Pre-freeze Motility Research Articles

Development of in vitro tests to predict fertility of bulls

The overall objective was to develop an in vitro test to predict fertility of bulls in the field. We investigated the bull effect on in vitro embryo production, zona binding and acrosome reaction, and the correlation of this effect to field fertility meas ured by 60–90 d non-return rate. Frozen semen from three separate ejaculates of eight unrelated young bulls, obtained from an artificial insemination (AI) center, was used. On thawing, ejaculates from each bull were pooled, motile sperm were selected and (a) subjected to immunofluorescent assay at 0 and 4 h of incubation in capacitation medium to assess acrosome status, (b) used in an in vitro fertilization assay system to assess cleavage and blastocyst production rates, and (c) sperm-zona binding assay was carried out to determine the number of sperm bound to the zona pellucida of mature oocytes. Percentage of pre-freeze motile sperm (PrFM) and non-return rate data were obtained from the AI center. PrFM, percentage of acrosome reacted sperm at 0 h (AR1), increase in percentage of acrosome reacted sperm after 4 h (InAR) and sperm-zona binding rates (ZB) differed (P < 0.05) among sperm samples obtained from different young bulls. Significant correlations (P < 0.05) were observed between PrFM and AR1 (r = -0.31), InAR (r = 0.36), and ZB (r = 0.32). AR1 was negatively correlated to ZB (r = -0.27) and cleavage rate (r = -0.20), InAR was positively correlated with ZB (r = 0.31) and cleavage rate (r = 0.26). None of the in vitro tests was correlated with non-return rate. These findings indicate that along with pre-freeze motility, a combination of in vitro tests including the percentage of spontaneously acrosome reacted sperm at thawing, might be useful in predicting bull field fertility. Such a combination of assays, however, has yet to be determined. Key words: Field fertility, acrosome reaction, zona binding, IVF, fertility assay

Semen collection, cryopreservation and artificial insemination in the dromedary camel

Collection of semen with a bovine artificial vagina (AV) was attempted with each of 14 camels over a period of 2 years. Semen samples were evaluated, extended and cryopreserved. Frozen thawed semen, diluted cooled semen or whole semen was used to inseminate some female camels which were induced to ovulate with hCG. Males ejaculated semen into the AV in 74.6% collection attempts. The male copulated for at least 200 s in 62.9% attempts. The remaining copulations were of shorter duration. Similarly, 49.3% ejaculates were at least 3 ml of semen. Libido and donation of semen improved from December onwards and reached a peak after mid January with peak performance persisting until April. It declined during May. The majority of camels had lost libido and refuse to donate semen by the end of May. Camel semen is in gel form. While 35.9% of 203 semen samples exhibited no individual sperm motility, 28.5% exhibited low to fair grade individual sperm motility and only 35.4% exhibited >50% sperm motility. Differences existed between animals ( P<0.01) and months ( P<0.05) of collection, while effect of copulation time was not significant. Mass motility was not observed in camel semen. Individual sperm motility develops after liquefaction of semen. Addition of caffeine but not chymotrypsin improved the individual motility. The mean live percent sperm count and normal acrosome were 73.3±1.0 and 92.0±0.5, respectively. Only 51.1% of 45 semen samples with pre-freeze motility of >50% and 25% of 16 semen samples from low pre-freeze motility group with an overall success of 44.2% of 61 semen samples were successfully preserved. Wide variation was observed in the freezability of semen from different males. Attempts to impregnate female camels with liquid semen, frozen thawed semen and whole semen after hCG induced ovulation resulted in 0/10, 1/13 and 4/10 pregnancies.

