Influence of epididymal maturation on the capacity of hamster and rabbit spermatozoa for complement activation

Hamster and rabbit spermatozoa released from the epididymis were tested for the ability to activate complement via the alternative pathway. While hamster spermatozoa were more active, the spermatozoa of both species reduced complement activity in homologous and also human serum previously adsorbed to remove sperm antibodies, and they bound C3 in the presence of EGTA + Mg2+. Hamster spermatozoa from the caput epididymidis were more anticomplementary and bound more C3 than did cauda spermatozoa and, though less marked, a similar difference was evident between caput and cauda spermatozoa from the rabbit epididymis.     

Flow sorting of X and Y chromosome-bearing spermatozoa into two populations

The only established difference on which to base the separation of X and Y chromosome-bearing spermatozoa is chromosomal constitution. This difference is quantifiable both from chromosome morphology (karyotype) and from DNA content. Flow cytometric techniques were used to measure relative DNA content of the X and Y populations and to flow-sort spermatozoa from Chinchilla laniger. Epididymal spermatozoa were recovered in PBS, fixed in 80% ethanol, treated with papain and dithioerythritol, and stained for DNA with Hoechst 33342. Sperm nuclei were analyzed and sorted on an EPICS V flow cytometer/cell sorter, modified specifically for spermatozoa. Two clearly resolved peaks (coefficient of variation < 1.5%) with approximately 7.5% difference in DNA content between X and Y chromosome-bearing spermatozoa were evident. Sperm nuclei were sorted from a portion of the X and Y peaks at a rate of 55 nuclei/sec for each population. Purities of individual X and Y populations averaged 95% as determined by reanalysis of the sorted populations. Successful sorting of Chinchilla X and Y chromosome-bearing spermatozoa into separate populations may aid in the identification of a biochemical marker that could be used to discriminate between the two sperm populations and lead to a practical procedure for sexing spermatozoa.     

On-Chip Cryopreservation: A Novel Method for Ultra-Rapid Cryoprotectant-Free Cryopreservation of Small Amounts of Human Spermatozoa

Cryopreservation of human spermatozoa free from cryoprotectant can avoid toxicity caused by highly concentrated cryoprotectant and a series of specific carriers have been previously explored, except for PDMS chip. Our study is aimed at exploring a novel device for ultra-rapid cryopreservation of small numbers of spermatozoa without cryoprotectant based on polydimethylsiloxane (PDMS) chips. Spermatozoa from 25 healthy men were involved in this study, comparing on-chip cryopreservation with different micro-channel height (group A: 10 µm height, group B: 50 µm height, group C: 100 µm height) and conventional freezing (group D) in liquid nitrogen for 72 h. The viability, motility, DNA integrity by comet assay and acrosome integrity by fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining of frozen-thawed spermatozoa of each group were compared. The motility and viability of post-thawed spermatozoa was significantly decreased than that of pre-freezing spermatozoa. There was no difference of viability and motility of frozen-thawed spermatozoa between group A and D, while viability and motility of group B and C decreased compared to group A. Comet assay showed that no matter for group A or D, there was no difference of CR, TL, TD and OTM between pre-frozen and post-thawed spermatozoa. There was no difference of CR, TL, TD and OTM of post-thawed spermatozoa between group A and group D neither, while spermatozoa DNA damage was more serious in group B and group C with increasing height of micro-channel compared with group A. The proportion of intact acrosome of post-thawed spermatozoa in group A was the highest when compared with group B and group C, though similar to that of group D. In conclusion, PDMS chip with 10 µm height micro-channel is ideal for ultra-rapid cryopreservation of small quantity of spermatozoa without cryoprotectant.     

Buffalo and cattle hybrid embryo development is decreased by caffeine treatment during in vitro fertilization

Water buffalo are renowned for difficulties in the implementation of assisted reproductive technologies, with both males and females being problematic. In this study, we used cattle oocytes to assess the effect of treatments with heparin and caffeine on buffalo spermatozoa and subsequent fertilization and embryo development in vitro. There was no significant difference between buffalo and bovine spermatozoa in the events associated with fertilization. Fertilization of cattle oocytes with buffalo spermatozoa resulted in 7.8% of oocytes developing into hybrid embryos. A difference in the developmental capability of hybrid embryos compared with the cattle control was observed. This has not been previously reported. The subsequent transfer of a limited number of hybrid embryos did not produce a viable pregnancy. However, control treatments in this experiment also failed to achieve pregnancy, so objective data is not available to provide conclusions about the developmental competence of the buffalo and cattle hybrid embryos. Optimal spermatozoa capacitation treatments achieved 61% fertilization and 21% zygote cleavage into two cell embryos. There was no significant difference in fertilization or development due to heparin or spermatozoa concentrations. However, treatment of buffalo and cattle spermatozoa with caffeine significantly decreased embryo cleavage but also tended to decrease embryo development to the blastocyst stage. These studies suggest that problems with reproduction in buffalo may reside with biological mechanisms associated with the oocyte that are often complicated by poor male reproductive performance. Selection for bull fertility would prevent some of these complications.     

