Effect of glycerol and cryopreservation on oocyte penetration by human spermatozoa

A poor penetration rate of glycerol-treated, cryopreserved human spermatozoa as compared to untreated fresh control, was observed in the zona-free hamster oocyte test. Similarly, glycerol treatment of freshly ejaculated spermatozoa depressed the penetration rate unless the culture medium also contained glycerol. Immediately after thawing, glycerol-treated, cryopreserved spermatozoa possessed adequate progressive motility, but their incubation in glycerol-free culture medium caused a severe reduction in motility. Even if the same number of progressively motile, cryopreserved, glycerol-treated spermatozoa as unfrozen spermatozoa were added to the eggs, a much lower penetration rate was obtained by the treated spermatozoa. It is concluded that spermatozoa develop a glycerol dependence and that removal of glycerol from the surrounding medium, as most likely occurs when spermatozoa pass through the cervix, reduces both the motility and the ability of spermatozoa to become capacitated and fuse with oocytes. Thus, glycerol is not an optimal cryopreservative agent. Further, the decreased oocyte penetration rate of glycerol-treated, cryopreserved spermatozoa is due to other factors besides the decrease in sperm motility.     

Annexin V binding to plasma membrane predicts the quality of human cryopreserved spermatozoa

Cryopreservation-induced stress may result in membrane injury with consequent decrease of sperm motility and fertilizing capacity. This study has investigated the relationship between human spermatozoa tolerance to cryopreservation and the loss of plasma membrane asymmetry, especially translocation of phosphatidylserine (PS) from the inner to the outer leaflet. The prospective study was performed on semen samples from 31 men. Conventional characteristics of 20 semen were analysed, before and after cryopreservation as well as externalization of PS assessed by annexin V-staining in combination with the propidium iodide which stains dead cells. The fertilizing capacity was evaluated by a zona free hamster egg penetration test in 11 freeze/thaw spermatozoa samples. The percentages of vital annexin V-negative and annexin V-positive spermatozoa in post-thaw samples were significantly lower than in pre-freeze ones (10 +/- 3 vs. 25 +/- 5% and 22 +/- 3 vs. 34 +/- 4% respectively), while the percentages of dead spermatozoa annexin V-negative and annexin V-positive had increased (42 +/- 4 vs. 23 +/- 4% and 23 +/- 4 vs. 16 +/- 2% respectively). The percentages of progressive and total motile spermatozoa were significantly correlated with the percentage of vital annexin V-negative spermatozoa before as well as after cryopreservation. Furthermore, recovery of motile spermatozoa after freeze/thaw was strongly correlated (p < 0.002) with the proportion of vital annexin V-negative spermatozoa in fresh semen. The percentage of penetration of zona-free hamster eggs was correlated (p < 0.02) with the percentage of live annexin V-negative cryopreserved spermatozoa capacitated for 3 h. These findings provide evidence that annexinV-binding staining in combination with PI brings additional information to predict the outcome of cryopreserved ejaculated sperm and may be used as a novel and reliable marker to study the freeze/thaw process.     

The motility of demembranated human spermatozoa is inhibited by free calcium ion activities of 500 nmol/L or more

A number of studies have demonstrated that high calcium ion activities inhibit sperm motility, but little is known about the effect of different calcium activities close to the physiological range. Therefore, we investigated whether raising calcium activities within the submicromolar range would inhibit the motility of demembranated human spermatozoa. Spermatozoa were demembranated with Triton X-100 and motility was measured objectively by computer assisted semen analysis. Motility, reactivated by 1 mol adenosine 5'-triphosphate (AlphaTauP)/L, was short lived, with maximum activity only sustained for about 1 min. Reactivated motility was not affected by 50 micromol cAMP/L. The amplitude of lateral head displacement was significantly greater at room temperature than at 37 degrees C, but there were no significant differences between the percentage of sperm motile or their velocity at the two temperatures. The calcium buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) at 1 mmol/L was included in the demembranation-reactivation medium, and free calcium ion activities were calibrated using the fluorescent calcium probe Fura-2. Calcium ion activities of > or =500 nmol/L significantly inhibited the percentage of demembranated-reactivated spermatozoa that were motile, and the velocity and lateral head displacement of these cells. The range of intracellular calcium activities in spermatozoa from 24 cryopreserved ejaculates was 110-534 nmol/L; roughly twice the value in fresh spermatozoa. Therefore, calcium ion activities in the range observed in cryopreserved spermatozoa can inhibit the activity of demembranated human spermatozoa.     

