Cryopreservation in different concentrations of glycerol alters boar sperm and their membranes.

To test the hypothesis that glycerol would concomitantly affect sperm membrane structure and the function of the intact cells, boar semen (4 ejaculates from 4 boars) was cryopreserved in an egg yolk extender with 0%, 2%, 4%, or 8% glycerol in 0.5-mL straws using previously derived optimal cooling and thawing rates. Increasing glycerol concentrations increased spermatozoal progressive motility immediately after thawing and after 2 hours at 43 degrees C, but decreased the percentage of sperm with normal acrosomal morphology. The mathematical products of the motility and acrosomal integrity scores (MOT x NAR index) were low in 0% and 8% glycerol, and significantly higher in 2% and 4% glycerol. The fluidity of sperm-head plasma membranes, a measure of molecular interaction, was assessed with the lipid probes trans-parinaric acid and cisparinaric acid (tPNA, cPNA), during a 2.5-hour incubation with or without 1 mM Ca2+. Membrane fluidity detected by each probe differed significantly, indicating the presence of at least 2 domains whose constituent molecules had unique dynamics. Behavior of each domain was radically altered by cryopreservation. Increasing glycerol concentration caused a variably faster loss of fluidity in the cPNA domain, and had highly variable effects on fluidity change over time in the tPNA domain. Normal acrosomal ridge (NAR) and the MOT x NAR index correlated significantly with the fluidity of the more mobile cPNA domain (+/- 1 mM Ca2+), supporting the hypothesis of an interrelationship of glycerol concentration during cryopreservation with sperm membrane structure and cell function. The MOT x NAR index may be a useful guide in choosing optimal cryoprotectant concentrations.

Storage of boar semen.

The problems, aspects and methods of liquid storage and freeze-thawing of boar semen are discussed and a review is given on examination of spermatozoa by the recent fluorescent staining methods.

Behaviour of blastomere nuclei fused to mouse oocytes is affected by oocyte enucleation and age.

The influence of oocyte age and presence of oocyte meiotic apparatus on the behaviour of introduced blastomere nuclei was evaluated. Blastomeres from 4-cell mouse embryos were fused to intact (metaphase II) oocytes, demi-oocytes (nucleate) or cytoplast (anucleate). Fusion and simultaneous activation of the recipient oocytes were accomplished by a single electrical pulse at 20 or 24 h post human chorionic gonadotrophin (hCG) administration. The hybrids were fixed for evaluation 2 h after fusion. There was no difference in the behaviour of blastomere nuclei in whole oocytes and demi-oocytes. Most nuclei fused to the nucleate recipients at 20 h underwent breakdown of nuclear membrane (NMBD), chromosome condensation and consequently proceeded to telophase, in parallel with the resident meiotic chromosomes. Following fusion to cytoplasts, only a small portion of the blastomere nuclei underwent chromosome condensation and the vast majority (83%) of the nuclei remained in interphase. The influence of oocyte age on nuclear behaviour was assessed in oocyte-blastomere hybrids prepared by simultaneous fusion and activation at 20 and 24 h post-hCG administration. The introduced nuclei proceeded to telophase in 63% of the hybrids constructed at 20 h, but in only 28% of those constructed at 24 h. We conclude that nuclei introduced into aged or enucleated oocytes at the time of activation are predominantly remodelled in their interphase configuration.

Development of whole and demi-embryos of mice in culture and in vivo after supercooled storage.

Demi-embryos (produced by destroying 1 or 2 blastomeres of 2- or 4-cell embryos, respectively) and intact mouse embryos were cultured to the blastocyst stage, stored at -5 degrees C for 48 h, then cultured for 24 h and transferred into pseudopregnant recipients. Supercooled storage did not impair the developmental potential of whole or demi-embryos in vitro, nor was there a difference between whole and demi-embryos with respect to growth in vitro. Similarly, there was no effect of supercooling on development of intact or demi embryos after transfer into pseudopregnant recipient mice, but fewer recipients of demi-embryos remained pregnant (P < 0.05). This was considered to be partly due to the lesser ability of demi-embryos to maintain luteal function and establish pregnancy.

Parthenogenetic development of activated in vitro matured bovine oocytes.