INVESTIGATION OF FERTILIZING CAPACITY OF CRYOPRESERVED SPERMATOZOA FROM PATIENTS WITH CANCER

Purpose There are few published reports concerning fertilization and pregnancy outcomes achieved with cryopreserved spermatozoa from cancer patients. Controversy exists regarding the value of sperm banking for these patients before therapy, whether the spermatozoa are viable after long-term storage and whether they can fertilize the ovum. We assess fertilization and pregnancy outcomes achieved with cryopreserved spermatozoa from cancer patients using assisted reproductive techniques. Materials and Methods We studied 10 cancer patients who transferred cryopreserved semen specimens from our sperm bank to outside in vitro fertilization programs for assisted reproductive technique. Of these patients 5 had Hodgkin's disease, 2 testicular cancer, 1 leukemia and 2 prostate cancer. The length of specimen storage ranged from 14 to 135 months (median 49, interquartile range 24 and 82). Results The median pre-freeze motility was 44% (interquartile range 36 and 55%) and the median total sperm count was 31.1 × 10 6 (interquartile range 6.3 and 53.9 × 10 6). At 24 hours after banking the median post-thaw motility was 11% (interquartile range 6 and 35%) and the median total sperm count was 6.6 × 10 6 (1.2 and 17.1 × 10 6). A total of 18 cycles of assisted reproductive technique were performed among 10 couples with an overall pregnancy rate of 50% per couple, with 2 deliveries, 1 ongoing pregnancy and 2 miscarriages. The pregnancy rate per cycle of in vitro fertilization and intracytoplasmic sperm injection was 36.4% with an implantation rate of 13%. Conclusions These results indicate that poor quality cryopreserved spermatozoa from cancer patients, irrespective of the length of storage, may provide successful results with the latest micromanipulative techniques such as intracytoplasmic sperm injection.

Optimal utilization of cryopreserved human semen for assisted reproduction: recovery and maintenance of sperm motility and viability.

Our purpose was to evaluate sperm motility and viability and the maintenance of these parameters in already cryopreserved semen samples following repeated freezing/thawing cycles. Human spermatozoa were subjected to five cycles of cryopreservation/thawing. Recovery of sperm motility and viability and the proportion of viable nonmotile sperm were determined up to 6 hr after thaw. Sperm motilities (prefreeze motility, 70.1%; n = 9 samples) after each of five freeze/thaw cycles were 24.4, 8.0, 3.5, 1.5 and 1.8%. The recovery of sperm viability was higher than that of motility after each cycle: 39.1, 25.3, 22.6, 17.8, and 16.5%. Recoveries of motility and viability were improved if the thawed samples were left in the original cryopreservation medium prior to refreezing vs. if a washing/ resuspension step was included. The recovery of sperm motility in the first thawing cycle was indicative of the expected motile sperm recovery in the second thawing cycle. Cryopreserved semen that is intended to be reused in future assisted reproduction treatments should be thawed only once and aliquoted in the original freezing medium before refreezing. The recovery of sperm motility and viability in the second thawing cycle, thus the applicability of the sample in conventional in vitro fertilization or intracytoplasmic sperm injection may be anticipated in > 90% of the samples. In view of intracytoplasmic sperm injection it is important that sperm viability is maintained better than motility; after the first, second, and third thawing cycles the ratios of motile:nonmotile viable sperm were 1:1, 1:4, and 1:7, respectively.

Artificial insemination and in-vitro fertilization using donor spermatozoa: a report on 15 years of experience

Donor insemination (DI) using cryopreserved semen commenced at The Royal Women's Hospital in 1976. Over the next 15 years we performed 5953 treatment cycles to achieve 816 pregnancies (13.7% per cycle) and 706 live births. In-vitro fertilization (IVF) using donor spermatozoa commenced in 1986. Over the next 5 years we performed 303 treatment cycles for 185 couples. Including subsequent transfer of cryopreserved embryos, a total of 33% of couples achieved a successful pregnancy by IVF. Statistical analysis indicated that, for DI pregnancies, the most important semen variable was the percentage post-thaw motility, whilst for normal fertilization in IVF it was the pre-freeze motility. These results may be explained by the compensatory effects of post-thaw processing of spermatozoa for IVF, but not for DI in our clinic.

Effects of cancer on spermatozoa quality after cryopreservation: a 12-year experience

To determine whether type of cancer and response to treatment was related to prefreeze or post-thaw semen quality and to predict post-thaw sperm motility from prefreeze motility. Retrospective study. Tertiary care institution. One hundred six cancer patients cryopreserving their semen specimens. Computer-assisted semen analysis was performed before and after cryopreservation on each patient specimen. The relationship of sperm motility and motion characteristics to type of cancer and patient's response to treatment. Prefreeze and post-thaw semen quality did not differ between patients presenting with testicular cancer and Hodgkin's disease. Patients with leukemia or advanced soft tissue cancer had a higher prefreeze and post-thaw motility and higher total and motile sperm count than testicular and Hodgkin's disease patients. A prefreeze sperm motility of > or = 15% could predict a post-thaw motility of > 10%. Prefreeze or post-thaw semen quality in cancer patients is not affected (except the prefreeze motile sperm count within the testicular cancer patients) by the type of disease. Prefreeze motility can predict post-thaw motility. Cryopreservation of semen should be offered to cancer patients irrespective of the type of disease.