Adenosine triphosphate levels in human spermatozoa

ATP levels in human spermatozoa have been positively correlated with good motility. This has given rise to the impression that good ATP levels per se are related to good motility, i.e., the higher the ATP level, the better the motility and fertilizing potential. There was no direct correlation between motility percentage, forward progression, viability percentage, and ATP levels (when expressed per 1 million spermatozoa) in the general population. This finding was not unexpected since other factors, such as defects of the microtubules and viscosity of the semen, could affect the motility in some patients. However, when semen from individual patients was assessed, the motility percent, viability, and ATP concentration decreased by comparable levels over a 4-h period postejaculation. Semen samples with normal counts of spermatozoa (greater than 20 X 10(6)/ml) had higher levels of ATP than samples from patients with oligozoospermia. Spermatozoa from patients whose semen contained less than 0.60 X 10(-2) nmol ATP/1 million spermatozoa showed a rapid drop in motility over a 4-h period compared with semen samples where the motility remained above 10% motile over this period, the latter samples having ATP levels averaging 3.30 X 10(-2) nmol/1 million spermatozoa.     

Relationships between computerized measurements of motion of frozen-thawed bull spermatozoa and fertility

A computerized system (CellSoft, CRYO Resources, Ltd.) was validated using video tapes of frozen-thawed bull spermatozoa diluted in filtered (0.2 micron) egg yolk-citrate extender (8 X 10(6) spermatozoa/ml) and analyzed at 30 frames/sec for the percentage of motile spermatozoa (greater than or equal to 20 microns/sec) and linear velocity of motile spermatozoa. Virtually all motile spermatozoa were detected and debris rarely were classified as immotile spermatozoa if the extender had been filtered. Variation about the mean for percent motile cells was similar when only 12 rather than 20 or 30 frames/field were analyzed. Use of 20 frames/field was adequate to determine the percentage of motile bull spermatozoa. Five mixtures of live and killed spermatozoa were analyzed (four bulls) to evaluate accuracy. Percent motile spermatozoa was correlated (r = 0.97) with the ratio of live:killed spermatozoa. Mean linear velocity of motile spermatozoa was similar for each mixture (P greater than 0.05). To further evaluate accuracy, percent motile spermatozoa was determined by computer and by "track motility" (20 samples; 0 to 63% motile spermatozoa); values were correlated (r = 0.95). The system was precise (CV of 6% based on triplicate analyses of the same samples) and reasonably accurate for evaluating bull sperm motility if the extender had been filtered and 20 to 25 fields (greater than or equal to 200 spermatozoa) were evaluated. Correlations between measurements of sperm motion and fertility were studied using cryopreserved semen from two fertility trials. For the first, 75-day nonreturn rate data for 20 samples of bull semen (10 bulls) were not significantly correlated with evaluations made by CellSoft. For the second fertility trial, the competitive fertility index (a measure of relative fertility) for nine bulls was correlated (r greater than or equal to 0.68; P less than 0.05) with percent motile spermatozoa, linear velocity and straight-line velocity. Multiple correlations based on six characteristics evaluated by CellSoft, at 0 or 1.5 hours, and the competitive fertility index were greater than or equal to 0.94. Based on the latter data, the system may facilitate prediction of the relative fertility of bull spermatozoa.     

Catalase improves motility,vitality and DNA integrity of cryopreserved human spermatozoa

Cryopreservation of human spermatozoa offers a pre‐therapeutic possibility of preserving progenity in patients with testicular tumours. We aimed to investigate effects of cryopreservation and addition of catalase on sperm motility, vitality and DNA integrity in fresh and swim‐up spermatozoa. Semen samples were collected from 50 fertile men. Each sample was divided into two parts. First part was subdivided into two equal aliquots: both cryopreserved with and without catalase. The second part was subdivided into two equal aliquots: both processed by swim up and then cryopreserved with or without catalase. Semen analyses, sperm vitality and sperm DNA integrity were performed. Sperm concentration showed significant decrease while percentage of progressive motility, sperm vitality and % of DNA damage showed significant increase in processed and cryopreserved processed samples when compared with fresh and cryopreserved fresh samples. There was no significant difference in sperm concentration while there was significant increase in % of progressive motility and sperm vitality and % of DNA damage showed significant decrease in samples with catalase when compared with samples without catalase (either fresh or processed). Catalase supplementation (fresh and processed) during cryopreservation results in better post‐thawing percentage of progressive motility and percentage of sperm vitality and improved DNA integrity.     