Bovine oocytes matured in vitro for 26 hours were electrically stimulated 1) by a single pulse (Treatment A); 2) by 3 pulses 30 minutes apart (Treatment B); 3) by a single pulse followed by 5 minutes of incubation in the stimulation medium (Treatment C); or 4) by a single pulse at 27 hours of maturation (Treatment D). The oocytes were then cultured for up to 8 days to assess parthenogenetic activation and development. Each electrical stimulation consisted of a 60-mus square wave pulse of 2.5 or 3.6 kV/cm. Treatment A was less effective than the other treatments (P<0.05), activating 47 or 59% of oocytes at 2.5 or 3.6 kV/cm, respectively. However, there were no differences due to voltage nor among the other treatments, which activated 64 to 78% of the oocytes. The cleavage rate, 28 to 38%, was not affected by the activation treatment, but development to the 8-cell stage or beyond was greater after activation with the higher voltage. While the numbers of morulae or blastocysts resulting from any given treatment were too small to support meaningful statistical comparison, the results indicate that bovine parthenogenotes produced in vitro are capable of development to the blastocyst stage.

The effect of warming velocity on motility and acrosomal integrity of boar sperm as influenced by the rate of freezing and glycerol level.

The effect of thawing velocities ranging from 10 degrees C/min to 1,800 degrees C/min on the motility and acrosomal integrity of boar spermatozoa frozen at 1 degree C/min (suboptimal), 5 degrees C/min, and 30 degrees C/min (optimal) rate was studied with the sperm suspended for freezing in diluent containing 2, 4, or 6% of glycerol (v/v). The influence of thawing on sperm survival depends on the rate at which the sperm had been frozen. In semen frozen at a suboptimal rate of 1 degree C/min, the percentage of motile sperm (FMP) initially fell to 3.5-4.0% when the thawing rose to 200 degrees C/min, but, with further increases in thawing rate, increased and reached peak values (10.3-11.0% FMP) after thawing at 1,800 degrees C/min. The percentage of sperm with normal apical ridge (NAR) also increased moderately with thawing rate, but the degree of improvement decreased as the glycerol level was increased. In semen frozen at 1 degree C/min, acrosomal integrity (NAR) was best maintained in 2% glycerol, reaching 22.9% NAR after thawing at 1,800 degrees C/min. In semen frozen at the optimal rate of 30 degrees C/min, the increases in thawing rates above 200 degrees C/min substantially improved motility. Motility was generally higher in semen protected by 4 or 6% glycerol, with the peak values of 44 or 46% FMP, respectively, after thawing at 1,200 degrees C/min. The proportion of sperm with NAR also increased with thawing rate, but as in the case of suboptimally frozen sperm it was influenced negatively by the glycerol concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of ejaculated boar semen after freezing in 2 or 6% glucerol for in vitro fertilization of porcine oocytes matured in vitro.

Two concentrations of glycerol in a freezing diluent were tested with respect to the in vitro fertilizing capacity of frozen-thawed boar spermatozoa which, before exposure to oocytes, were subjected to 3 methods of fractionation. These were 1) the upper fraction, 2) the swim-up and 3) percoll gradinet-centrifugation. The highest proportions of motile spermatozoa were obtained by the swim-up procedure, while acrosomal integrity was best preserved by the upper fraction procedure. Raising the glycerol concentration from 2 to 6% (v/v) during freezing decreased the proportion of spermatozoa with a normal apical ridge. Spermatozoa separated by the upper fraction method showed the greatest penetration of oocytes and produced the highest incidence of polyspermy. The glycerol level affected penetration and polyspermy only with spermatozoa separated in a percoll gradient, where the higher level of glycerol increased oocytes penetration and polyspermy. Pronuclei formation was influenced by the separation procedure and by the glycerol concentration in the freezing diluent. The results indicate that frozen boar semen can be used for in vitro fertilization more successfully than fresh semen since penetration by frozen upper fraction spermatozoa was similar to, the degree of polyspermy was lower, and the formation of two pronuclei was greater (P<0.01) than in oocytes exposed to fresh semen.

The effect of supercooling on the developmental capacity of mouse embryos.

The effect of supercooled storage (at subzero temperatures without ice formation) on compacted mouse morulae and early blastocysts was studied. The embryos were equilibrated with one of three storage solutions containing 1, 3, or 6% each of methanol and glycerol and cooled to -2, -5, -10, or -15 degrees C and stored for up to 24 h to assess the effect of subzero storage at different temperatures and concentrations of the permeating cryoprotectants on embryo survival. Early blastocysts showed substantially greater survival than morulae and, in general, survival of embryos of either stage increased with the concentration of cryoprotectant, while the proportion of embryos surviving decreased with decreasing storage temperature and with increased duration of storage.

New thermal stress test to assess the viability of cryopreserved boar sperm.