Effect of cryopreservation on semen quality in patients with testicular cancer

Objectives Current techniques in cryopreservation of human semen substantially decrease sperm quality. In addition, the pregnancy rate using cryopreserved sperm obtained from testicular cancer patients is lower than when sperm from normal fertile men is used. However, it is still unclear whether cryopreserved sperm from these patients is inherently defective or if the sperm loses its motility after thawing. This study was undertaken to assess the effect of cryopreservation on the quality and motion characteristics of semen from patients with testicular cancer before definitive therapy compared with semen from normal volunteers. Methods We compared the sperm quality before and after cryopreservation in samples from 34 patients with testicular cancer and 30 normal volunteers who were referred for sperm banking over a 7-year period. The effects of cancer stage and histologic type on various semen parameters were also examined. A computer-assisted semen analysis was performed before and after cryopreservation on each specimen. The nitrogen vapor technique using Test yolk buffer with glycerol as a cryoprotective agent was used for cryopreservation. The motile sperm count and motion characteristics (motility, velocity, linearity, amplitude of the lateral head movement, motility index) were analyzed before and after cryopreservation and compared between the groups. Results Semen quality did not significantly differ among patients with Stage I, II, or III cancer. However, semen quality tended to be poorer at higher cancer stages. In general, semen quality was better among patients with pure seminomas than with pure embryonal tumors; quality was worst among patients with mixed germ cell tumors. However, 71.4% of patients with mixed tumors presented with Stage III disease, whereas all patients with seminomas presented with Stage I disease. Significant differences were also seen in prefreeze motility (median, 42% [interquartile range, 24 to 51] versus 60.5% [range, 49 to 73]; P = 0.0004) and motile sperm count (6.7 × 10 6/mL [range, 3.4 to 14.4] versus 50.0 [range, 24.6 to 72.0]; P = 0.0001) in patients compared with controls, respectively. The motile sperm count and percent motility significantly decreased in both patients and controls after cryopreservation ( P = 0.0001). However, the percentage decline in motile sperm count and motion characteristics after cryopreservation did not differ significantly between patients and controls ( P > 0.01). Conclusions We conclude that the effect of cryopreservation on sperm quality in patients with testicular cancer is identical to its effect on sperm from normal fertile men. Differences in values after preservation are explained by poor semen characteristics before freezing; semen quality declines with more extensive disease. Stage I patients also had poorer quality than control subjects. Thus, we recommend that routine sperm banking be encouraged among all patients with testicular cancer before the initiation of specific medical treatment. We also recommend that future efforts be focused on improving the technique of sperm banking.

Evaluation of different cooling rates, equilibration periods and diluents for effects on deep-freezing, enzyme leakage and fertility of taurine bull spermatozoa

We studied the effects of 2 diluents (Tris and milk), 4 cooling rates (10°C/30°C to 5°C for 1 or 2 h), 2 equilibration periods (0 and 2 h) and their interactions on the freezability, glutamic oxaloacetic transaminase (GOT) leakage and fertility of frozen-thawed semen in 18 ejaculates from 3 Friesian bulls. The means of pre- and post-freezing motility, GOT leakage and fertility rates (52.81% based on follow up of 267 inseminated cows) were significantly (P<0.01) influenced by the bulls, cooling rates & equilibration periods, but not by diluents or the interactions studied. The mean prefreeze motility of spermatozoa following 1 h of cooling from 10°C to 5°C was significantly lower (60.38%) and that after 2 h of cooling from 30°C to 5°C was higher (72.38%) than 2 h of cooling from 10°C to 5°C (66.57%) or 1 h of cooling from 30°C to 5°C (67.96%). The mean post-thaw motility observed following 2 h of prefreeze cooling was, however, significantly greater (45%) than after 1 h of cooling (35%) for both the initial temperatures. Leakage of GOT pre- and post-freezing was significantly less following 2h of cooling from 30°C to 5°C (17.26 and 27.36 μmole/L) than after 1 h of cooling from either 10°C (19.71 and 30.13 μmole/L) or 30°C (18.95 and 29.58 μmole/L) and 2 h of cooling from 10°C to 5°C (21.43 and 34.48 μmole/L). The conception rates for semen frozen at the above cooling rates (66.13, 48.65, 56.67 and 42.25%, respectively) were inverse to GOT leakage. An equilibration period of 2 h over that of 0 h at 5°C adversely affected the prefreeze motility and GOT leakage, but it significantly improved postthaw motility (44.03 vs 35.49%) and fertility rates (57.86 vs 47.24%). These findings suggested that both Trisand milk-based diluents were equally efficacious for cryopreservation of bovine semen, and that slow cooling of semen straws over a period of 2 h from 30°C to 5°C as compared with faster cooling rates or a lower initial temperature (10°C), plus at least 2 h of equilibration time at 5°C were essential for optimal freezability, lower enzyme leakage & higher fertility rates within the tropics.