Correlation between the motility of frozen–thawed epididymal spermatozoa and the outcome of intracytoplasmic sperm injection

The purpose of this study was to investigate if the outcome of ICSI was influenced by epididymal sperm motility in frozen-thawed specimens. A total of 18 ICSI treatment cycles using spermatozoa retrieved by microsurgical epididymal sperm aspiration (MESA) were analysed retrospectively. Cryopreservation of epididymal spermatozoa was performed when enough epididymal aspirates were collected. Sixty-nine out of 126 oocytes injected with spermatozoa retrieved by MESA were fertilized, giving a fertilization rate of 54.8%. Out of 18 embryo transfer cycles, 6 (33.3%) achieved pregnancies. Fresh epididymal spermatozoa were used in 5 cycles while frozen-thawed epididymal spermatozoa were used in 13 cycles for ICSI. The fertilization rates were 68.6% (35/51) in the former group and 45.3% (34/75) in the latter group, respectively. There was a significant difference between the two groups (p < 0.05). In ICSI treatments using fresh epididymal spermatozoa, the cells used for injection were all motile. However, motile epididymal spermatozoa could be used in only five ICSI treatment cycles after freeze-thawing. In 6 cycles, only immotile sperm were used for injection of frozen-thawed spermatozoa. The fertilization rate in each group was 68.4% (13/19) and 31.6% (12/38), respectively. There was a significant difference between these groups (p < 0.01). These results indicate that the outcome of ICSI was influenced by sperm motility in frozen-thawed epididymal specimens. When no sperm motility could be recovered after freeze-thawing even with chemical treatments, consideration should be given to retrieving fresh epididymal spermatozoa again to achieve a better fertilization rate in such patients.     

ATP and ADP content of human ejaculated spermatozoa

ATP and ADP were measured by a bioluminescent method in human ejaculated spermatozoa from 31 normal donors and oligozoospermic patients. ATP and ADP expressed for the sperm total number were significantly correlated ( P < 0.001). ATP and ADP levels were found significantly correlated with the total motile spermatozoa and with the total viable cells (respectively P < 0.001 and P < 0.001 for ATP and P < 0.001 and P < 0.001 for ADP). ATP/ADP ratio was found significantly lower in subjects with sperm motility less than 50% ( P = 0.024) and total viable cells less than 80% ( P = 0.026).     

PC3 prostasomes对精细胞上游筛选后活力的影响

目的 探讨从PC3前列腺癌细胞株中分防出的prostasome样颗粒（PC3prostasome）对上游筛选后精细胞活力的。方法 将新鲜或冻融精细胞置入含PC3prostasome(0.10mg/ml或0.25mg/ml）及人血白蛋白（HSA，10mg/ml）的EBSS液中上游筛选后，测试比较活力的差别。结果 新鲜标本不同孵育时间和孵育1h不同参数间比较，PC3prostasome0.10mg/ml组促精细胞活力人中于HSA组（P＜0.01），且此作用可持续6h。冻融复温标本PC3prostasome组恢复活力的细胞比HSA组增加63％（P＜0.001）。结论 PC3prostasome具有与精浆prostasome相同的刺激精细胞活力作用，且此作用强于HSA。     