A new, rapid, thermal stress test for assessing the viability of boar semen, requiring only 45 min of incubation at 42.5 degrees C, was developed and compared with a widely used stress test of 180 min incubation at 37 degrees C. The shorter procedure was found to have the same discriminatory ability as the standard test in assessing the effects of freezing conditions on the percentage of spermatozoa remaining motile. Neither test was able to show differences in the kinetic rating of motile sperm after freezing in relation to the glycerol concentration present during freezing. However, the new test had a greater ability to distinguish the effects of different concentrations of glycerol, over the range of 0 to 6%, and to reveal different degrees of acrosomal damage sustained during freezing. The longer procedure was unable to distinguish among glycerol concentrations from 0 to 4% with respect to acrosomal damage and produced an overall lower proportion of sperm having a normal apical ridge. The new thermal stress test thus has the advantages of greater sensitivity and more rapid execution over the test hitherto in widespread use.

[New pathogenetic aspects of the relations between viral and microbial infections and peripheral vasculopathies]. / Quelques nouveaux aspects pathogéniques des relations entre certaines infections à virus et inframicrobiennes et des vasculopathies périphériques.

The immunofluorescence technique using antisera against some viruses and inframicrobes allowed the detection of pathogens in altered vascular tissues (arteritis and phlebitis-phlebectasia). Pathomorphological aspects and some dehydrogenase activities in these patients were also investigated.

Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straws.

The interaction of glycerol concentrations of 0-10% and cooling rates from 1 to 1,500 degrees C/min with boar spermatozoa motility and acrosomal integrity (proportion of spermatozoa with normal apical ridge) was studied after thawing 0.5 ml straws at a constant rate. While increasing the glycerol concentration from 0 to 4% progressively improved motility, the percentage of spermatozoa with a normal apical ridge gradually decreased. The magnitudes of the respective changes depended on cooling rate. A peak value of 48.1% and rating 3.8 were obtained in semen protected with 4% glycerol, frozen at 30 degrees C/min. Increasing the glycerol levels above 6% resulted in a gradual decrease in motility. The proportion of spermatozoa with normal apical ridge was highest in semen protected with 0-1% glycerol after cooling at 30 degrees C/min (64.4% and 66.1%, respectively), but at these glycerol concentrations the percentage of motile spermatozoa was low. At the 30 degrees C/min cooling rate, the decline in the proportion of cells with normal apical ridge due to increasing the glycerol levels to 3 and 4% was relatively slow (57.3% and 49.4%, respectively). Cooling at 1 degrees C/min was detrimental to acrosomal integrity, which decreased with increasing glycerol concentration, in contrast to increasing motility, which even at its maximum, remained low. The direct plunging of straws into liquid nitrogen (1,500 degrees C/min) resulted in damaged acrosomes in all spermatozoa with the total loss of motility. Balancing motility and acrosomal integrity, freezing boar semen protected with 3% glycerol by cooling at 30 degrees C/min resulted in optimal survival for boar semen frozen in 0.5 ml French straws.

Continuous live-dead discrimination of ram sperm during freezing.

Use of the dye amaranth (Color Index 16185) as a supravital stain for ram sperm is described. At a concentration of 0.4% in diluted semen, the dye was completely excluded by motile sperm and had no effect on sperm motility. The nuclei of immotile sperm were stained pink by amaranth. The decrease in sperm motility during 24-h storage at 5 degrees C was accompanied by a corresponding increase in stained sperm nuclei. The presence of the dye during freezing had no effect on sperm cryosurvival but tended to reduce sperm motility during post-thaw incubation. Insemination of ewes with fresh semen containing amaranth or with semen frozen in the presence of amaranth resulted in pregnancies in 7/10 ewes in each group, compared to 6/9 in the case of ewes inseminated with fresh semen without dye.

The effect of glycerol-related osmotic changes on post-thaw motility and acrosomal integrity of ram spermatozoa.

