , Wang Jian-Ping
Published: January 2021

In this study, we extend the analyses done on sputtered BixSe1-x based accumulation mode FETs. Previously, we studied the basic electrical and leakage properties of these FET devices. We extend our analyses to obtain key parameters of the BixSe1-x (x = 0.44) film at various gate voltages. We start by extracting the sheet carrier density and bulk mobility for different gate voltages, using the Drude model with the previously obtained semi-empirical relationship between carrier concentration and bulk mobility for BixSe1-x. The change in sheet carrier density is a result of accumulation or depletion or majority carriers from the BixSe1-x/SiO2 interface, which show hysteretic behavior. This allows us to calculate the surface sheet carrier density and the quasi-static capacitance at various gate voltages. We use a simple capacitive model to separate the capacitance originating from the gate and the film bulk. The capacitance from the film bulk is due to the surface charge thickness and is directly dependent on the Debye length. From the change of capacitance, with respect to gate voltage, we were able to identify the characteristics of the conduction band edge and the bulk band gap/Dirac cone.
Local inspection of magnetic properties in GdMnIn by measuring hyperfine interactions
Authors: Cabrera-Pasca G. A. , Magno J. F. , Ferreira W. L. , Campos A. C. , Bosch-Santos B. , Sales T. S. N. , Pereira L. F. D. , Burimova A. , Saxena R. N. , Freitas R. S. , Carbonari A. W.
Published: January 2021

GdMnIn is reported to crystallize in the hexagonal MgNi2-type structure presenting a spin-glass behavior with no magnetic order attributed to the triangular spin frustration of magnetic ions. In the present work, FC-ZFC magnetization, specific heat and AC susceptibility measurements along with the local magnetic exchange measured by hyperfine interactions at In sites are used to investigate the magnetic behavior in GdMnIn compound. The ZFC-FC magnetization curves exhibit an inflection which was ascribed to the antiferromagnetic transition at TN= 145 K. These curves also give an indication of thermomagnetic irreversibility at 118 K, which along with the absence of inflection in specific heat results might be associated to spin-glass behavior. Results of AC susceptibility and magnetic hyperfine field measured using 111In(111Cd) probe nuclei carried out by perturbed angular correlations (PAC) technique did not show evidence of spin-glass behavior. The thermomagnetic irreversibility in FC-ZFC curves along with results of hyerfine interactions suggest the presence of magneto-crystalline anisotropy effects and a weak long-range coupling in GdMnIn.
Cryogenic motor with two radial AMBs in liquid nitrogen
Authors: Komori M. , Kato H. , Asami K. , Sakai N.
Published: January 2021

A cryogenic motor with active magnetic bearings (AMBs) which works in liquid nitrogen is discussed in this paper. The cryogenic motor is composed of two AMBs, four displacement sensors, a permanent magnet (PM) motor, etc. In order to check the stability for the rotor, FFT (fast Fourier transform) analysis for the levitated rotor in the radial direction is performed. Displacement of rotor in the axial direction is studied during rotation.
Spin injection characteristics of Py/graphene/Pt by gigahertz and terahertz magnetization dynamics driven by femtosecond laser pulse
Authors: Idzuchi H. , Iihama S. , Shimura M. , Kumatani A. , Mizukami S. , Chen Y. P.
Published: January 2021

Spin transport characteristics of graphene have been extensively studied so far. The spin transport along the c-axis is however reported by rather limited number of papers. We have studied spin transport characteristics through graphene along the c-axis with permalloy(Py)/graphene(Gr)/Pt by gigahertz (GHz) and terahertz (THz) magnetization dynamics driven by femtosecond laser pulses. The relatively simple sample structure does not require electrodes on the sample. The graphene layer was prepared by chemical vapor deposition and transferred on Pt film. The quality of the graphene layer was characterized by Raman microscopy. Time-resolved magneto-optical Kerr effect is used to characterize gigahertz magnetization dynamics. Magnetization precession is clearly observed both for Pt/Py and Pt/Gr/Py. The Gilbert damping constant of Pt/Py was 0.015, indicating a spin pumping effect from Py to Pt. The Gilbert damping constant of Pt/Gr/Py was found to be 0.011, indicating that the graphene layer blocks spin injection. We also performed the measurement of THz emission for Pt/Py and Pt/Gr/Py. While a THz emission is clearly observed for Pt/Py, a substantial reduction of THz emission is observed for Pt/Gr/Py. With these two different experiments, and highly anisotropic resistivity of graphite, we conclude that the vertical spin transport is strongly suppressed by the graphene layer.
Environmental impacts on sperm and oocyte epigenetics affect embryo cell epigenetics and transcription to promote the epigenetic inheritance of pathology and phenotypic variation
Authors: Nilsson Eric , Ben Maamar Millissia , Skinner Michael K.
Published: January 2021