Semen characteristics of the captive Indian leopard, Panthera pardus

Semen samples from 11 Indian leopards (Pantherapardus) from 3 different zoos in India were collected by electroejaculation. A computer-aided semen analyzer (CASA) was used for assessing the quality of the semen vis-à-vis sperm motility. The volume of the ejaculate, sperm density, and the number of motile and morphologically normal spermatozoa were found to be 1.57 +/- 1.26 mL, 55.78 million +/- 38.67 million per mL, 57.05% +/- 16.96% and 71.92% +/- 15.32%, respectively. Although the spermatology varied between individuals in the study, Box-Whisker-plot analysis suggested that the distribution was normal (P > .05). The ejaculated sperm were cryopreserved after diluting in test-yolk buffer. The post-thaw motility was 32.14% and did not differ at 30 or 60 days after cryopreservation. CASA indicated that the progressive velocity (VSL) of cryopreserved spermatozoa was decreased and, as a consequence, they moved more slowly than the neat (VSL 76.3 microm/sec in neat vs 53.8 microm/sec in cryopreserved spermatozoa) and the trajectories were less planar. However, both cryopreserved and neat spermatozoa penetrated the zona-free hamster oocyte with equal efficiency (79% neat vs 80% cryopreserved). The study also reports application of CASA for feline spermatozoa and provides information for the first time on the spermatology of the Indian leopard. This baseline data could be used in captive breeding programs. The results are compared and discussed with the available information on other felines.     

L- carnitine in idiopathic asthenozoospermia: a multicenter study

Summary. The aim of the study described here was to evaluate any possible effect of L-carnitine on spermatozoal motility in a group of patients with unexplained asthenozoospermia in four different infertility centres. One hundred patients received 3 g d⁻¹ of oral L-carnitine for 4 months. Sperm parameters were studied before, during and after this treatment. Motility was also studied by means of a computer-assisted sperm analysis.
The results of the study indicate that L-carnitine is able to increase spermatozoal motility, both in a quantitative and in a qualitative manner (per cent motile spermatozoa increased from 26.9±1.1% to 37.7 ± 1.1% [ P < 0.001]; per cent spermatozoa with rapid linear progression increased from 10.8 ± 0.6% to 18.0 ± 0.9% [ P < 0.001]; mean velocity increased from 28.4 ± 0.6 μm s⁻¹ to 32.5 ± 0.8 μm s⁻¹ [ P < 0.001]; linearity index increased from 3.7 ± 0.1 to 4.1±0.1 [ P < 0.001], especially in the subgroup of patients with poor rapid linear progression of spermatozoa (per cent of motile spermatozoa increased from 19.3± 1.9% to 40.9± 1.4% [ P < 0.001], and per cent of spermatozoa with rapid linear progression increased from 3.1±0.4% to 20.3±1.6% [ P < 0.001]) An increase in spermatozoal output was also observed (total number of ejaculated spermatozoa increased from 142.4 ± 10.3 10⁶ to 163.3 ± 11.0 × 10⁶ [ P < 0.001]). The authors conclude that oral administration of L-carnitine may improve sperm quality at least in patients with idiopathic asthenozoospermia.     

Correlation between defective motility (asthenospermia) and ATP reactivation of demembranated human spermatozoa

Human spermatozoa treated with 0.05% Triton X-100 to remove the cell membranes became immotile but flagellar movement was reinitiated by addition of 0.06 to 1 mM adenosine triphosphate (ATP). The percentage of spermatozoa showing flagellar movement 2 minutes after addition of 1 mM ATP from men with idiopathic asthenospermia (mean +/- SD, 34 +/- 15%), oligozoospermia (17 +/- 21%), sperm autoimmunity (17 +/- 12%), vasoepididymostomy (20 +/- 22%), or idiopathic zero motility (0%) was significantly lower than with spermatozoa from normal men (54 +/- 12%). The percentage of reactivated spermatozoa was correlated with the proportion of live cells (Eosin Y stain, r = 0.602, P less than 0.001), percentage of motile cells (r = 0.761, P less than 0.001), and motility index (r = 0.759, P less than 0.001) in the same semen samples. When expressed relative to initial sperm motility, the proportion reactivated was similar for idiopathic asthenospermia (85%) and normospermia (82%). Thus, failure to produce ATP does not appear to be a frequent cause of low sperm motility in man.     

