The experiments presented here identify several factors that affect survival (motility) of cryopreserved mouse spermatozoa after freezing and thawing. Among these factors are: (i) the temperature at which spermatozoa are collected, (ii) the cooling rate to 0°C and (iii) the warming rate from −196°C to ambient. When excised epididymides were cooled to near 0° (1–4°C) and spermatozoa collected and mixed with cryoprotectant at that temperature, motilities after subsequent freezing and thawing were 8–10 times higher than when the spermatozoa were collected from the epididymides at 22°C. In addition, the survival rates of spermatozoa warmed at rates ranging from 150 to 2000°C min−1 were about five times higher than those in suspensions warmed at about 7500°C min−1. The combination of a low collection temperature and the lower warming rates resulted in approximately 50% motility relative to unfrozen controls. Motility was reduced to 6–8% when the collection temperature was 22°C, and to approximately 10% when frozen suspensions of spermatozoa collected in the cold were rapidly warmed from −196°C. When spermatozoa collected at 22°C were abruptly cooled to 0°C, 40–80% of the cells suffered an irreversible loss of motility after warming. In contrast, when spermatozoa were cooled to 0°C at 1°C min−1 and warmed (either rapidly or slowly), motilities were similar to those of uncooled controls (75–90%). These findings indicate sensitivity to cold shock. Finally, the addition of raffinose or sucrose to the suspending medium did not affect the survival of the spermatozoa cooled slowly, but it did increase the survival of spermatozoa that were rapidly cooled to 0°C (55–60% versus 25–30%).
Jun Tao, Junying Du, F. W. Kleinhans, E. S. Critser, P. Mazur and J. K. Critser
D. Y. Gao, J. J. McGrath, Jun Tao, C. T. Benson, E. S. Critser and J. K. Critser
A perfusion technique using micropipette methodology was developed to determine quantitatively the membrane transport properties of mammalian oocytes. This method eliminates modelling ambiguities inherent in microdiffusion, a closely related technology, and should prove to be especially valuable for study of the coupled transport of water and cryoprotectant through mammalian oocytes and embryos. The method is described and evidence given for validity of the method for the simple case of uncoupled flow of water through the mouse oocyte membrane. The zona pellucida of a mouse oocyte was held by a micropipette with an 8–10 μm diameter tip opening and perfused by hyperosmotic media. The kinetic volume change of the cell was videotaped and quantified by image analysis. Experimental data and mathematical modelling were used to determine the hydraulic conductivity of the oocyte membrane (L p) found to be 1.05, 0.45 and 0.26 μm min−1 atm−1 at 30°C, 22°C and 12°C, respectively. The corresponding activation energy, E a, for L p was calculated to be 13.0 kcal mol−1. These values are in agreement with data obtained by other techniques. One of the major advantages of this technique is that the extracellular osmotic condition can be changed readily by perfusing a single cell with a prepared medium. To study the response of the same cell to different osmotic conditions, the old perfusion medium can be removed easily and the cell reperfused with a different medium. The second advantage is that the time required for mixing the original cell suspension and perfusion medium is minimized, allowing for accurate control of the extracellular osmolality and ensuring accuracy of the subsequent mathematical formation. This technique also has wide applicability in determining the membrane transport properties of mammalian oocytes, embryos and other cell types.
J. A. Gilmore, Junying Du, Jun Tao, A. T. Peter and J. K. Critser
A series of six experiments was conducted to determine the fundamental cryobiological properties of boar spermatozoa to develop optimal approaches for cryopreserving this important cell type. In the first experiments, boar spermatozoa samples were diluted in various osmolalities of experimental solutions (185–900 mOsmol kg−1) to provide hypo-, iso-, and hyperosmotic conditions. Equilibrium cell volumes (Expts 1 and 2) were measured after exposure for 3 min and the change in cell volume was measured over time using an electronic particle counter (Expt 3). The isosmotic cell volume was found to be 26.3 ± 0.39 μm3 (mean ± sem; n = 5). Over this range of osmolalities, boar spermatozoa behaved as linear osmometers (a linear volume versus 1/osm plot, r = 0.99) with an osmotically inactive cell fraction of 67.4 ± 4.5%. The rate of water permeability (L p) was determined to be 1.03 ± 0.05 μm min−1 atm−1, which was consistent within and among donors (P > 0.130). A second series of experiments was performed to determine the effect of temperature and osmolality on boar sperm motility (Expt 4), and the effect of osmolality on the integrity of the sperm plasma membrane and its temperature dependence. Plasma membrane integrity was measured before and after boar spermatozoa were returned to an isosmolality (Expt 6). Motility was not affected at 30°C, relative to that at room temperature, but was significantly decreased (P < 0.05) at 8°C and 0°C (yielding a relative reduction to 85% and 35% of original motility, respectively; n = 6). Sperm motility was not significantly decreased (P > 0.05) until the osmolality reached 210 mOsmol kg−1, at which time motility began to decrease from 95% to 10% of the original value at 90 mOsmol kg−1. The integrity of the plasma membrane of boar spermatozoa was found to be dependent on temperature, donor and osmolality, decreasing significantly (P < 0.05) below room temperature, and below 185 mOsmol kg−1 (P < 0.05). There was no significant difference (P > 0.10) in the integrity of the plasma membrane of the samples before and after returning to 290 mOsmol kg−1, indicating that osmotic damage occurs during the initial change from isosmotic to hyposmotic media. These osmotic characteristics could be used to determine optimal conditions for cryopreservation of boar spermatozoa.