Previous studies have demonstrated that exposure to environmental factors can cause epigenetic modifications to germ cells, particularly sperm, to promote epigenetic and transcriptome changes in the embryo. These germ cell and embryo cell epigenetic alterations are associated with phenotypic changes in offspring. Epigenetic inheritance requires epigenetic changes (i.e. epimutations) in germ cells that promote epigenetic and gene expression changes in embryos. The objective of this perspective is to examine the evidence that germ cell epigenome modifications are associated with embryo cell epigenetic and transcriptome changes that affect the subsequent development of all developing somatic cells to promote phenotype change. Various epigenetic changes in sperm, including changes to histone methylation, histone retention, non-coding RNA expression and DNA methylation, have been associated with alterations in embryo cell epigenetics and gene expression. Few studies have investigated this link for oocytes. The studies reviewed herein support the idea that environmentally induced epigenetic changes in germ cells affect alterations in embryo cell epigenetics and transcriptomes that have an important role in the epigenetic inheritance of pathology and phenotypic change.
Dry storage of mammalian spermatozoa and cells: state-of-the-art and possible future directions
Authors: Loi P. , Anzalone D. A. , Palazzese L. , Dinnyés A. , Saragusty J. , Czernik M.
Published: January 2021

This review provides a snapshot of the current state-of-the-art of drying cells and spermatozoa. The major successes and pitfalls of the most relevant literature are described separately for spermatozoa and cells. Overall, the data published so far indicate that we are closer to success in spermatozoa, whereas the situation is far more complex with cells. Critical for success is the presence of xeroprotectants inside the spermatozoa and, even more so, inside cells to protect subcellular compartments, primarily DNA. We highlight workable strategies to endow gametes and cells with the right combination of xeroprotectants, mostly sugars, and late embryogenesis abundant (LEA) or similar ‘intrinsically disordered’ proteins to help them withstand reversible desiccation. We focus on the biological aspects of water stress, and in particular cellular and DNA damage, but also touch on other still unexplored issues, such as the choice of both dehydration and rehydration methods or approaches, because, in our view, they play a primary role in reducing desiccation damage. We conclude by highlighting the need to exhaustively explore desiccation strategies other than lyophilisation, such as air drying, spin drying or spray drying, ideally with new prototypes, other than the food and pharmaceutical drying strategies currently used, tailored for the unique needs of cells and spermatozoa.
102 Age-related differences in seminal parameters and expression of fertility marker genes in Marwari stallion
Authors: Chaudhary A. K. , Purohit G. N. , Mehta J. S. , Ravi S. K. , Talluri T. R.
Published: January 2021

The objective of the present study was to observe age-related differences in seminal parameters and expression of fertility marker genes in Marwari stallions. Semen was collected from 9 Marwari stallions (6 ejaculates per stallion) of 3 different age groups (2 to 4 years, GI; 4 to 6 years, GII; and >6 years, GIII) twice a week using an artificial vagina by allowing the stallions to mount a mare. The collected ejaculate was divided into 2 parts. Part A was used for evaluation of various seminal parameters (colour, consistency, total volume, gel volume, gel-free volume, pH, progressive sperm motility, sperm concentration, sperm viability, total sperm morphological abnormalities, plasma membrane integrity, acrosomal integrity, and DNA integrity). Part B was centrifuged to obtain the sperm pellet for DNA extraction. Six fertility-related marker genes (SPATA1, SP17, PLCz, CRISP3, UBB, and PRM1) were examined in the spermatozoa DNA using PCR. Expression levels of these genes were also studied using quantitative real-time PCR. Data obtained were analysed statistically by one-way or two-way analysis of variance using the SPSS computer program (version 20.0; IBM Corp.). No significant difference was observed among the GI, GII, and GIII stallions for seminal parameters, except mean sperm concentration, which was lowest in 2- to 4-year-old stallions (146.06±11.50 million mL−1), intermediate in stallions >6 years old (182.03±8.51 million mL−1), and highest in stallions of 4 to 6 years (270.92±9.12 million mL−1; P<0.01). The study demonstrated that all six fertility-related genes showed differential expression in stallions from the GI, GII, and GIII groups. In addition, expression levels of the same genes varied across individuals. Expression of SPATA1, SP17, PLCz, CRISP3, UBB, and PRM1 genes was reduced in stallions below 4 years of age compared with older stallions. This result suggests that expression of these genes increases with age, although possibly only up to a certain age. We inferred that stallions around 4–6 years of age can be considered optimum to use for breeding purposes, provided seminal parameters are normal.
12 Artificial insemination and embryo transfer results in ewes during a long daylength period
Authors: Mittleider T. , Collins S. , Gibbons P. , Gibbons J.
Published: January 2021