Improvement of post-thaw sperm motility in poor quality human semen

To find alternative cryopreservation methods to improve the post-thaw fertilizing capacity of poor quality human sperm. Controlled clinical study. Fertility clinic of a teaching hospital. Men with poor quality semen samples, i.e., asthenozoospermia (< 40% motile sperm) and/or oligozoospermia (< 20 x 10(6) sperm/mL). Fertile sperm donors were used for comparison. Semen samples were divided into four aliquots and slowly diluted 1:1 with: [1] n-tris (hydroxymethyl) methyl-2-amino ethane sulfonic acid (TES) and tris (hydroxymethyl) aminomethane (Tris)-citric acid-egg yolk buffer with 12% glycerol (TEST), [2] TEST+CryoSeeds (Cell Systems, Ltd., Cambridge, UK), [3] TEST + 10 mM dithiothreitol (DTT), or [4] TEST+CryoSeeds + 10 mM DTT. Cryovials were frozen using slow staged cooling and static vapor freeze and stored at -196 degrees C. The frozen aliquots were randomly thawed and, after 15 minutes at 37 degrees C, motion analysis was performed. The percent motility after freeze-thaw in TEST was significantly decreased to 42 +/- 5% of prefreeze motility (P < 0.001). Addition of CryoSeeds with holding at -5 degrees C for 10 minutes resulted in 47 +/- 6% of prefreeze motility, which was not different than TEST alone. Addition of DTT to TEST significantly improved post-thaw motility over TEST alone to 71 +/- 7% of initial motility (P < 0.01). The combination of CryoSeeds and DTT further improved post-thaw motility to 80 +/- 10% of initial motility, which was not different than the neat semen. The present results suggest that DTT, a reducing agent that prevents oxidation of sulfhydryl groups, protects poor quality spermatozoa from excessive cryodamage. Thus, DTT along with seeding may be a useful addition when long-term storage of poor quality semen is crucial for maintaining reproductive potential.

Human spermatozoal tail hypo-osmotic swelling test, motility characteristics in hypotonic saline, and survival of spermatozoa after cryopreservation.

Normozoospermic semen samples (n = 82) were examined to investigate whether the degree of sperm tail swelling in hypo-osmotic medium (fructose and sodium citrate; 150 mOsm/l), and motility characteristics after a 15-min exposure to hypotonic saline (sodium chloride; 150 mOsm/l) could predict the cryosurvival rate (% post-thaw motility/% pre-freeze motility x 100%) of spermatozoa after cryopreservation by the liquid nitrogen vapour freezing method using the TEST-glycerol-egg yolk buffer. The CellSoft automated semen analyser was used to analyse sperm motility in pre-freeze and post-thaw semen samples, and after exposure to hypotonic saline. Sperm tail hypo-osmotic swelling and sperm motility in pre-freeze semen showed no significant correlations (P > 0.05) with the cryosurvival rate. There were significant correlations (P < 0.05) between the cryosurvival rate and the following sperm motility parameters in hypotonic saline: % motility (r = 0.2846), motility index (% motility x curvilinear velocity; r = 0.2809) and % decrease in motility index from the baseline value in semen (r = 0.3378). The % decrease in motility index after hypotonic saline treatment was significantly less (P < 0.05) in the normal (> or = 50% cryosurvival rate; mean +/- SEM 5.9 +/- 3.2%; n = 33) compared with the subnormal (< 50% cryosurvival rate; 27.3 +/- 4.8%; n = 49) cryosurvival groups. This parameter was also determined, by multivariate discriminant analysis, to be capable of classifying each pre-freeze semen sample into either cryosurvival group with 69.5% accuracy.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation and Treatment of Infertility in Spinal Cord Injured Men Through Rectal Probe Electroejaculation