J. K. Critser, M. J. Lindstrom, M. M. Hinshelwood and E. R Hauser
Summary. Angus and Angus crossbred heifers were ovariectomized, treated with oestradiol implants and randomly assigned to (1) the natural photoperiod of fall to spring for 43°N latitude or (2) extra light simulating the photoperiod of spring to fall. Weekly blood samples were taken for 6 months (fall to spring equinox). All heifers were cannulated every 4 weeks and blood samples were taken for 4 h at 15-min intervals. Sera were assayed for LH, FSH, prolactin and oestradiol. In samples taken weekly, serum LH and FSH concentrations were higher while serum prolactin was lower in heifers exposed to natural photoperiod. There was a photoperiod × time interaction for both FSH and prolactin with concentrations diverging as photoperiod diverged. Circulating concentrations of oestradiol were not different between groups. In samples taken every 4 weeks at 15-min intervals, baseline concentrations of LH and FSH and LH pulse amplitude were higher while prolactin pulse frequency was lower in heifers exposed to natural photoperiod. There was a photoperiod × time interaction for each of these pulsatile characteristics. The correlation between LH and prolactin concentrations estimated from the 15-min samples differed between the two photoperiod treatment groups. The pooled correlation coefficient (r) was −0·12 under natural photoperiod and +0·50 under extra light. There was also a photoperiod × time interaction with negative correlations occurring when photoperiod was decreasing and positive correlations occurring when photoperiod was increasing. These results support the hypothesis that photoperiod alters serum concentrations of LH, FSH and prolactin in cattle.
J. K. Critser, B. W. Arneson, D. V. Aaker, A. R. Huse-Benda and G. D. Ball
Summary. A series of 4 experiments was conducted to examine factors affecting the survival of frozen–thawed 2-cell mouse embryos. Rapid addition of 1·5 m-DMSO (20 min equilibration at 25°C) and immediate, rapid removal using 0·5 m-sucrose did not alter the frequency (mean ± s.e.m.) of blastocyst development in vitro when compared to untreated controls (90·5 ± 2·7% vs 95·3 ± 2·8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (−0·3°C/min) at −40°C with subsequent rapid thawing (∼1500°C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at −80°C with subsequent slow thawing (+ 8°C/min) (36·8 ± 5·6% vs 63·9 ± 5·7%). When slow cooling was terminated between −40 and −60°C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below −60°C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below −60°C and thawed slowly. However, when slow cooling was terminated at −32°C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to −80°C and thawed slowly (52·4 ± 9·5%, 59·5 ± 8·6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages. However, for the 2-cell stage, to achieve high survival rates with rapid thawing the temperature at which slow cooling is terminated is greater (∼ −32°C) than values reported for later stages.
Keywords: mouse; 2-cell embryos; freezing rate; thawing rate; development to blastocysts
Junying Du, Jun Tao, F. W. Kleinhans, P. Mazur and J. K. Critser
Experiments were conducted to determine the water volume and osmotic behaviour of mouse spermatozoa using an electron paramagnetic resonance technique using the spin label tempone, and the broadening agent potassium chromium oxalate. After a swim-up procedure, an average water volume of 43.3 μm3 of individual spermatozoa was obtained at 290 mosmol. If a water compartment of 59% is assumed, the total volume of mouse spermatozoa is 73.4 μm3. A plot of the relative water volume of mouse spermatozoa versus the reciprocal of buffer osmolality (Boyle van't Hoff plot) is linear in the range 250–900 mosmol of sodium chloride solutions (r 2 = 0.96). The Boyle van't Hoff plot intercept indicates that 13% of the spin-label accessible isotonic water is osmotically inactive.
J. K. Critser, T. M. Block, S. Folkman and E. R. Hauser
Summary. Angus and Angus crossbred prepubertal heifers were ovariectomized and randomly assigned to either increasing light simulating the photoperiod of the vernal equinox to the summer solstice (I) or decreasing light simulating the photoperiod of the autumnal equinox to the winter solstice (D) for 43°N latitude. Three blood samples were taken each week for 14 weeks, the first at 11:00 h and two others 2 days later, 1 h before lights on (dark), 1 h before lights off (light). At the end of 14 weeks 4 heifers from each treatment group were cannulated and samples were taken for 12 h at 15-min intervals, 6 h in the light and 6 h in the dark. All sera were assayed for LH, FSH and prolactin. In addition, the samples taken at 15-min intervals were assayed for melatonin. In samples taken weekly at 11:00 h circulating concentrations of LH and prolactin were higher among animals in Group I, while FSH concentrations were not different between Groups D and I. In samples collected weekly in the light or the dark, LH and prolactin concentrations were higher in Group I animals. However, prolactin concentrations were higher and LH concentrations tended to be higher in samples taken in the dark. FSH concentrations were not different between either D or I or dark and light. In samples taken at 15-min intervals the prolactin baseline was higher and pulse amplitude tended to be higher for Group I animals. Neither LH nor FSH pulse characteristics differed between I and D; however, LH baseline and LH pulse amplitude were higher in the dark. Melatonin pulse amplitude was higher among animals in Group D and higher in serum collected in the dark. These results suggest that photoperiod alters circulating concentrations of LH and prolactin and alters pulsatile release of LH, prolactin and melatonin in the prepubertal heifer.