Sheep are polyestrous, short-day breeders (∼11h of daylength), and exhibit oestrus approximately every 16–17 days during the breeding season, usually in late September to late December in the Northern Hemisphere. Progressive sheep producers often use assisted reproductive techniques such as laparoscopic AI and ovarian hyper-stimulation, embryo collection, and embryo transfer (ET) to increase genetic gain, and strive to have early December lambs to target specific show markets, which dictates AI or ET during the late summer. This field trial compared pregnancy rates following AI or ET in July and August (∼14h of daylength) in southwest Virginia (36–38′12″ N). Ewes (AI, n=83; ET recipients, n=33) were synchronized using a modified Ovsynch protocol involving intravaginal progesterone implants for 14 days, prostaglandin F2α (intramuscular) 48h before expected oestrus, and PG600 (IM) and gonadotrophin-releasing hormone (intramuscular) 52 to 54h and 16h before AI, respectively. Ewes were subjected to AI (frozen/thawed semen) regardless of whether they displayed signs of oestrus, and ewes selected as embryo recipients were subjected to a similar protocol but instead received 1 or 2 embryos (based upon the number of viable embryos produced per embryo donor) 6 days following the AI of the embryo donors. Ovarian hyper-stimulation of the embryo donors (n=13) was enabled by twice-daily FSH injections [totalling 350–455IU of Folltropin V (10–13mL)] for the 4 days before AI. Six days following AI, embryos were recovered surgically from the embryo donors (n=13) and yielded an average (±s.e.m.) of 6.6±1.2 total ova, 4.7±1.1 transferable quality embryos, and 1.9±0.8 unfertilized ova per collection. Pregnancy was detected using transrectal ultrasonography at ∼30 days of gestation and the pregnancy rates were analysed using Chi-squared. There was a tendency (P=0.092) for more pregnancies to be established following ET (22/33; 66%) compared with AI (41/83; 49%). There was no statistical relationship between AI ewes or ET recipient ewes that became pregnant relative to whether they displayed signs of oestrus or not. Embryo transfer was a more successful approach to produce pregnancies in ewes compared with AI during long daylength periods in this field trial. Further, ova from hyper-stimulated embryo donor ewes experienced a very high fertilization rate. Future studies will evaluate the ova capability directly via laparoscopic aspiration of ovarian follicles and IVF and evaluation of hyper- and non-hyper-stimulated follicular waves (using transrectal ultrasonography) and endocrine dynamics during different long and short daylength periods. Extending the opportunity to generate embryos and offspring regardless of daylength will assist aggressive sheep producers in reaching their reproductive, financial, and genetic goals.
120 High variation in prediction of developmental competence of bovine oocytes based on visual examination
Authors: Raes A. , Pascottini O. B. , Opsomer G. , van Soom A.
Published: January 2021