A total of 18 men older than 29 years with spinal cord injury was for fertility potential with testing of semen obtained by rectal probe electroejaculation. After fertility testing, including sperm penetration assay, semen cryopreservation and sperm antibody status, 6 of the 18 men proceeded with their partners to use rectal probe electroejaculation in efforts to conceive. Sperm was obtained in 16 of 18 cases. Average ejaculate total sperm count (306 million) was good but motility (22%) was poor. Adequate sperm retrieval after processing yielded a normal sperm penetration assay in 4 of 16 cases (25%) in which sperm were obtained. Favorable semen cryopreservation (greater than 33% of pre-freeze motility noted after thaw) was present in 5 of 16 cases (31%). Live births were achieved in 2 of 6 couples attempting conception. Despite the typically poor sperm motility noted in electroejaculates, rectal probe electroejaculation can result in pregnancies from couples involving spinal cord injured men. The sperm penetration assay data indicate that pregnancy should be achievable in at least 25% of spinal cord injured couples. Achieving these conceptions requires a team approach involving a urologist/andrologist, gynecologist/reproductive endocrinologist and a sperm-processing laboratory.

Effect of various cooling rates (from 30°C to 5°C) and thawing temperatures on the deep-freezing of [formula omitted][formula omitted] and [formula omitted][formula omitted] semen

A total of 36 semen ejaculates, six from each of three Holstein-Friesian bulls and three Murrah buffalo bulls, were frozen in tris citric acid-fructose-egg-yolk-glycerol diluent after 1 hour of equilibration to study the effect of various cooling rates (15, 30, 60 and 120 minutes from 10° to 5°C vs a control sample cooled for 120 minutes from 28° to 5°C) and thawing temperatures ( 40° C 60 seconds , 60° C 15 seconds and 80° C 5 seconds ) on prefreeze and post-thaw sperm motility. Sperm motility differed significantly (P < 0.01) between various cooling rates in both the Holstein-Friesian bull semen and the Murrah buffalo semen at prefreezing, immediately post-thawing, and after 1 hour of post-thaw incubation at 38°C. Post-thaw sperm motility and survival at 38°C were significantly (P<0.01) higher in Holstein-Friesian bulls at 60°C and 80°C than at 40°C (39.79±2.46% and 38.15±2.18% Vs 35.16±2.19%, and 20.22±2.14% and 19.05±2.05% vs 14.83±1.64%, respectively). In Murrah buffalo bulls the recovery percentage and survival rate increased significantly (P<0.01) with the increase in temperature from 40°C to 80°C (41.72±2.45%, 47.45±2.09% and 51.61±2.06%; and 9.22±1.47%, 11.79±1.63% and 12.27±1.53%, respectively). Prefreeze motility did not differ between cattle and buffalo bulls (64.97±1.08% Vs 67.11±0.89%, respectively) but post-thaw motility was significantly (P<0.01) higher in the buffalo (46.93± 1.39% Vs 37.70±1.32%). While incubation survival was higher in the cattle (18.04±1.16% Vs 10.96±0.89%). A fast cooling rate was found to be detrimental for cattle spermatozoa, whereas the post-thaw buffalo sperm motility deteriorated very quickly at 38°C. The influence of species-by-cooling rate interaction was significant (P<0.01) for post-thaw motility and survival rate, but the species-by-thawing or cooling-by-thawing interactions were not significant. These results suggest that a cooling rate of 2 hour either at 10°C or 28°C is essential for cattle semen. However, buffalo semen can be frozen successfully after 30 minutes of cooling at 10°C. A thawing temperature of 60°C yielded a higher sperm motility rate than 40°C. Thus, our findings can be applied under tropical conditions for the successful freezing-thawing of bovine semen provided conception rates are not affected adversely.

