Effects of cell concentration during cryopreservation on the post-thaw quality of Santa ines ram sperm.

BACKGROUND: Non-surgical artificial insemination techniques for sheep may benefit from larger numbers of sperm in the insemination dose because the ewe cervix is convoluted and often cannot be traversed with an insemination gun resulting in deposition of the sperm at the os cervix. OBJECTIVE: To compare a range of sperm concentrations when cryopreserving semen from Santa Ines rams and determine the effects of this on post-thaw quality. MATERIALS AND METHODS: One ejaculate from each ram (n = 10) was diluted to four sperm concentrations to obtain the following groups: G-400, G-800, G-1200, and G-1600 x 106 sperm/mL. The semen samples were packaged in 0.25 mL straws, cooled to 5 degree C, cryopreserved in liquid nitrogen vapor, thawed in a water bath (40 degree C per 20 s), and were analyzed for computerized kinetics, capacitation and acrosome integrity, and plasma membrane integrity of sperm. RESULTS: The G-400 treatment resulted in samples with the highest linearity and progressive motion (P < 0.05) and had significantly greater plasma membrane integrity, and lower capacitation and acrosome reaction rates compared to G-1600 (P < 0.05). Overall, use of the G-400 treatment resulted in better kinetics, less plasma membrane damage and less early capacitation. However, despite reducing the ejaculate yield and increasing the costs of the semen freezing process, the G-800 and G-1200 treatments make a greater absolute number of sperm with good kinetics, plasma membrane integrity and capacitation status available. CONCLUSION: Ram sperm concentration impacts cryopreservation, and higher concentrations may be advantageous if a single artificial insemination protocol is desirable. doi.org/10.54680/fr22610110812.

Modification of trout sperm membranes associated with activation and cryopreservation. Implications for fertilizing potential.

We investigated the effects of two trout sperm activation solutions on sperm physiology and membrane organization prior to and following cryopreservation using flow cytometry and investigated their impact on in vitro fertility. Overall, frozen-thawed samples had greater phospholipid disorder when compared with fresh samples (high plasma membrane fluidity; P < 0.0001) and sperm activated with water also had high plasma membrane fluidity when compared to sperm activated with Lahnsteiner solution (LAS; P < 0.0001). Following cryopreservation water activated samples had membranes with greater membrane protein disorganization compared with LAS but the membrane protein organization of LAS samples was similar to samples prior to freezing (P < 0.0001). Post-thaw water activation resulted in significant increases in intracellular calcium compared to LAS (P < 0.002). In vitro fertility trials with frozen-thawed milt and LAS activation resulted in greater fertility (45%) compared to water activated samples (10%; P < 0.0001). Higher fertility rates correlated with lower intracellular calcium with water (R(2) = -0.9; P = 0.01) and LAS (R(2) = -0.85; P = 0.03) activation. Greater plasma membrane phospholipid (R(2) = -0.89; P = 0.02) and protein (R(2) = -0.84; P = 0.04) disorder correlated with lower water activation fertility rates. These membrane organization characteristics only approached significance with LAS activation in vitro fertility (P = 0.09, P = 0.06, respectively). Potentially the understanding of sperm membrane reorganizations and the physiology associated with activation following cryopreservation may enable users in a repository or hatchery setting to estimate the fertilizing potential of a sample and determine its value.

Biobanking genetic resources: challenges and implementation at the USDA National Animal Germplasm Program.

There is adequate infrastructure in the US to identify and acquire germplasm from the major beef and dairy cattle and swine breeds. However, when we venture outside these species, the same tasks become more difficult because of a lack of breed associations, databases that include genotypic and phenotypic data and low numbers of animals. Furthermore, acquisition of germplasm from non-cattle and non-swine species can be difficult because these animals are often not located near the National Animal Germplasm Program, which makes collection and preservation of the samples in a timely manner that much more complicated. This problem is compounded because not all preservation protocols are optimised for field collection conditions or for all types of germplasm. Since 1999, the USDA National Animal Germplasm Program has worked to overcome these obstacles by developing policies, procedures and techniques in order to create a germplasm repository for all agricultural species (wild and domesticated) in the US. Herein, we describe these activities and illustrate them via a case study on how our efforts collecting Navajo-Churro sheep have created a secure backup of germplasm and how we specifically overcome these issues as they relate to rare and minor breeds of agricultural species.