The quality of an oocyte is often represented by its morphological characteristics. Stringent selection of bovine cumulus–oocyte complexes (COCs) for invitro embryo production (IVP) is regularly based on parameters such as colour and granulation of the ooplasm, density of the surrounding cumulus cells, brightness of the zona pellucida, among other less common parameters. The link between developmental competence and morphology of the COC has been demonstrated in literature in group culture system. However, this has not been yet examined in an individual invitro production system. This study investigates the ability of IVP researchers to predict whether an oocyte will develop into a blastocyst at Day 8 of invitro culture in an individual production system. A set of 29 pictures of immature bovine COCs was presented in duplicates to eight bovine IVP researchers with different institutional backgrounds. The observers were asked if they would select each oocyte for further IVP processing, assuming that only the oocytes with the highest developmental potential can proceed, and thus a stringent selection is warranted. These 29 immature oocytes were matured, fertilized, and cultured individually invitro (using conventional methods) so that their ability to develop into a blastocyst at Day 8 post-fertilization was known. The ability to reach the blastocyst stage was stated as the gold standard and compared with the answers of the observers using kappa statistics and sensitivity (Se) and specificity (Sp) tests. Kappa (κ) for interobserver agreement was κ=0.01 (Se=50%, Sp=53%; positive predictive value=14%, negative predictive value=87%) with 50% accuracy of prediction. The intraobserver agreement was κ=0.69, with κ=1 referring to 100% agreement (Se=87%, Sp=83%), with an accuracy of 85%. The ability of trained embryologists to predict the fate of oocytes’ developmental competence was accurate for only half of the presented oocytes. Additionally, the proportion of false positive (1 − Sp) and false negative (1−Se) predictions was equally distributed. The low positive predictive value might be due to the lower development rates in individual culture systems versus group culture systems, whereas observers are mainly trained to select oocytes for group culture. This study demonstrated the subjective nature of the oocyte selection process that is based on visual examination of COC morphology because interobserver agreement was almost nonexistent. Interpretation of the morphology within an observer remains consequent, represented by the high intraobserver agreement. Consequently, there is a need to develop a new model for the objective determination of oocyte quality for individual IVP. The implementation of machine learning algorithms could objectively enhance oocyte selection and artificial reproductive technologies in extension.
129 Polar body extrusion following exposure of pig oocytes to different invitro maturation media
Authors: Mphaphathi M. L. , Honneysett K. M. , Maduwa T. T. , Pohler K. G. , Nedambale T. L.
Published: January 2021

The invitro maturation (IVM) rate of pig oocytes is a critical step for subsequent invitro embryo production. The objective of the present study was to compare different IVM media (NCSU 23, NCSU 37, TCM-199, pFF, BO-IVM commercial, and Bovine IVM) on polar body extrusion in pig oocytes following 44h of maturation duration. Ovaries from sows were collected from the local slaughterhouse and transported in warmed (38°C) saline to the laboratory, within 2h following slaughter. The slicing method was used to retrieve oocytes from the ovaries. Collected oocytes (n=353) were selected and washed three times in TCM-199+10% fetal bovine serum. The oocytes were then subjected to IVM as per treatment groups for 44h. Following maturation period, oocytes were removed from each maturation medium and denuded of granulosa cells by vortexing. The oocytes polar body extrusion was evaluated with the aid of OosightTM Imaging Sytem connected to an inverted research microscope. Treatment means were compared using Fisher’s protected least significant difference t-test. Polar body extrusion was 30.0, 18.5, 32.8, 22.1, 32.1, and 27.0% for BO IVM commercial, Bovine IVM, NCSU 23, NCSU 37, TCM-199, and pFF media, respectively (P>0.05). The NCSU 23 and TCM-199 had numerically higher percentages of oocytes with extruded polar body compared with other treatments groups (P>0.05). The lowest (18.5%) polar body extrusion was recorded in Bovine IVM medium. In conclusion, all the IVM media tested can be used during IVM of pig oocytes, with NCSU 23 and TCM-199 having numerically higher polar body extrusion percentages.
135 Differential gene expression of bovine long noncoding RNAs in single oocytes aspirated from small and large follicles
Authors: Current J. , Baldwin H. , Zhang M. , Yao J.
Published: January 2021

In mammals, maternal mRNAs accumulate in the oocyte and play important roles during early embryogenesis in embryonic genome activation. Proper development is dependent upon regulation of these maternal transcripts while simultaneously activating the embryonic genome. Long noncoding RNAs (lncRNAs) regulate various cellular processes, including modulation of gene expression and epigenetic status. Recently, lncRNAs have been functionally characterised as key regulators of embryonic genome activation in humans and mice. Our laboratory identified 1535 lncRNAs in bovine oocytes using RNA-sequencing. The objective of this study was to verify our RNA-seq data and if present, characterise the expression of six highly abundant lncRNAs (2160.1, 5379.1, 18208.1, 23721, 25823.1, and 34876.2) in single oocytes aspirated from varying size follicles at different developmental stages. Single oocytes were aspirated from small (<4mm) and presumably oestrogen active (6–18mm) follicles and were denuded at the germinal vesicle (GV) or MII stage. MII stage was determined by cumulus expansion and the extrusion of the first polar body. Real-time quantitative PCR analysis, using RPL-19 as a housekeeping gene analysed using the standard curve method, revealed detectable expression for each lncRNA in single oocytes from small (SF) and presumably oestrogen active (EA) follicles at both stages. Average expression of each lncRNA from SF was used as a calibrator in normalization. Effect of follicle size and maturity stage and their interaction on the lncRNA expression were examined using a two-factorial ANOVA followed by Tukey’s HSD post hoc test. In five lncRNAs (2160.1, 5379.1, 18208.1, 25823.1, and 34876.2), maturity stage was significant (P<0.05). Moreover, we found significant interaction of stage and size in expression of lncRNA 25823.1 (P<0.05). SF oocytes at the GV stage had the highest relative expression of lncRNA 25823.1. Specific differences were detected between SF MII and SF GV oocytes, indicating higher expression in the earlier developmental stage (P<0.05). The SF oocytes at MII had a 0.63-fold decrease in relative expression with respect to the SF oocytes at GV. In addition, we found a tendency for significant interaction between maturity stage and follicle size in the expression of lncRNA 5379.1 (P<0.1). LncRNA 5379.1 exhibited the same expression pattern as lncRNA 25823.1 with SF oocytes at MII having a 0.75-fold decrease in relative expression relative to SF oocytes at GV. In summary, both lncRNA 25823.1 and 5379.1 had higher expression in oocytes aspirated from small follicles during the earlier developmental stage. The literature indicates that follicle size can be an indicator of oocyte quality and that RNAs accumulated or degraded during maturation are crucial to achieve developmental competence. Therefore, lncRNAs 25823.1 and 5379.1 shows promise to be linked with oocyte quality.
138 Effect of different temperatures on invitro maturation rates on cattle oocytes
Authors: Sebopela M. D. , Mphaphathi M. L. , Sithole S. M. , Nedambale T. L.
Published: January 2021