Adenosine triphosphate (ATP) in human spermatozoa

The seminal adenosine triphosphate (ATP) content was determined by bioluminescence after treatment with trichloroacetic acid (TCA) in 81 semen samples 1.5 h after ejaculation obtained from men attending our fertility clinic, and selected to contain either 20% or less spermatozoa with good progressive motility (n = 22), or 60% or more spermatozoa with good progressive motility (n = 59) (Study I), and in 18 semen samples from fertile men 30 min and 3.5 h after ejaculation (Study II). The latter samples were divided into 2 equally large groups according to sperm motility. In Study I the mean sperm ATP concentration was significantly higher in the semen samples with bad motility (0.63 nmol per living spermatozoa x 10(-6)) than in semen samples with good motility (0.39 nmol per living spermatozoa x 10(-6); P less than 0.01). In Study II the ATP concentration per living spermatozoa was also lower in the group with the best motility in comparison with the spermatozoa with lower motility (P less than 0.01), both 30 min and 3.5 h after ejaculation. During the 3-5 h incubation the sperm ATP concentration decreased by 21% (P less than 0.01) in the former group of samples but remained unchanged in the latter group. The results indicate that, in semen samples with highly motile spermatozoa, the consumption of ATP is higher than in semen samples with impaired sperm motility. It is therefore essential that the time between ejaculation and ATP measurement is as short as possible to obtain comparable results. Repeated ATP measurements in combination with an analysis of the number of living spermatozoa, may provide further information on the fertilizing capacity of spermatozoa.     

Teratospermic and normospermic domestic cats: ejaculate traits, pituitary-gonadal hormones, and improvement of spermatozoal motility and morphology after swim-up processing

Electroejaculate traits, testicular volume, and circulating FSH, LH, and testosterone concentrations were compared between two populations of domestic cats consistently producing either a high (greater than 60%, normospermic) or low (less than 40%, teratospermic) incidence of structurally normal spermatozoa/ejaculate. The effects of semen dilution in Biggers, Whitten and Whittingham (BWW) or modified Krebs Ringer bicarbonate (mKRB) medium and swim-up processing on sperm viability and duration of motility in vitro also were assessed. Ejaculate volume, percent sperm motility, sperm progressive motility, motile spermatozoa/ejaculate, testes volume, and mean serum FSH and LH concentrations were similar (P greater than 0.05) between normospermic and teratospermic cats. However, sperm concentration/ml of ejaculate was greater and circulating testosterone levels were lower in teratospermic males. Swim-up processing increased (P less than 0.05) percent sperm motility, progressive motility, and the number of structurally normal sperm cells recovered and also prolonged the duration of sperm motility in both cat populations. In teratospermic ejaculates, swim-up separation increased the proportion of morphologically normal spermatozoa recovered by more than two-fold. Diluting cat semen with either BWW or mKRB increased flagellar bending in both normospermic and teratospermic cats. The sperm motility characteristics of only the teratospermic ejaculates were influenced by medium type; mKRB increased percent sperm motility and progressive motility whereas BWW had no effect. Compared with undiluted raw ejaculates, the duration of sperm motility was improved 18- to 24-fold by diluting semen in either BWW or mKRB medium followed by swim-up processing. This study demonstrates that the electroejaculate characteristics of domestic cats vary markedly and that some males consistently produce high proportions of morphologically abnormal spermatozoa. Diminished serum testosterone concentrations and normal pituitary secretion of FSH and LH in teratospermic males suggest that there is an inverse relationship between gonadal androgen production and pleiomorphic spermatozoa in the domestic cat. The swim-up procedure is effective for recovering motile, structurally normal spermatozoa from teratospermic cats.     

Comparison of fresh and cryopreserved human sperm attachment to bovine oviduct (uterine tube) epithelial cells in vitro.