Quantitative analysis of the effect of freezing on donor sperm motion kinetics

One hundred ninety-two semen specimens from 14 donors were analyzed on a CellSoft Semen Analyzer (CRYO Resources, New York, NY) before and after freezing. Mean post-thaw motility decreased by 52%. Correlation of the percent decrease in post-thaw versus prefreeze motility was significant but of poor predictive value. Comparisons of the percent change in post-thaw motility and sperm motion kinetics between four discrete ranges of prefreeze motility (32% to 66%, 67% to 76%, 77% to 84%, 85% to 94%) revealed that the effect of freezing on sperm cell survival was equivalent between all ranges. However, significant differences occurred between these ranges for curvilinear velocity, straight line velocity, and mean amplitude of lateral head displacement but not for linearity nor beat cross frequency. All correlations between prefreeze and post-thaw motion variables were significant but closest for curvilinear velocity and straight line velocity. Furthermore, correlations of prefreeze versus post-thaw velocity measurements were strongest for those cells within the 65% to 85% range of prefreeze motility. We suggest that sperm survival is independent of prefreeze motility. However, velocity kinetics appear stable after freezing for those specimens that had an initial motility of 65% to 85%.

Effects of Equex STM and equilibration time on the pre-freeze and postthaw motility of equine epididymal spermatozoa

Epididymal spermatozoa from castrated stallions were recovered, frozen, and thawed using techniques adapted from those used with ejaculated semen. An average of 7.93 × 10 9 spermatozoa were recovered per stallion, with a range of 1.45 to 16.82 × 10 9. Average prefreeze (postequilibration) motility was 32.0% ± 1.4 SEM. Prefreeze and postthaw motilities of epididymal spermatozoa were found to vary significantly between different stallions (P < 0.0001). No differences in prefreeze or postthaw motilities were seen between prefreeze equilibration times of 30 and 120 min. Prefreeze motility did rise, however, during the first 30 min of equilibration. Inclusion of the surfactant Equex STM a a Sodium and Triethanolamine Lauryl Sulfate, Nova Chemical Sales, Inc., Scituate, MA. in the extender had no significant effect on prefreeze nor postthaw motilities. In the motility of prefreeze sperm, interactions were found between the horse used and equilibration time and also between the horse used and the inclusion of Equex STM in the extender. No such interactions were seen with postthaw motilities. Postthaw motility averaged 6.4% ± 0.5 SEM or 20.7% ± 1.6 SEM of the prefreeze motility.

Pregnancy following in vitro fertilization using cryopreserved semen from a man with testicular teratoma

Semen for cryopreservation was collected in a man with a testicular teratoma after unilateral orchidectomy but before chemotherapy which rendered him azoospermic. After two years artificial insemination using this semen in his wife failed repeatedly. The semen quality on thawing was extremely poor in terms of sperm motility. A pre freeze motility of 90 per cent was reduced to 2 per cent, and the movement was graded as sluggish. Using the techniques of semen and oocyte preparation and in vitro fertilization, a number of cleaving embryos was produced. A pregnancy was established after four of these embryos were replaced in the wife. The pregnancy aborted spontaneously, but a subsequent course of treatment resulted in an on-going twin pregnancy. The potential of in vitro fertilization for overcoming the poor quality of semen after storage by cryopreservation from men with testicular neoplasms is discussed.

Semen characteristics as a function of age in 833 fertile men

The relationship between age and semen characteristics has been studied; any effect due to the influence of the length of abstinence preceding ejaculation was eliminated. There is an improvement in semen characteristics up to 25 years of age, followed by a leveling off and a subsequent decrease. This variation is not significant as far as the sperm count, semen volume, and the total number of spermatozoa are concerned. The variation, although small, is highly significant for the morphologic characteristics and prefreeze and postthaw motility. The values for the older subjects were significantly lower for postthaw motility in the group 36 to 40 years of age, in the group 41 to 45 years of age for morphologic normality, and in the group 46 to 50 years of age for prefreeze motility. The lower values in the group 21 to 25 years of age are particularly noticeable with regard to morphologic characteristics. The same curve is encountered in the variation with age of each abnormal form, but the most marked variation is found in the increased percentage of coiled tails, which first appears in the group 36 to 40 years of age.

Motility characteristics of human sperm, nonfrozen and cryopreserved.