Climate change represents a major stressful environmental condition that compromises the reproductive efficiency of animals. The temperature affects various components of the maturation processes in cattle oocytes, this includes disruptions of oocyte development and maturation. Therefore, the study aimed to compare the effects of different incubation temperatures (32.5, 33.5 34.5, 35.5, 36.5, 37.5, 38.5, 39.5, 40.5, 41.5, 42.5, and 43.5°C) on the maturation rate of cattle oocytes. Cattle ovaries were collected from a local abattoir and transported to the laboratory in thermo flask at 37°C. Oocytes were recovered from ovaries using the aspiration method and matured using 500 uL of TCM-199 medium supplemented with 10% fetal bovine serum, FSH, LH, and E2 covered with mineral oil. The maturation rate of oocytes was determined by the extrusion of the first polar body after 22h of IVM period. The total number of 80 oocytes were matured in each group and replicated 4 times. Data were analysed using GenStat® statistical program (VSN International). Comparisons were considered significantly different (P<0.05) using Fisher’s protected least significant difference test. The oocyte polar body extrusion at 32.5 (0±0), 33.5 (4.5±5.2), 34.5 (22.0±1.4), 35.5 (29.5±7.6), 36.5 (41.5±3.1), 37.5 (55.5±4.1), 38.5 (62.2±3.3), 39.5 (49.5±3.3), 40.5 (17.3±6.4), 41.5 (10.3±5.7), 42.5 (8.2±5.9), and 43.5°C (5.7±7.8) (P<0.05) was recorded. The highest percentages of oocytes with polar body extrusion were at 37.5°C (55.5±4.1) and 38.5°C (62.2±3.3) and differed significantly from all other treatment groups (P<0.05). Temperature lower than 37.5°C and higher than 39.5°C did not intensify oocyte polar body extrusion. We concluded that the temperatures of 37.5°C and 38.5°C were suitable for IVM of cattle oocytes with acceptable polar body extrusion rates compared with other temperatures.
139 Effect of lycopene supplementation in maturation medium on production of reactive oxygen species and post-vitrification quality of bovine blastocysts
Authors: Sidi S. , Pascottini O. B. , Angel-Velez D. , Dolatabad N. A. , Residiwati G. , Van Damme P. , Bawa E. K. , Van Soom A.
Published: January 2021