A simple vitrification method for cryobanking avian testicular tissue.

Cryopreservation of testicular tissue is a promising method of preserving male reproductive potential for avian species. This study was conducted to assess whether a vitrification method can be used to preserve avian testicular tissue, using the Japanese quail (Coturnix japonica) as a model. A simple vitrification method that included dimethyl sulphoxide, ethylene glycol, and sucrose as cryoprotective agents, and allowed the storage of tissue in a sealed macrotube was applied to the testicular tissue from 1-wk-old Japanese quail. The vitrified tissue was warmed at room temperature or at 40°C. After warming, tissue was implanted onto the chorioallantoic membrane of 8- to 9-d-old chicken embryos and the vascularization of the grafts was evaluated. When compared with fresh tissue, the tissue that had been warmed at 40°C showed no difference in vascularization. The tissue that had been warmed at room temperature was significantly less vascularized than the fresh tissue. Vitrification of testicular tissue and storage in macrotubes provide a promising model for preservation and recovery of male germplasm of avian species.

Comparative costs of programmes to conserve chicken genetic variation based on maintaining living populations or storing cryopreserved material.

1. There have been substantial losses of chicken lines kept for research in recent years and the objective of this research was to critically review alternative methods of preserving genetic resources. 2. The costs of programmes using living populations, semen cryopreservation and reconstitution, and ovary and semen cryopreservation and reconstitution were evaluated over 20 years using biological parameters of cryopreservation and population reconstitution that were derived from the literature. 3. Keeping live populations was most cost effective for periods of up to three years, but keeping live populations is increasingly difficult to justify with longer periods and any research population that will not be used within five years should be cryoconserved and in situ maintenance discontinued. 4. The rapid reconstitution possible using ovaries and semen would allow the inclusion of cryopreserved material in a short-term research project with the cost of recovery included in the budget. The low cost of cryoconservation suggests that all avian material should be conserved and reconstituted when needed for research.

Inseminating fresh or cryopreserved semen for maximum efficiency: implications for gene banks and industry.

Developing gene bank germplasm collections for animal genetic resources requires establishing germplasm collection goals, that consider capturing the genetic diversity of the population in question and the amount of germplasm required for its reconstitution or other purposes, or both. Computing collection goals for chickens is complicated, compared with mammalian species, due to the multiple chances a single insemination of semen has to fertilize an egg. To address this issue, fertility data were used in conjunction with econometric procedures for determining production efficiency and diminishing returns. Experimental treatments consisted of inseminating fresh semen intravaginally (FIV), frozen-thawed semen inseminated intramagnally (FTIM), and frozen-thawed semen inseminated intravaginally (FTIV). Analysis revealed that the maximum efficiency for a single insemination was at postinsemination d 6, 8, and 3 for FIV, FTIM, and FTIV, respectively. But, additional benefit from a single insemination can be garnered by continuing to collect and incubate eggs to d 11, 17, and 11 for FIV, FTIM, and FTIV, respectively. By extending the insemination interval, the number of fertile eggs can be increased by 62 (FIV), 62 (FTIM), and 48% (FTIV). The ramifications of these results are profound when placed in the context of germplasm collection for gene banks. By using the FTIM treatment, the number of germplasm samples needed to secure a chicken breed, at the 150% level, can be reduced from the FAO projection of 2,454 to 386 straws (0.5 mL). Such a change represents a substantial reduction in collection, processing, and storage costs for gene banks. For industry, the results suggest that extending the time interval between inseminations will yield more fertile eggs and create opportunities to increase the number of hens mated to a rooster.

Evaluation of glycerol removal techniques, cryoprotectants, and insemination methods for cryopreserving rooster sperm with implications of regeneration of breed or line or both.