Formation of a prefertilization sperm reservoir in mammals is thought to occur via sperm cell attachment to fallopian tube or oviduct epithelial cells (OEC). Recent data suggests that such an interaction also occurs for human sperm in the fallopian tube. We have previously validated an in vitro sperm-OEC coculture system utilizing bovine OEC monolayers to study postejaculatory human sperm physiology. This study was done to evaluate aspects of human sperm attachment to OEC in coculture and to determine if such attachment and subsequent sperm survival differ between fresh and cryopreserved human sperm. In experiment 1, aliquots of fresh (n = 4) or cryopreserved sperm (n = 3) from normospermic donors were placed into coculture with OEC monolayers at dilutions ranging from 2 x 10(5) to 15 x 10(6) sperm per well. Numbers of each type of sperm attaching to OEC at each concentration were determined. In experiment 2, fresh and cryopreserved sperm from the same donors (n = 4) were put into OEC coculture to observe numbers attaching and subsequent survival time for each sperm type. Sperm attachment to OEC occurred in a linear, dose-dependent manner for fresh and cryopreserved sperm in experiment 1, both as a function of total sperm numbers and as a function of numbers of motile sperm applied (R2 > or = 0.79). However, cryopreserved sperm attached to the OEC at a slower rate than fresh (as a function of the average increase in the number of sperm attaching per unit increase in the number of sperm applied; P < 0.05), with an overall lower percentage of the total and motile sperm applied attaching to OEC (P < 0.01) for cryopreserved versus fresh sperm. Fewer cryopreserved sperm also attached to the OEC, as compared with fresh sperm, in experiment 2 (P < 0.05), even after correcting for motility differences between the sperm types. Sperm survival time in coculture was also decreased for cryopreserved sperm as compared with fresh sperm (P = 0.005). Understanding the kinetics of sperm and OEC interactions may be useful for developing improved cryopreservation protocols or bioassays of sperm function.     

The Effect of Caffeine on Guinea Pig Epididymal Spermatozoa: Motility and Fertilizing Capacity

Guinea pig epididymal spermatozoa, when taken from different segments of the epididymis, are known to differ markedly in their in vitro motility, metabolism and morphology. In this report, the fertilizing capacity of epididymal spermatozoa isolated from proximal and distal segments and treated with caffeine was tested. Twenty six guinea pig sows were inseminated 16–26 h after parturition with isolated epididymal spermatozoa. In 15 cases the spermatozoa were treated with caffeine (10 mM), and 11 cases served as controls. Sperm characteristics (mean ± SE) were: proximal sperm; concentration 48 ± 3.9 millions/ml, vitality 94 ± 0.9%, motility 42 ± 5.3%. Distal sperm: concentration 161 ± 21 million/ml, vitality 94 ± 0.9% and motility 86 ± 3.0%. Caffeine increased significantly the motility of proximal spermatozoa (by 59.5%). These suspensions of spermatozoa were used for intraperitoneal artificial insemination.
The impregnation rate of untreated proximal sperm was zero (0 out of 4), compared with 83% (5 out of 6) for the caffeine treated sperm. The impregnation rate of untreated distal segment sperm was 42.8% (3 out of 7) compared with 100% (9 out of 9) for caffeine treated sperm.     

Influence of cryopreservation on the microelectrophoretic motility (EPM) of human spermatozoa

The net surface charge of spermatozoa represents a characteristic of the plasmamembrane. It can be evaluated indirectly, by the measurement of the sperm electrophoretic motility (EPM). The EPM of washed human spermatozoa was determined in the microelectrophoresis to investigate three problems: (a) intra- and interindividual variation coefficients of EPM of cryopreserved spermatozoa, (b) effects of cryopreservation medium, temperature shock and cryoprotection on the EPM, (c) relationship between the EPM and the cryotolerance of the spermatozoa. The inter- and intrain-dividual variation coefficients of the sperm EPM of 24 cryopreserved semen samples of 6 fertile semen donors amounted to 7% and to 6%, respectively. The cryopreservation did not cause a significant change of sperm EPM in contrast to the temperature shock in liquid nitrogen. Temporary incubation of spermatozoa with egg yolk decreased their EPM. A significant correlation between EPM and the cryotolerance ascertained by the alteration of sperm motile efficiency could be found. After contact with egg yolk spermatozoa achieved a behaviour that corresponded to a better cryotolerance.     