Excessive production and accumulation of reactive oxygen species (ROS) may cause embryo damage associated with oxidative stress. Lycopene, a natural antioxidant, can scavenge singlet oxygen and is one of the most effective antioxidants among carotenoids. We evaluated the effects of supplementation of lycopene (antioxidant), menadione (prooxidant), and their combination during invitro oocyte maturation on ROS generation in matured oocytes and the quality of vitrified-warmed embryos. Cumulus–oocyte complexes, collected from the slaughterhouse, were matured in groups of 60 in 500μL of TCM-199 medium+50mg mL−1 gentamycin+20ng mL−1 epidermal growth factor, for 22h at 38.5°C in 5% CO2 in air and then supplemented with (1) 0.2μM lycopene, (2) 5μM menadione, (3) 0.2μM lycopene+5μM menadione (L+M), or (4) not supplemented (control). Fertilization and embryo culture were performed similarly for all the groups. In the first experiment, ROS measurement (n=236; via fluorescent microscopy) was performed in denuded, matured oocytes incubated in 5μM CellROX® Green (ThermoFisher Scientific) for 1h. Fluorescent intensity was measured in Image-J. In the second experiment, embryos in the blastocyst stage (n=143) were vitrified as previously described by Ortiz-Escribano et al. (2017 Biol. Reprod. 96, 288-301). Vitrified blastocysts were then warmed and washed in decreasing concentrations of sucrose and incubated for 2 days in culture medium [50µL of synthetic oviductal fluid (SOF)+(5g mL−1 insulin, 5g mL−1 transferrin, 5ng mL−1 selenium)]. The quality of vitrified-warmed blastocysts was assessed using a differential staining as described by Wydooghe et al. (2011 Anal. Biochem. 416, 228–230). The effects of pro- and antioxidant supplementation on oocyte fluorescent intensity and embryo quality parameters were fitted in linear mixed-effects models, and results are expressed as least squares means and standard errors. The fluorescent intensity for ROS was lower (P<0.05) in lycopene (10.06±2.92) than in menadione (16.8±2.92). No differences (P>0.05) in ROS intensity values were found among the other groups [control (13.5±2.92) and L+M (13.7±2.90)]. Total cell number (TCN) was similar (P>0.05) in lycopene (153±2.95), L+M (143±4.59), and control (145±3.67) but lower (P<0.05) in menadione (134±6.08). Lesser numbers of apoptotic cells (AC) and AC/TCN values (P<0.05) were recorded in lycopene (4.12±3.07 and 2.71±2.21) compared with control (6.18±3.82 and 4.31±2.75), L+M (6.00±4.79 and 4.22±3.45), and menadione (7.75±6.33 and 5.82±4.56). For the remaining embryo quality parameters, no differences were found (P>0.05). In conclusion, lycopene supplementation during invitro oocyte maturation effectively scavenged free radicals, lowering oxidative stress and improving embryo quality post-vitrification and warming.
14 Telomere length in cloned camels produced by somatic cell nuclear transfer is not different from that in their naturally produced counterparts
Authors: Wani N. A. , Kumar K. P.
Published: January 2021

There are controversial reports on the restoration of eroded telomere length in offspring produced by somatic cell nuclear transfer (SCNT) in different animal species. To the best of our knowledge, no earlier studies report telomere length in naturally produced or cloned animals in any of the camelid species. Therefore, the present study was conducted to estimate the telomere length in dromedary camels produced by SCNT, and their age-matched naturally produced counterparts by terminal restriction fragment (TRF) analysis. Genomic DNA was extracted from venous blood collected from 6 cloned animals (aged 3, 12, and 24 months), and their age-matched counterparts by a conventional phenol/chloroform protocol. Denatured and neutralized DNA was blotted onto a positively charged nylon membrane and fixed by baking at 80°C for 3h. After washing in a prewarmed digoxigenin (DIG) easy hybridization solution at 42°C for 1h, DNA hybridization was carried out using a telomere (TTAGGG)n-specific, DIG-labelled hybridization probe (Roche Diagnostics, Germany) at 42°C for 4h. Stringent washes were carried out at the same temperature, followed by chemiluminescence reaction. The signals were captured using the Azure Biosystems C600 gel documentation system. Molecular weights of the unknown TRF bands on the gel were calculated using known molecular weight marker provided by Telo TAGGG Telomere Length Assay Kit (Roche Diagnostics). A TeloTool program from MATLAB software with a built-in probe intensity correction algorithm was used for TRF analysis. The experiment was replicated 3 times, and the data, presented as mean±s.e.m., were analysed using a 2-sample t-test (Minitab statistical software). No difference (P>0.05) was found in the mean telomere length of cloned camels compared with their naturally produced age-matched counterparts (21.7±0.3 vs. 22.1±0.3; 21.9±0.3 vs. 22.1±0.3; 22.2±0.5 vs. 22.0±0.1; 20.5±0.5 vs. 22.5±0.7; 20.1±0.1 vs. 22.5±0.7; 21.7±1.1 vs. 22.6±0.2), respectively. In conclusion, this is the first study where telomere length has been reported in naturally produced and cloned dromedary camels produced by SCNT. We found that telomere lengths in cloned camels were similar to those of their age-matched naturally produced counterparts, suggesting that the camel cytoplast reprograms the somatic cell nucleus and restores the telomere length to its totipotency stage.
143 Therapeutic efficacy and safety of adipose tissue-derived mesenchymal stem cells in treating mastitis and metritis in dairy cattle
Authors: Singh R. , Bhaskar V. , Saini S. , Kumar A. , Thakur A. , Kumar S. , Malakar D.
Published: January 2021