A series of experiments was designed to evaluate the quality of cryopreserved rooster sperm and its fertility so that programs needing to bank germplasm and recreate animals can do so utilizing a minimal amount of cryopreserved semen. In experiment 1, rooster semen from the National Animal Germplasm Program genebank was thawed and glycerol was removed using a discontinuous Accudenz column or by stepwise dilution. The postthaw sperm motilities, plasma membrane integrity, and concentration were determined before and after deglycerolization. Line differences in postthaw sperm concentration and progressive motility were observed before deglycerolization (P<0.05). After glycerol removal, the sperm that was centrifuged through Accudenz had greater total motility (37 vs. 33% sperm; P<0.05), but use of the stepwise dilution method recovered more sperm per milliliter (320.4x10(6)) compared with the Accudenz method (239.2x10(6) sperm; P<0.05; range across 6 lines of 165.7 to 581.0x10(6) sperm/mL). In experiment 2, rooster semen was cryopreserved using Lake's diluent containing either dimethyl acetamide (DMA) or glycerol as the cryoprotectants. Postthaw analysis revealed that the samples cryopreserved with glycerol survived freezing better, determined by total motility (47.8 and 15.1% glycerol and DMA samples, respectively; P<0.05) and annexin V analyses (1.6 and 11.3% membrane-damaged sperm for glycerol and DMA samples, respectively; P<0.05). Differences in sperm motilities (total and progressive motility) and velocities (path velocity, straight-line velocity, curvilinear velocity) were observed between the 2 cryoprotectant treatments once the glycerol had been removed from those samples cryopreserved with glycerol, of which the glycerol samples had significantly more motile sperm and higher velocities (P<0.05). The fertility of the samples frozen using the 2 cryoprotectants was tested using a single insemination (intravaginal or intramagnal) of 200x10(6) sperm and the fertility (number of live embryos) was evaluated over 18 d. Overall, the intravaginal inseminations had lower fertility than the intramagnal inseminations (P<0.05). In the intravaginal inseminations, the sperm cryopreserved using DMA resulted in lower fertility, but there were no differences in fertility in the intramagnal inseminations due to cryoprotectant (P>0.05). These results indicate that reasonable postthaw sperm quality and fertility can be derived using cryopreserved rooster semen. By utilizing this information, estimations can be made for storing sufficient material for line or breed, or both, recreation programs.

Ubiquitination and its influence in boar sperm physiology and cryopreservation.

Recent reports document the potential use of the ubiquitin protein as an indicator of mammalian sperm quality or fertility, based on poor morphology, sperm count, and other cellular qualities. However, its influence on cellular physiologic mechanisms and boar sperm cryopreservation are unknown. The objective of this research was to determine the influence of boar sperm ubiquitination (n=12 boars) on motility (using CASA), and flow cytometry and fluorescent probes (in parentheses) to evaluate mitochondrial activity (JC-1), plasma and acrosomal membrane integrity (PI and FITC-PNA), membrane fluidity (M540), and chromatin stability (TUNEL) for fresh and frozen-thawed samples. The effects of ubiquitination (determined flow cytometrically) on the ability of frozen-thawed boar sperm to capacitate (FLUO-3AM) and acrosome react (FITC-PNA) were also investigated using flow cytometry. Cryopreservation induced a decrease in the percentage of sperm that were ubiquitinated from 29 to 20% (P<0.0001), but no significant effects of ubiquitin on sperm quality (motility, membrane integrities and organization) were detected. The ability of sperm to capacitate and acrosome react was influenced by ubiquitination. Samples with more ubiquitinated boar sperm were able to maintain plasma membrane integrity (PMI) better and have fewer live acrosome-reacted cells over 120 min of induced capacitation (P<0.05). In conclusion, frozen-thawed ubiquitinated boar sperm were better able to survive the physical stresses of induced capacitation, yet were still capable of capacitating and acrosome reacting, which may enable use of this assay for in the vitro evaluation of the quality of boar sperm.

The post-thaw quality of ram sperm held for 0 to 48 h at 5 degrees C prior to cryopreservation.