Fertility and its relationship to motility characteristics of spermatozoa in ewes after cervical, transcervical, and intrauterine insemination with frozen-thawed ram semen

The fertility of ewes after artificial insemination and the relationship between fertility and motility characteristics assessed by a computerized motility analysis system were examined with ram semen frozen in diluents reported to improve postthaw motility. The percentages of motile and progressive spermatozoa were better when frozen in proline- or glycine betaine-containing or HEPES-based, rather than Tris-based, diluents (P < 0.01). The fertility of spermatozoa frozen in diluents containing proline or glycine betaine was slightly reduced, whereas when both compatible solutes were present, the reduction was more pronounced, in comparison with semen frozen in Tris- or HEPES-based diluents (9.5 versus 71.1 and 66.6%; P < 0.01). Fertility of frozen-thawed spermatozoa was higher after laparoscopic insemination than after cervical or transcervical insemination (P < 0.01). Similarly, higher fertility was obtained after cervical insemination with fresh than with frozen-thawed semen (32.4 versus 11.3%; P < 0.01). Furthermore, loss of embryos was lower after laparoscopic insemination of ewes with semen frozen in a Tris diluent than with semen frozen in proline diluents, in glycine betaine diluents, or in proline-plus-glycine betaine diluents (0.0 versus 26.0, 38.5, and 60.0%; P < 0.001). A wide variation in the postthaw percentage of motile (31.6-59.7%) and progressive (22.6-43.1%) spermatozoa and in the fertility of spermatozoa from individual rams was also observed after laparoscopic (29.2-59.7%) or cervical insemination (8.7-30.5%). Postthaw motility results from immediately after thawing and fertility results from experiments where intrauterine insemination was performed with semen frozen in proline- or glycine betaine-containing or HEPES- or Tris-based diluents were pooled and subjected to a pairwise correlation procedure. The correlation analysis showed relationships between some of the motility characteristics (P < 0.01), but there were no relationships between the motility characteristics and fertility.     

Catalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity

The potential protective effect of catalase supplementation during in vitro culture of frozen/thawed bull spermatozoa was investigated. Frozen/thawed semen collected from three fighting bulls was diluted in phosphate buffered saline (PBS) and incubated at 37 °C under different experimental conditions: Control, Catalase (CAT) (200 U/mL), Oxidant (OXI) (100 μ m Fe²⁺/1 m m ascorbate), and Catalase + Oxidant (CAT/OXI). We assessed sperm motility, acrosomal integrity, viability and chromatin status (SCSA®) at 0, 2 and 6 h of incubation. Our results showed that catalase abolished the effect of the oxidant, protecting spermatozoa against reactive oxygen species, and improving both sperm motility and chromatin status during incubation. The OXI treatment significantly reduced the percentage of motile sperm after 6 h of incubation. The statistical model also showed that there were differences in sperm motility between CAT/OXI (20.8 ± 2.9%) and OXI (11.6 ± 7.6%) ( p  < 0.001). There were no significant effects of OXI on sperm viability, acrosomal status or proportion of abnormal tails. %DFI (spermatozoa with moderate or high DNA Fragmentation Index) was significantly higher on OXI ( p  < 0.001). Catalase prevented DNA fragmentation even in the presence of the oxidant (%DFI: 30.3 ± 0.8% OXI vs. 17.4 ± 0.7% CAT/OXI). We conclude that catalase supplementation after thawing could protect bull spermatozoa against oxidative stress, and it could improve media used for processing thawed spermatozoa.     

Kruger strict morphology and post-thaw progressive motility in cryopreserved human spermatozoa

The purpose of this prospective study was to evaluate Kruger strict morphology and conventional semen analysis in predicting cryosurvival and the progressive motility recovery rate of frozen spermatozoa. Our study included 56 semen samples with >10 million spermatozoa per ejaculate. The main outcome measures were conventional semen analysis, strict morphology analysis by the Kruger method, cryosurvival rate and post-thaw sperm motility. A significant reduction in sperm motility after cryopreservation was demonstrated. The freeze-thawing process caused a 66% reduction in rapid progressive motile spermatozoa, a 45% reduction in slow progressive motile spermatozoa and a 2% reduction in nonprogressive motile spermatozoa. The cryosurvival and progressive motility recovery rates were not correlated with parameters of conventional semen analysis, such as sperm concentration, motility, WHO morphology and total motile count, but the progressive motility recovery rate was significantly correlated with the percentage of spermatozoa exhibiting Kruger normal morphology (P = 0.028). The recovery rate of rapidly progressive motility was profoundly decreased compared with slow progressive motility following the frozen-thaw procedure of semen. Kruger strict morphology assessment was a better predictor of the progressive motility recovery rate following the freezing-thaw procedure than parameters of conventional semen analysis.