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.

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.

Spinal cord hypothermia without systemic hypothermia.

BACKGROUND AND PURPOSE: Hypothermia has been shown to be beneficial in the setting of acute SCI. However, widespread use has been hindered by the need for systemic hypothermia as the vehicle for achieving spinal cord hypothermia. This study demonstrates that localized spinal cord hypothermia can be achieved via a percutaneous approach while maintaining systemic normothermia. MATERIALS AND METHODS: Five Yucatan swine underwent catheterization of the subarachnoid space and infusion of room temperature, chilled, and iced PL solutions into the cervical spinal canal, with drainage from the lumbar canal. Thermocouples were placed within the spinal cord and in the subarachnoid space and recorded during infusions and recovery from hypothermia. RESULTS: Results demonstrated that hypothermia as low as 16.8°C is feasible in the spinal cord with retention of systemic normothermia, with strong (r = 0.95) correlation between the spinal cord temperature and the CSF temperature. Degrees of cooling varied with flow rates and with infusate temperature. CONCLUSIONS: While the data are preliminary in a small group of animals, the ability to rapidly create a wide range of controlled spinal cord hypothermia while preserving normal body temperature warrants wider exploration. The study also indicates that further investigation of the hypothesis that CSF temperature monitoring may be an acceptable surrogate for direct spinal cord temperature monitoring should be pursued.

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.

Boar sperm quality in lines of pigs selected for either ovulation rate or uterine capacity.

Selection for 11 generations in swine for ovulation rate (OR) or uterine capacity (UC) resulted in significant changes in component traits of litter size. Our objective was to conserve the unique germplasm for the future and to characterize sperm quality as a correlated response to the selection criterion imposed compared with an unselected control line (CO). Boars representing genetic diversity available in all 3 lines were produced in 2 farrowing seasons. Season 1 was born in September 2005 and was sampled for semen characteristics in October 2006. Season 2 was born in March 2006 and was sampled for semen characteristics in February and March 2007. Each boar (n = 60) was collected twice. The sperm-rich fraction was obtained, and volume and concentration of sperm cells were measured to estimate total sperm production. Each ejaculate was extended 1:3 (vol/vol) with Androhep Plus (Minitube, Verona, WI) and was packed for shipping to the National Animal Germplasm Program laboratory for processing into frozen straws. Semen quality was measured by computer-assisted semen analysis at 3 semen processing points: fresh (FR), 24 h after extender added (E), and postthaw (PT). A mixed model ANOVA was applied to the data. Fixed effects of farrowing season, line, and 2-way interactions were fitted. The random effect of boar (n = 60) within farrowing season and line was used to test line differences. Sperm concentration was not different (P = 0.18) among the lines (0.594 × 10(9), 0.691 × 10(9), and 0.676 × 10(9) cells/mL for CO, OR, and UC lines, respectively). However, significance (P = 0.04) was detected for the volume of the sperm-rich fraction, greatest for OR (86.4 mL), intermediate for UC (75.5 mL), and least for CO (70.2 mL). Line differences were thus detected (P = 0.02) for total sperm production per ejaculate, greatest for OR (54.9 × 10(9)), intermediate for UC (48.7 × 10(9)), and least for CO (40.5 × 10(9)). A larger percentage of progressively motile sperm and greater estimates of sperm velocity only at processing point E (P < 0.01) were detected in favor of CO. Estimates of motility, velocity, and other parameters of sperm movement measured on E processing points were positively correlated with the same estimates obtained PT, but the magnitude was low to moderate (r range -0.03 to 0.23). Thus, selection for component traits of female reproduction had a favorable effect on total sperm production of boars.

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.

Genetic structure and diversity among sheep breeds in the United States: identification of the major gene pools.

Understanding existing levels of genetic diversity of sheep breeds facilitates in situ and ex situ conservation activities. A comprehensive evaluation of US sheep breeds has not been previously performed; therefore, we evaluated the genetic diversity among and within 28 US sheep breeds. Both major and minor breeds were included in the analysis and consisted of 666 animals from 222 producers located in 38 states. The level of within-breed genetic diversity was variable and not dependent upon status of a breed as a major or minor breed. Bayesian cluster analysis indicated the breeds were grouped more by physiological differences (meat vs. wool production) rather than geographic origin. Results suggest several actionable items to improve in situ and ex situ conservation. The results clearly identify breeds in need of increased in situ and ex situ management (e.g., Hog Island and Karakul) and allow several suggestions for in situ management of flocks. Conversely, several of the breeds appear genetically similar and therefore require less emphasis on collecting germplasm samples for the gene bank. Commercially important breeds (e.g., Rambouillet and Suffolk) were found to have substantial variation, which should enable breeders to proceed, unencumbered by genetic diversity concerns, with selection strategies that maximize profit.

Implications of the pH and temperature of diluted, cooled boar semen on fresh and frozen-thawed sperm motility characteristics.

Boar semen is typically collected, diluted and cooled for AI use over numerous days, or frozen immediately after shipping to capable laboratories. The storage temperature and pH of the diluted, cooled boar semen could influence the fertility of boar sperm. Therefore, the purpose of this study was to determine the effects of pH and storage temperature on fresh and frozen-thawed boar sperm motility end points. Semen samples (n = 199) were collected, diluted, cooled and shipped overnight to the National Animal Germplasm Program laboratory for freezing and analysis from four boar stud facilities. The temperature, pH and motility characteristics, determined using computer automated semen analysis, were measured at arrival. Samples were then cryopreserved and post-thaw motility determined. The commercial stud was a significant source of variation for mean semen temperature and pH, as well as total and progressive motility, and numerous other sperm motility characteristics. Based on multiple regression analysis, pH was not a significant source of variation for fresh or frozen-thawed boar sperm motility end points. However, significant models were derived which demonstrated that storage temperature, boar, and the commercial stud influenced sperm motility end points and the potential success for surviving cryopreservation. We inferred that maintaining cooled boar semen at approximately 16 °C during storage will result in higher fresh and frozen-thawed boar sperm quality, which should result in greater fertility.