The effects of holding diluted ram semen at 5 degrees C for up to 48 h prior to cryopreservation were investigated. Semen from six rams was collected by electro-ejaculation in the autumn and again from six different rams in the spring. The sperm concentration and motility were determined using spectrophotometry and computerized automated semen analysis, respectively. Samples were diluted at 23 degrees C to 400 x 10(6)cells/ml in a one-step Tris-egg yolk-glycerol (5%, v/v) media, cooled to 5 degrees C over 2h and maintained at 5 degrees C for the duration of the experiments. Aliquots were loaded into 0.5 ml French straws at 0, 24 or 48 h after cooling, frozen in liquid nitrogen vapor for 12-13 min, 4.5 cm above the liquid nitrogen, and plunged into liquid nitrogen for storage. After thawing, autumn samples frozen after 0, 24, or 48 h of storage exhibited similar percentages of motility (29, 31, 36%, respectively), progressively motility (16, 15, 17%, respectively), plasma membrane integrity (28, 35, 29%, respectively) and live acrosome-reacted cells (0.4, 0.6, 0.8%, respectively; P>0.05). In addition, the quantity of sperm that bound to hen's egg perivitelline membranes after being held at 5 degrees C for 0, 24, or 48 h was not significantly different when the values were expressed as means of the quantity of sperm (155, 177, 106 sperm, respectively) or as the proportion of sperm inseminated (0.39, 0.49, 0.34, respectively; P>0.05). Likewise, ram sperm collected in the spring and frozen at 0, 24 and 48 h after cooling had similar (P>0.05) total motility (21, 25, 20%, respectively), progressive motility (14, 15, 11%, respectively), plasma membrane integrity (26, 33, 31%, respectively) and live acrosome-reacted cells (3.7, 3.5, 3.2%, respectively; P>0.05). The 0 h holding time had significantly less sperm bound to a hen's egg perivitelline membrane compared to the 48 h holding time (250 and 470 sperm, respectively) although the 24h holding time was not different from the 0 or 48 h holding time (281 sperm; P<0.05) but analysis of the proportion of the total sperm inseminated resulted in no significant differences observed (P>0.05). These results indicate that ram sperm can be held at 5 degrees C for up to 48 h prior to freezing with no injurious effects on motility, membrane integrity, or fertilizing potential as indicated by membrane binding ability.

A method for cryopreserving chicken primordial germ cells.

This study established a method for preserving chicken primordial germ cells (PGC) that enables long-term storage in liquid N. Gonads were harvested from stage 27 chick embryos and pooled in groups of 5, 10 (10E), or 20 embryos, contributing gonads to the cell suspension. The gonadal cells, including PGC, were then frozen in 1 of the following cryoprotectant treatments: 2.5% dimethyl sulfoxide (DMSO), 5% DMSO, 10% DMSO, 2.5% ethylene glycol (EG), 5% EG, 10% EG, and 0% cryoprotectant as a control. The cells were liberated and frozen in a biosecure cryopreservation straw at a rate of -1 degrees C/min until reaching -85 degrees C and were then plunged into liquid N (-196 degrees C), in which they were stored until analysis. Flow cytometry was used to analyze the PGC post-thaw. The PGC marker stage-specific embryonic antigen-1, which was detected with goat antimouse IgM fluorescein isothiocyanate, was used to label all PGC, and propidium iodide was used to detect cells with compromised cell membranes. There was an interaction effect for the number of viable PGC per individual embryo (P < or = 0.05). The highest level (183.6 +/- 28.4) of viable PGC per individual embryo was observed for 10% EG with 10E and was significantly higher (P < or = 0.05) than cryopreservation in 2.5% DMSO with 10E and 20 embryos, 2.5% EG with 10E, 5% EG with 10E, and all 0% cryoprotectant treatments. No statistical interaction (P > 0.05) was observed for the percentage of viable PGC. However, the highest percentage (80.6%) was observed at 10% EG with 10E. It was demonstrated that PGC were successfully frozen, and the most effective treatment was 10% EG with 10 embryos/straw.

Enhanced fertility prediction of cryopreserved boar spermatozoa using novel sperm function assessment.

Due to reduced fertility, cryopreserved semen is seldom used for commercial porcine artificial insemination (AI). Predicting the fertility of individual frozen ejaculates for selection of higher quality semen prior to AI would increase overall success. Our objective was to test novel and traditional laboratory analyses to identify characteristics of cryopreserved spermatozoa that are related to boar fertility. Traditional post-thaw analyses of motility, viability, and acrosome integrity were performed on each ejaculate. In vitro fertilization, cleavage, and blastocyst development were also determined. Finally, spermatozoa-oviduct binding and competitive zona-binding assays were applied to assess sperm adhesion to these two matrices. Fertility of the same ejaculates subjected to laboratory assays was determined for each boar by multi-sire AI and defined as (i) the mean percentage of the litter sired and (ii) the mean number of piglets sired in each litter. Means of each laboratory evaluation were calculated for each boar and those values were applied to multiple linear regression analyses to determine which sperm traits could collectively estimate fertility in the simplest model. The regression model to predict the percent of litter sired by each boar was highly effective (p < 0.001, r(2) = 0.87) and included five traits; acrosome-compromised spermatozoa, percent live spermatozoa (0 and 60 min post-thaw), percent total motility, and the number of zona-bound spermatozoa. A second model to predict the number of piglets sired by boar was also effective (p < 0.05, r(2) = 0.57). These models indicate that the fertility of cryopreserved boar spermatozoa can be predicted effectively by including traditional and novel laboratory assays that consider functions of spermatozoa.