Motility and penetration competence of frozen-thawed miniature pig spermatozoa are substantially altered by exposure to seminal plasma before freezing

The objective was to determine if exposure of spermatozoa to seminal plasma before freezing decreases its freezability, assessed by percentage motile cells (using computer-assisted semen analysis) and in vitro penetration ability (using in vitro fertilization and chlortetracycline fluorescence assessment). Ejaculated spermatozoa from miniature pigs were washed by centrifugation within 20 min after collection, then incubated in seminal plasma or modified Hulsenberg VIII diluents (mHM). When the spermatozoa were cryopreserved, spermatozoa incubated in seminal plasma before freezing had significantly lower post-thaw motility than spermatozoa incubated in mHM. The incubation of spermatozoa in seminal plasma also significantly prevented frozen-thawed spermatozoa from penetrating the oocytes. The second experiment, using unfrozen spermatozoa, was to determine if the incubation of spermatozoa with seminal plasma reduced penetration ability before freezing, resulting in a significantly lower penetration rate after freezing (compared with spermatozoa incubated without seminal plasma). The penetration competence of unfrozen spermatozoa was significantly decreased by incubation in seminal plasma, but no difference in motility was observed between spermatozoa exposed to seminal plasma versus mHM. We concluded that ejaculated seminal plasma contained some factor(s) that modified the sperm before freezing and reduced the freezability and post-thaw penetration competence of spermatozoa.     

Phosphatidylserine Membrane Translocation in Human Spermatozoa: Topography in Membrane Domains and Relation to Cell Vitality

The complex structure of the human spermatozoa membrane comprises five topographic domains. Transmembrane asymmetry of the distribution of phospholipids including phosphatidylserine (PS) is considered a marker of cell activity. The objective of the study was to determine which cytomembrane domains of human spermatozoa are involved in PS membrane translocation and to identify the possible relationship of PS translocation with spermatozoa morphology and vitality. In normozoospermic semen of 35 donors, annexin-V labeling with fluorescein determined PS translocation. Propidium iodide staining distinguished between vital and dead spermatozoa. Three types of PS membrane translocation have been distinguished: (1) in the midpiece, (2) in the acrosomal part and (3) simultaneously in the midpiece and acrosomal part. In morphologically normal vital spermatozoa, PS translocation occurred in the midpiece but never in the equatorial region. In dead spermatozoa, simultaneous PS translocation in the midpiece and acrosomal part was most often observed. The difference between proportions of, respectively, vital and dead spermatozoa presenting PS translocation located in different domains was significant (P < 0.0001). In vital cells, there was no difference in PS translocation prevalence between morphologically normal and abnormal spermatozoa (P > 0.05). The strict relation of PS translocation to specific membrane domains indicates functional specificity. It seems doubtful to include this phenomenon in physiological mechanisms of elimination of abnormal spermatozoa.     

Preliminary results of hemizona assay (HZA) as a fertility test for canine spermatozoa