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.

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.

Preoperative embolization of cerebral arteriovenous malformations with onyx.

BACKGROUND AND PURPOSE: Preoperative embolization facilitates the surgical management of complex cerebral arteriovenous malformations (cAVMs). This analysis aims to investigate the risks for preoperative cAVM embolization with Onyx. MATERIALS AND METHODS: We retrospectively analyzed clinical data of all patients who underwent embolization with Onyx as a preoperative treatment of cAVMs at our institution since 2005 (US Food and Drug Administration [FDA] approval). Patients with arteriovenous fistulas were excluded. A total of 107 patients were treated for cAVMs during the study period. Of those patients, 41 underwent cAVM embolizations with Onyx in 82 procedures. RESULTS: After the embolization, the cAVM diameter was reduced from 3.71 +/- 1.55 cm to 3.06 +/- 1.89 cm (P < .05). Median volume reduction was 75%. Complete occlusion with embolization alone was achieved in 4 (10%) cAVMs. The recurrence rate for completely occluded cAVMs was 50% (2 patients). A total of 71% of the 41 patients treated with Onyx underwent surgery, and 15% underwent radiosurgery. There were 9% who have not yet received definitive treatment of their residual cAVMs. A new permanent neurologic deficit occurred in 5 patients (6.1% per procedure or 12.2% per patient). CONCLUSIONS: A considerable risk for a permanent neurologic deficit remains for cAVM embolization with Onyx. The risk has to be carefully weighted against the benefit of volume reduction in the treatment of cAVMs.

Review of current and emerging therapies in acute ischemic stroke.

The statistics for stroke in the USA reads like a familiar ad slogan cited in most papers pertaining to acute ischemic stroke (AIS). Stroke is the third leading cause of death in the USA. While stroke ranks third among all causes of death, behind diseases of the heart and cancer, it is the leading cause of serious long-term disability in the USA.(1) Approximately 795 000 people, 87% of whom are ischemic, suffer from stroke each year in the USA.(2) That means that on average, every 40 seconds someone within the USA develops a stroke. For 2009 the combined direct and indirect cost of stroke, from hospitalization and rehabilitation to institutionalization, is estimated at $68.9 billion within the USA.(2).

Retrograde crossing stent placement strategies at the basilar apex for the treatment of wide necked aneurysms: reconstructive and deconstructive opportunities.

Neck remodeling devices such as the Neuroform or Enterprise greatly facilitate the endovascular treatment of wide necked basilar apex aneurysms. The complex anatomy of the basilar apex affords opportunity for antegrade, multiple crossing and retrograde device placement strategies to facilitate coil embolization. A retrograde approach is possible in the presence of a posterior communicating artery large enough to allow device navigation. Our experience with a retrograde device placement strategy in three patients is reported. In two patients, device positioning extended from one P1 segment of the posterior cerebral artery to the other across the basilar apex. In one patient, device positioning extended from the P1 segment of the posterior cerebral artery across the basilar apex into the opposite superior cerebellar artery. All patients underwent reconstructive or deconstructive uncomplicated coil embolization after device placement with stable aneurysm occlusion on follow-up angiography. In appropriate anatomic situations, retrograde stent placement across the basilar apex through a posterior communicating artery may represent a preferred strategy for wide necked basilar apex aneurysms.

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.

Influence of patient age and stenosis location on wingspan in-stent restenosis.

BACKGROUND AND PURPOSE: Wingspan is a self-expanding, microcatheter-delivered microstent specifically designed for the treatment of symptomatic intracranial atherosclerotic disease. Our aim was to discuss the effect of patient age and lesion location on in-stent restenosis (ISR) rates after percutaneous transluminal angioplasty and stenting (PTAS) with the Wingspan system. MATERIALS AND METHODS: Clinical and angiographic follow-up results were recorded for all patients from 5 participating institutions. ISR was defined as >50% stenosis within or immediately adjacent (within 5 mm) to the implanted stent and >20% absolute luminal loss. For the present analysis, patients were stratified into younger (55 years) age groups. RESULTS: ISR occurred at a rate of 45.2% (14/31) in the younger group and 24.2% (15/62) in the older group (odds ratio, 2.6; 95% confidence interval, 1.03-6.5). In the younger group, ISR occurred after treatment of 13/26 (50%) anterior circulation lesions versus only 1/5 (20%) posterior circulation lesions. In the older group, ISR occurred in 9/29 (31.0%) anterior circulation lesions and 6/33 (18.2%) posterior circulation lesions. In young patients, internal carotid artery lesions (10/17 treated, 58.8%), especially those involving the supraclinoid segment (8/9, 88.9%), were very prone to ISR. When patients of all ages were considered, supraclinoid segment lesions had much higher rates of both ISR (66.6% versus 24.4%) and symptomatic ISR (40% versus 3.9%) in comparison with all other locations. CONCLUSION: Post-Wingspan ISR is more common in younger patients. This increased risk can be accounted for by a high prevalence of anterior circulation lesions in this population, specifically those affecting the supraclinoid segment, which are much more prone to ISR and symptomatic ISR than all other lesions.

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.

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.