Inferring regional brain activity from evoked potential fields on the scalp.

A new method is described to calculate epicortical potential fields from scalp fields based on linear algebra. It requires detailed anatomical information, for each subject, obtained from MR images. The calculation is validated in a physical model of the human head and applied to human subjects. The results suggest that the method yields reliable epicortical fields that help to localize evoked cortical activity in humans.

How the brain integrates affective and propositional language into a unified behavioral function. Hypothesis based on clinicoanatomic evidence.

Recent publications suggest that the right hemisphere dominates in modulating the affective components of language. Disorders of language form right-sided focal brain lesions have been called "aprosodias" and can be classified in a manner similar to the aphasias. We describe a patient with motor aprosodia who subsequently died and underwent neuropathologic examination. From the neuropathologic findings and recent observations concerning the neurology of depression, we hypothesize that the motor integration of propositional and affective language takes place in the brainstem, whereas their higher-order integration takes place via the callosal connections between Wernicke's area on the left and its homologue on the right. Direct application of these functional and anatomic relations can help clinicians to properly interpret the often incongruous and disparate behavioral and language responses encountered in brain-damaged patients.

Brain blood flow SPECT in temporary balloon occlusion of carotid and intracerebral arteries.

It is important to determine preoperatively which patients can tolerate permanent occlusion of a cervical internal carotid or cerebral artery when such a procedure may be necessary to treat cerebrovascular or neoplastic lesions. Here we report our experience in combining temporary intra-arterial balloon occlusion with concomitant cerebral blood flow imaging in preoperative evaluation of such patients. Forty-two patients with a variety of cerebrovascular and neoplastic lesions underwent trial balloon occlusion of an internal carotid or intracerebral artery. Eight patients developed both neurologic symptoms as well as brain perfusion defects during trial occlusion. Nine others developed only perfusion defects. The remainder were asymptomatic and had negative scans. Brain blood flow imaging during intra-arterial balloon occlusion identified 17 patients potentially at risk for developing postsurgical ischemic deficits. Treatment alternatives to acute arterial sacrifice were developed for these patients.

Does electrothrombosis occur immediately after embolization of an aneurysm with Guglielmi detachable coils?

Current options for treating cerebral aneurysms include surgical clipping and placement of Guglielmi detachable coils (GDCs). The latter system is reported to induce acute aneurysmal occlusion by a mechanism of electrothrombosis. We report our observations in the case of a ruptured aneurysm treated with GDCs and then surgically exposed 2 hours later. The lack of thrombus within the aneurysm and around the coils led us to question the mechanism of action of GDCs.

Idiopathic ischemic cerebral infarction in childhood: depiction of arterial abnormalities by MR angiography and catheter angiography.

BACKGROUND AND PURPOSE: We report our experience with MR imaging, MR angiography, and catheter angiography in children with acute idiopathic cerebral infarction and suggest that catheter angiography may still play an important role in this setting. METHODS: During the past 8 years, 18 children with idiopathic cerebral infarction underwent MR imaging and catheter angiography; 17 were also studied with MR angiography. MR imaging was done within 34 hours after onset of hemiplegia or seizures or both. Sixteen patients underwent catheter angiography within 36 hours of MR imaging; 12 studies were performed within 22 hours. Two patients underwent catheter angiography, in both cases within 72 hours. Infarcts were compared with arterial abnormalities seen at catheter angiography, and the results of MR angiography were compared with those seen at catheter angiography. RESULTS: Comparing MR angiography with catheter angiography, we found the positive predictive value of MR angiography for arteriopathy was 100%, with a negative predictive value of 88%. MR angiography was equivalent to catheter angiography in the detection and depiction of proximal middle cerebral artery disease; however, depiction of disease in the internal carotid artery (ICA) and detection of peripheral embolic disease were better with catheter angiography than MR angiography. CONCLUSION: Basal ganglia lesions associated with ICA disease by MR angiography should probably be studied with digital subtraction angiography, as MR angiography did not depict the length and severity of ICA disease as well as catheter angiography did. Hemispheric infarcts should be studied with catheter angiography, as emboli may occur in the absence of heart disease; the circle of Willis may be uninvolved with embolic disease, and MR angiography is not sensitive to emboli in small peripheral intracranial arteries.