Ram semen, collected by artificial vagina, was diluted and processed for long-term storage as described by P. S. Fiser, L. Ainsworth, and R. W. Fairfull (Canad. J. Anim. Sci. 62, 425-428, 1982). The concentration of the cryoprotectant, glycerol, was adjusted to 4% in the diluted semen prior to freezing by a one-step addition at 30 degrees C (Method 1), by cooling the semen to 5 degrees C and addition of the glycerol gradually over 30 min (Method 2), by one-step addition of glycerol prior to equilibration for 2 hr (Method 3), or by cooling to 5 degrees C, followed by a holding period of 2 hr at 5 degrees C, and the one-step addition of glycerol just prior to freezing (Method 4). After thawing, the glycerol concentration of the semen was reduced by stepwise dilution from 4 to 0.4% over 15 or 30 min or by a one-step ten-fold dilution. The average post-thaw percentage of motile spermatozoa was significantly lower after addition of glycerol by Method 1 (39.9%) than when the glycerol was added by the other three methods (range, 44.0-46.4% averaged over the glycerol dilution). The average post-thaw percentage of intact acrosomes (61.2%), highest in semen in which the glycerol was added by Method 2, was not significantly different from those in which glycerol was added to semen by Methods 3 and 4, but it was significantly higher than that found in semen in which the glycerol was added by Method 1 (54.4%). However, when averaged over the method of glycerolation, the post-thaw percentage of motile spermatozoa (range, 43.7-44.2%) and the percentage of intact acrosomes (range, 56.8-59.5%) did not differ significantly in semen subjected to gradual decrease in glycerol concentration and diluent osmolality (over 15 and 30 min) or by a one-step, 10-fold dilution. These data indicate that post-thaw survival of spermatozoa can be influenced by the way in which glycerol is added prior to freezing. However, post-thaw spermatozoa motility and acrosomal integrity can be maintained even after a rapid decrease in glycerol concentration such as that which accompanies insemination or dilution of semen for assessment of motility.

Evaluation of a new diluent and different processing procedures for cryopreservation of ram semen.

Ram semen was processed for freezing after initial dilution with a modified Tris-fructose diluent. Two aliquots were processed by cooling gradually to 5 degrees C, further dilution, equilibration and freezing in 0.5 ml straws either in pressurized liquid nitrogen (LN(2)) vapor (Method A) or on a block of dry ice (Method B). A third aliquot was cooled rapidly to 16 degrees C and then slowly to 5 degrees C, diluted further, equilibrated and frozen in straws in pressurized LN(2) vapor (Method C). The second dilution was carried out using a new diluent based on dextran-lactose. The diluted semen was equilibrated for 2 h before freezing. Semen was evaluated by artificial insemination (AI). The fertility of ewes bred by a double insemination with frozen-thawed semen processed by Methods A, B and C was 73% (n = 33), 67% (n = 30) and 80% (n = 30), respectively. In comparison, the fertility of ewes inseminated with fresh semen was 93% (n = 31). These preliminary data indicate an acceptable fertility can be achieved by AI with frozen-thawed semen processed using improved procedures.

The effects of rapid cooling (cold shock) of ram semen, photoperiod, and egg yolk in diluents on the survival of spermatozoa before and after freezing.

The effects of rapid cooling of semen (cold shock) from 30 degrees C to various temperatures above 0 degrees C on survival of ram spermatozoa suspended in diluents with or without egg yolk were assessed before and after freezing. Rapid cooling of extended semen from 30 to 15 degrees C had little or no effect on spermatozoa survival before or after freezing. Rapid cooling of extended semen from 30 degrees C to 10, 5, or 0 degrees C was accompanied by a progressive decrease in percentage of motile spermatozoa and percentage of intact acrosomes before freezing and a decrease in percentage of motile spermatozoa and after freezing. The ability of spermatozoa motile after cold shock to survive freezing and thawing, evaluated as cryosurvival, was not significantly (P greater than 0.05) affected by the temperature to which semen was cooled. The addition of egg yolk to the initial extender had a beneficial effect on percentage of motile spermatozoa particularly after rapid cooling of semen to 10 and 5 degrees C. Although egg yolk had little effect before freezing on semen rapidly cooled to temperatures above 15 degrees C and therefore not actually cold shocked, it substantially improved the subsequent survival of spermatozoa after freezing and thawing. Percentage of motile spermatozoa after cooling and after freezing was generally higher when the semen was collected during a decreasing photoperiod than during an increasing photoperiod.

The effect of thawing velocity on survival and acrosomal integrity of ram spermatozoa frozen at optimal and suboptimal rates in straws.

The effect of various thawing velocities on the motility and acrosomal maintenance of ram spermatozoa frozen at 20 degrees C/min (optimal) or 2 degrees C/min (suboptimal) was studied. The freeze-thaw motility and the percentage of intact acrosomes of spermatozoa frozen at 20 degrees C/min increased progressively with the thawing velocity. In semen frozen at 2 degrees C/min, motility of spermatozoa and the percentage of intact acrosomes declined drastically when the thawing velocity obtained in air at 20 degrees C was increased by thawing in water at 20 degrees C. Thawing at higher temperatures markedly increased both motility and acrosomal preservation, but the best results with semen frozen at 2 degrees C/min were lower than those obtained with semen frozen at 20 degrees C/min. The optimal freeze-thaw conditions for semen protected by 4% glycerol were freezing at 20 degrees C/min and thawing in water at 60 or 80 degrees C for 8 or 5 sec, respectively. Semen collected from rams exposed to a decreasing photoperiod exhibited higher motility after freezing and thawing than those exposed to an increasing photoperiod. However, there was no effect on acrosomal preservation after freezing at 20 degrees C/min.