Ejaculates (1651) obtained by masturbation from 88 donors were evaluated for count, motility, and kinetics. After the initial evaluation the ejaculates were frozen in liquid nitrogen, thawed 24 hr later, and assessed for postthaw motility and kinetics. The ejaculates were then divided into seventeen groups according to sperm count and evaluated to determine if direct relationships exist between prefreeze and thawed semen characteristics. The average sperm count was 151 X 10(6) sperm/ml. The freezing process resulted in a reduction of motility from a mean of 85% to a mean of 52%. Postthaw motility increased with sperm count up to 120 X 10(6) sperm/ml where there appeared to be no further effect. Linear regression analysis demonstrated a high correlation between prefreeze motility and postthaw motility (r = 0.947). Cryopreservtion of human spermatozoa results in a significant reduction of motility. A direct relationship exists between prefreeze and postthaw motility and prefreeze motility may be an important factor in evaluating the potential of an ejactulate to withstand cryopreservation.

Improved Motility Recovery of Human Spermatozoa after Freeze Preservation Via a New Approach

A new sperm isolation technique was studied in conjunction with the short-term freeze preservation of human spermatozoa. The isolation procedure yielded subpopulations of spermatozoa with very high percentage motility and progressive motility score, and which were virtually free of seminal debris. For 24 semen samples from 13 donors, the mean prefreeze values of percentage motility and motility score were 53% (2.8), 73% (3.1), and 88% (3.5) for the parent semen, 7.5% BSA (middle) fractions, and 17.5% BSA (bottom) fractions, respectively. These samples were used without a priori constraint on semen quality. Eleven samples were preselected on the basis of good motility in the semen. These yielded prefreeze motility values of 66% (3.2), 70% (3.4), and 87% (3.9) for the semen and two fractions, respectively. Sperm motility in the middle fractions was thus intermediate to that in the semen and in the bottom fractions, although closer to the former in these 11 cases. The sperm freeze preservation procedure involved dilution of the semen samples and separated sperm fractions with a cryoprotective semen extender, and freezing and thawing in a conventional manner. Post-thaw percentage motility, motility score, and percentage survival were substantially higher for the separated fractions than for the parent semen. For the 24 cases, the bottom fractions yielded mean values of 57% motility, 3.0 motility score, and 70% survival, in comparison with the respective values of 20%, 2.3, and 34% for the semen. In the 11 preselected cases, the bottom fractions yielded post-thaw mean values of 60% motility, 3.3 motility score, and 72% survival; the middle fractions yielded respective values of 45%, 3.1, and 64%; and the parent semen yielded respective values of 27%, 2.3, and 41%. It was concluded that the major factor in improving post-thaw motility recovery was the separation process as a whole, rather than the degree of separation.

Variable Motility Recovery of Spermatozoa Following Freeze Preservation

Semen from a random group of potentially fertile men and a small number of donors who had established an artificial insemination pregnancy was mixed with a cryoprotective agent and frozen by two different methods. After 1 week under liquid nitrogen (-196 degrees C), all samples were thawed and their motilities determined. The post-thaw motilities were compared with the prefreeze motilities. The mean motility recovery rate in the group of semen samples with high counts was better than that in the group with counts in the low-normal range. The recovery rate with the slow-freeze method was slightly higher than that with the quick-freeze technique. However, the wide range of post-thaw motility among the random donors (0 to 60%) and the donors of proven fertility (15 to 45%) demonstrated that prefreeze motility and/or paternity history was no guarantee of good motility recovery following freezing. At a time when semen banking is being contemplated for "fertility insurance" prior to vasectomy, it is apparent that a trial freeze is necessary before recommending freeze-preservation of his semen to the man contemplating permanent surgical sterilization.

Cervical mucus penetration in vitro by fresh and frozen-preserved human semen specimens.

A serious difficulty in the use of frozen-preserved human semen for donor insemination is our inability to predict, without rather extensive clinical trials, which specimens will be successful in producing conception and which will be generally infertile. Predictions cannot be made from the pre-freeze motility of the specimen (Behrman & Sawada, 1966) nor, in our experience, from the fertility of a donor's freshly ejaculated specimen. A technique for determination of cervical mucus penetration by spermatozoa using capillary tubes (Kremer, 1965) has been thoroughly investigated and found to have several advantages (Kremer, 1968). This capillary tube test has been shown to be a reasonably reliable predictor of the fertility of fresh human