Effects of inhaled stable xenon on cerebral blood flow velocity.

The effects of inhaled stable xenon gas on cerebral blood flow were studied with 23 transcranial Doppler examinations performed in 13 normal volunteers while breathing, 25, 30, or 35% xenon for 5 min. Doppler velocities from the middle cerebral artery rose significantly during inhalation in 85% of subjects and 78% of studies and decreased significantly in 15% of subjects and 17% of studies. These different velocity responses may represent different responses of pial vasculature to xenon. The mean velocity rise among those studies showing a significant increase was 38 +/- 3.6% (SEM). The velocity rise began 2 min after the start of xenon inhalation and increased rapidly, so that the velocities measured at the four times at which scans were obtained in our xenon CT protocol (0, 1.5, 3, and 5 min after the start of xenon inhalation) were significantly different. A consistent fall in the pulsatility of the Doppler waveform as the velocity increased provided evidence for xenon-induced vasodilation of the small-resistance vessels as the cause of the increase in flow velocity. Most subjects became mildly hyperventilated, so that the observed changes could not be attributed to hypercapnia. Inhalation of 25, 30, or 35% xenon for 5 min induces a delayed but significant rise in cerebral blood velocity. This suggests that cerebral blood flow itself may be rapidly changing during the process of xenon CT scanning. These changes may compromise the ability of the xenon CT technique to provide reliable quantitative measurements of cerebral blood flow.

Crossed cerebellar diaschisis and loss of consciousness during temporary balloon occlusion of the internal carotid artery.

We encountered a case of crossed cerebellar diaschisis during temporary balloon occlusion of the internal carotid artery in a 59-year-old woman. The rapidity of the diaschisis was unusual and SPECT scanning was adequate to show the early defect.

Endovascular problem solving with intravascular stents.

BACKGROUND AND PURPOSE: Intravascular stents are being used with increasing frequency in interventional neuroradiology. They provide the potential to expand the therapeutic capabilities of the endovascular therapist and stand to revolutionize endovascular intervention within both the intracranial and extracranial vessels. We present our application of stent technology to further the understanding of endovascular rescue from procedural complications and the solving of complex clinical problems. METHODS: Three patients underwent unplanned placement of intravascular stents. In two patients a stent was used to provide stabilization of an irretrievable intravascular device; in the third patient a stent was used to provide a scaffolding for proximal external carotid sacrifice. RESULTS: Stent deployment was successful in all patients. The intravascular devices stabilized by stent placement included unraveled fragments of a Guglielmi detachable coil (GDC) and a partially deployed coronary stent. Proximal external carotid sacrifice was achieved with the aid of a stent in one patient to control hemorrhage from recurrence of laryngeal cancer. No periprocedural neurologic complications were encountered. Six-month follow-up angiography in one patient showed only minimal myointimal hyperplasia induced by stent-stabilized GDC fragments adjacent to the internal carotid vessel wall. CONCLUSION: Stents can be used to provide stabilization of irretrievable intravascular devices or as a scaffolding for proximal vessel sacrifice. These applications may allow endovascular rescue of procedural complications and solve unique clinical problems.

Remote vascular catastrophes after neurovascular interventional therapy for type 4 Ehlers-Danlos Syndrome.

Type 4 Ehlers-Danlos Syndrome (EDS 4) is the most malignant form of Ehlers-Danlos Syndrome, often accompanied by neurovasacular complications secondary to vessel dissection or aneurysms. The fragile nature of connective tissue in these patients makes exovascular and endovascular treatment hazardous. We have treated four patients with EDS 4 over the last 8 years by using neuroendovascular procedures. Two of these individuals suffered remote vascular injuries around the time of their procedures and ultimately died. The circumstances surrounding their deaths will make up the body of this report.