Mastitis and metritis are two inflammatory diseases with high economic impact on dairy industry and farmers are losing revenues. Antibiotic treatment for these disease conditions typically achieve suboptimal outcomes. Moreover, emerging antibiotic resistance in mastitis and metritis worldwide has a severe effect on productivity, fertility, and lifespan of animals. A paradigm shift in treatment strategies is much needed for these animals. Attracting properties of mesenchymal stem cells may act as an alternative strategy for treating these diseases. In addition to their ability to differentiate into multiple types of cells, mesenchymal stem cells can orchestrate immune responses and modulate tissue microenvironments. In the present study, we evaluated the safety and efficacy of adipose tissue–derived mesenchymal stem cells (AT-MSC) for mastitis and metritis. A well-characterised adipose-derived stem cell line with 3–5 passages cells were used for treatment for these diseases. In a safety trial, mastitis- and metritis-infected cattle were administered with 1×106 cells of AT-MSC through different routes (local, IV, IV+local). Animals were clinically evaluated during 15 days of the experimental period and blood samples were collected for hemogram determination. The efficacy was checked under both invivo and invitro conditions. Invitro efficiency of AT-MSC was determined using a transwell plate experiment, in which AT-MSC were co-cultured with bacteria (Escherichia coli and Staphylococcus). Invivo efficiency was evaluated by administration of in 2mL (1×106 cells) of AT-MSC dose; then, somatic cell count (SCC) in milk of mastitic cattle and polymorphonuclear (PMN) cell count in cervical vaginal fluid (CVF) of metritic cattle were determined. Gene expression profiling of antimicrobial (cathelicidin, lipocalin, cystatin) and anti-inflammatory (IL-4, IL-6, IL-10) peptides were quantified in all groups: IV, local, IV+local, and control ATB (antibiotic). Allogenic AT-MSC did not induce any immunological rejection response in treated animals. A significant reduction of bacteria in CVF invitro when co-cultured with AT-MSC was observed. The PMN count was significantly reduced in CVF of the IV+local group. The SCC in milk of mastitic animals was decreased in the local (intramammary) group. Maximum expression of IL-6, IL-10, cathelicidin, lipocalin, and cystatin and angiopoietin genes were observed on Day 3 followed by Day 7 in the local group in case of mastitis. In metritis, expression of IL-6, IL-10, cathelicidin, lipocalin, cystatin, and angiopoietin were observed at Day 3 in the IV+local group. All mastitic and metritic cattle were completely and permanently cured within 30 days after treatment with mesenchymal stem cells. In conclusion, we provide initial evidence for the use of allogenic AT-MSC and their safety and efficacy to treat mastitis and metritis in dairy cattle.
146 Single injection of follicle-stimulating hormone before ovum pickup in lactating Holstein donors: Oocyte recovery and embryo production
Authors: Santos R. M. , Oliveira M. , Demetrio C. G. B. , Hasler J. H. , Fonseca J. C. , Demetrio D. G. B.
Published: January 2021

Lactating donor cows frequently have decreased oocyte quality, lower fertilization rates, and impaired early embryonic development due to their lactational metabolic challenges. Synchronization and stimulation of follicular growth before ovum pickup (OPU) has been used to improve oocyte quality and consequently, embryo production. The objective of this study was to evaluate the effects of a single injection of FSH before OPU on oocyte recovery and embryo production in lactating Holstein donors. During June and July 2020, 22 lactating Holstein donors (open, 40 to 90 DIM, producing >90 lbs of milk) from Ruann Dairy (Riverdale, CA) were randomly assigned to one of two treatments (crossover design). Donors did not receive any injections before OPU when assigned to the No FSH treatment. Treatment 1×FSH consisted of a single intramuscular (IM) injection of 140mg of FSH (Folltropin®, Vetoquinol) 36h after gonadotrophin-releasing hormone (GnRH; Fertagyl, Merck®, 129µg, IM). The FSH consisted of a 3.5-mL IM injection of 400mg of FSH diluted in 10mL of 0.5% hyaluronan (HA). OPU was performed 48 to 52h after FSH. All donors received both treatments on a 14-day interval. The recovered oocytes were fertilized with the same sexed female-sorted semen in both rounds (3 different sires were used). OPU, oocyte classification, IVM, IVF, and culture (IVC) were performed as described by Demetrio et al. (2020 Anim. Reprod. 17, e20200053). All oocytes went into IVM, except for degenerated occytes. The number of 4-cell (or more) embryos on Day 3 of IVC divided by the number of oocytes in IVC after IVF is defined as the cleavage rate. The number of blastocysts (early to hatched) on Day 7 of IVP divided by the number of oocytes in IVC after IVF is defined as the blastocyst rate. Poisson-normal (count data) and Logistic-normal (proportion data) models were used to analyse the data. Treatment, donor (random effect), and sire were included in the models. The results are summarised in Table 1. Oocyte recovery and embryo production were highly donor dependent. There were no differences in the number of recovered oocytes among treatments. Stimulation of the follicular growth before OPU with one single injection of FSH diluted in 0.5% HA 36h after GnRH improved oocyte quality, cleavage rates, blastocyst rates, embryo quality, and the total number of embryos per OPU in lactating Holstein donors.