Combined effects of glycerol concentration, cooling velocity, and osmolality of skim milk diluents on cryopreservation of ram spermatozoa.

The interaction of glycerol concentration from 0 to 16% and cooling velocity from 1 to 100 degrees C/min on freeze-thaw survival of ram spermatozoa was studied using a diluent based on 15% skim milk (450 mOs/kg water). Optimal spermatozoa survival (percentage motility and rating) was obtained with 4 to 6% glycerol and freezing rates of 10 to 100 degrees C/min. Similar results were obtained with 8% glycerol at freezing rates of 5 to 30 degrees C/min. Although the ram spermatozoa tolerated several cooling velocities at each glycerol concentration, increasing the concentration of glycerol resulted in a downshift in the range of optimal cooling velocities. Glycerol concentrations above 8% were toxic and contributed greatly to the progressive decrease in spermatozoa survival. Comparison of the 15% skim milk diluent (450 mOs/kg water) with a 19% skim milk diluent (600 mOs/kg water) showed that optimal cryosurvival was obtained with 4 to 6% glycerol and freezing rates of 10 to 100 degrees C/min with both diluents.

Effects of feeding white Leghorn hens diets that contain deoxynivalenol (vomitoxin)-contaminated wheat.

A short-term (10 weeks, Experiment 1) and a long-term experiment (24 weeks, Experiment 2) were done to determine effects of incorporating either white winter wheat, naturally contaminated with 1 mg deoxynivalenol (DON)/kg, or spring wheats, containing up to 6.5 mg DON/kg, into the diets of White Leghorn hens. Based on chemical analysis, the diets in Experiment 1 contained less than .05 to .7 mg DON/kg, while those in Experiment 2 contained from .2 to 4.9 mg/kg. Incorporation of winter or spring wheat in the experimental diets had no effect (P greater than .05) on feed intake and efficiency, egg production and yield, the number of soft shell and cracked eggs observed in the laying house, body weight at the completion of the experimental period, fertility, hatchability of fertile eggs, and the proportion of malformed embryos and pips. In addition, presence of DON-contaminated wheat did not influence (P greater than .05) the organ weight to body weight ratio for a randomly selected sample of hens necropsied at the completion of each experiment. There was little evidence of lesions in the oral cavity, esophagus, proventriculus and gizzard, hemorrhaging in the viscera or skeletal muscles, or of changes in the appearance of spleen, heart, and kidney. However, the livers from DON hens were fatty in appearance. Furthermore, vomiting (emesis), diarrhea, or changes in behaviour were not apparent and mortality, normally very low, was not increased during either experiment. Inverse linear relationships were obtained in Experiment 1 between dietary DON concentrations and egg weight (P less than .05), shell weight and thickness (P less than .01), and percent shell (P less than .05). Although egg and shell variables measured in Experiment 2 were not significantly influenced (P greater than .05) by DON treatment, trends towards lower values with higher dietary DON levels were evident. Egg specific gravity, nondestructive deformation, and quasistatic compression fracture strength of the egg's shell were not influenced (P greater than .05) by dietary DON levels. The results from these experiments indicate that laying hens can tolerate diets containing up to 5 mg DON/kg from white winter or spring wheat for extended periods of time without serious adverse effects on health and productivity.

The effect of glycerol concentration and cooling velocity on cryosurvival of ram spermatozoa frozen in straws.

The effect of varying the concentration of glycerol from 0 to 16% on the survival of ram spermatozoa frozen at increasing rates of cooling (1-100 degrees C/min) or by direct plunging of spermatozoa in 0.5-ml straws in liquid nitrogen was studied after thawing at a constant rate (in water at 39 degrees C for 30 sec). For each glycerol concentration, the ram spermatozoa tolerated a range of cooling velocities and the best survival rates (percentage motility and rating) were obtained when the glycerol concentration was 4 or 6% and when the rate of freezing ranged from 10 to 100 degrees C/min. No spermatozoa survived in any glycerol concentration following freezing in straws plunged into liquid nitrogen. In general, the range of cooling rates shifts to lower values as the glycerol concentration increases for optimum cryosurvival. However, the toxic effect of increasing the concentration of glycerol over 8% contributes greatly to the gradual decrease in cryosurvival of spermatozoa at these particular concentrations.