Table 1.
Oocyte recovery, cleavage rate, and embryo production results

Treatment
OPU
Oocytes per donor
Grade 1 and 2 oocytes
Cleavage rate (%)
Blastocyst rate (%)
Total embryos per OPU
Grade 1 embryos per OPU

No FSH
22
18.2
30a
69a
22a
3.4a
1.6a

1×FSH
22
17.5
34b
77b
34b
5.3b
3.3b

a,bValues with different superscripts in the same column differ (at least P<0.05).
20 Development and Oct4/Cdx2 gene expression of Puma concolor, Leopardus geoffroyi, and Panthera onca hybrid embryos produced using domestic cat oocytes
Authors: Rodriguez M. D. , Sestelo A. , Buemo C. , Ratner L. D. , Fernandez-Martin R. , Salamone D. F.
Published: January 2021

The development of assisted reproductive technologies (ARTs) in Felidae species has been proposed to guarantee the genetic viability of species that are on the verge of extinction. Investigation of sperm cryopreservation and IVF have been studied in several Felidae species, but the lack of viable homologous oocytes makes assessment of sperm fertilization capacity after cryopreservation difficult. We propose to evaluate the capability of Puma concolor (Pc), Leopardus geoffroyi (Lg), and Panthera onca (Po) cryopreserved sperm to induce domestic cat oocyte fertilization and subsequent pre-implantation embryo development. Additionally, we evaluated blastocyst quality by analysing the gene expression patterns of pluripotency (Oct4) and differentiation (Cdx2) related markers using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). After ovariectomy, domestic cat (Felis catus, Fc) cumulus–oocyte complexes were matured invitro at 38.5°C in a humidified atmosphere of 6.5% CO2 and 21% O2 for 22h. Cryopreserved sperm from Pc, Lg, and Po were used for heterologous IVF. The sperm was frozen using Tes-Tris–based diluent (TEST), and survival, sperm motility, and acrosome integrity were evaluated. The quality post-thaw was satisfactory. The survival was high, sperm motility (65%) did not decrease during incubation post-thaw, and sperm with intact acrosome was 62% for all species. Briefly, straws were thawed by exposing them to air for 10s and then immersing in a 37°C water bath for 30s. The contents of the straws were poured into prewarmed sterile 1.5-mL microtubes. The sperm suspension was diluted (1:3 vol/vol) by the slow (drop by drop) addition of a modified Tyrode’s solution (Pope et al. 2006 Methods Molec. Biol. 254, 227-244). For IVF, invitro matured oocytes (n=635) were co-incubated with 0.5×105 mL−1 motile spermatozoa at 38.5°C in a humidified atmosphere of 6.5% CO2 and 21% O2 for 18 to 20h. Presumptive zygotes were cultured invitro in 50-µL drops of modified Tyrode’s medium in a humidified gas mixture of 5% CO2, 5% O2, and 90% N2 at 38.5°C. Cleavage was determined 48h post-fertilization and blastocyst stage was evaluated at Day 8 and stored in RNA-Later at −20°C until RT-qPCR analysis. Data were analysed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc.), and differences were considered significant at P<0.05. We observed significantly lower cleavage rates of Po hybrid (hPo) embryos compared with hPc, hLg, and control Fc embryos (41% vs. 57%, 68%, and 58%, respectively). Additionally, we found that the blastocyst rate was higher using Lg sperm compared with sperm of other groups [hLg (50%) vs. hPc (29%), hPo (21%), and Fc (27%)].