The Hemizona assay (HZA) is considered to be an effective test for predicting the fertilizing potential of spermatozoa. It is a functional test that distinguishes the zona-binding capacity of spermatozoa from fertile and infertile males. The objective of this study was to validate the HZA for canine spermatozoa, as a test for diagnosing canine male fertility status. Various parameters that affect binding capacity were examined: the presence of an adequate number of capacitated and motile spermatozoa for an HZA, the influence of fertility status, sperm-binding variability within fertile dogs over 60 d, variability in sperm-binding capacity of different oocytes, the lower limit number of spermatozoa binding to a zona from the fertile control, and evaluation of HZI to determine the fertilizing capacity of spermatozoa. Hemizonae were obtained from frozen oocytes of spayed bitches. The oocytes were manually cut into nearly equal halves. Spermatozoa were capacitated by swim-up and 1 h incubation at 37 degrees C in modified Ham's F10 medium. Spermatozoa and hemizonae were co-incubated in 100-microL drops at 37 degrees C for 1 h. Spermatozoa from 7 fertile and 3 infertile dogs were used for this study. The optimal sperm concentration for hemizona insemination was 1 x 10(6)/mL capacitated and motile spermatozoa. A significant difference (P < 0.001) was found the number of tightly zonabound spermatozoa between fertile and infertile dogs. Although there was a small difference in zona binding capacity between ejaculates of the same fertile dog (44 +/- 18.24), the main cause for the difference mentioned above was that of poor zona pellucida-binding capacity of spermatozoa from infertile dogs. We found a maximum of 14.28% bad oocytes when we compared sperm samples from 3 fertile and 3 infertile dogs in 56 HZA replicates. To avoid the effect of bad zona on sperm binding we calculated 37 (95% CI) bound spermatozoa from infertile dogs in 56 replicates. Thus, an HZA experiment in which a control dog had < 37 zona bound spermatozoa was repeated. Based on a minimum of 37 bound spermatozoa for fertile males (controls), a differential zona-binding capacity and hemizona index (HZI) between fertile and infertile dogs and between 2 fertile dogs was determined. The binding differential between fertile and infertile dogs was 64.92 +/- 24.29, while between 2 fertile dogs it was 22.38 +/- 10.02 (P < 0.001). According to the HZI values, a value equal to or less than 41.11 indicated an infertile test dog, while an HZI value equal to or greater than 57.95 indicated a fertile test dog. Any value between these two could indicate either fertility or infertility. The evaluation of fertilizing potential of spermatozoa can be improved using the HZA protocols described above.     

RELATION BETWEEN THE ACROSOME REACTION AND FERTILIZATION IN THE SEA URCHIN. I. FERTILIZATION IN Ca-FREE SEA WATER WITH EGG-WATER-TREATED SPERMATOZOA. *

When spermatozoa are treated with egg-water and undergo the acrosome reaction, their fertilizing capacity is lost within 5 min. However, if insemination is carried out within 4 min after the egg-water treatment, there is no difference in fertilizing capacity between spermatozoa treated with egg-water and non-treated ones. With such spermatozoa, eggs can be fertilized even in the virtual absence of calcium, whereas with spermatozoa treated with Ca-free egg-water, no fertilization occurs under the same conditions. It is postulated that in normal fertilization the acrosome reaction has occurred before the attachment of the gametes. The failure of fertilization with normal spermatozoa in Ca-free sea water may be due to the failure of occurrence of the acrosome reaction.     

哺乳动物精子性别特异膜蛋白的研究进展

在精子形成过程中,存在性染色体特异基因的表达,这是X、Y精子膜蛋白差异形成的基础。尽管精子形成过程中形成的细胞间桥可能使精子细胞间共享基因表达产物,但雄性传递偏移现象和性别偏移现象的发生又证明两类精子间存在非共享蛋白,同时H-Y抗原表位的成功鉴定、精子分离实验结果以及性别特异蛋白的检测结果都肯定了精子膜蛋白差异的存在,只是这种膜蛋白的差异很小。蛋白分离技术的进步及技术间的优化集成,为精子间细微差异膜蛋白的分离提供了可能。     

Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa

Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-). Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa.     

Lipid changes during epididymal maturation in ram spermatozoa collected at different times of the year

The phospholipid and phospholipid fatty acid content of ram spermatozoa decreased during maturation in the epididymis but interpretation of the results was complicated by a possible seasonal factor. Loss of individual fatty acids was selective, resulting in an increase in unsaturation during maturation. Testicular spermatozoa and fluid collected directly into chloroform-methanol contained about 7 times more neutral lipid fatty acid than testicular spermatozoa collected for 6--18 h, separated from the rete testis fluid and then extracted; the difference was not due to lipid in the rete testis fluid. Thin-layer chromatography indicated that cholesterol esters and triglycerides were the neutral lipids which were not lost during collection. Epididymal spermatozoa contained only slightly less neutral lipid fatty acid than continuously collected testicular spermatozoa.     

In vitro fertility and motility characteristics of frozen-thawed boar epididymal spermatozoa separated by Percoll

Frozen-thawed epididymal spermatozoa from four boars were separated through a Percoll gradient, and motility characteristics and in vitro fertility were assessed. Percoll-separated spermatozoa had a significantly higher percentage of motile and progressively motile spermatozoa than those that were not separated (P < 0.0001). However, there were no clear differences in other motility parameters between Percoll-separated and un-separated spermatozoa. Furthermore, sperm agglutination was decreased by Percoll separation (P < 0.05). The effects of Percoll separation on in vitro fertility of spermatozoa differed among boars. In addition, there were large differences in fertility between sperm samples in vitro. Sperm samples, which indicate highly motile and progressively motile, did not always show high in vitro fertility. Furthermore, there was no distinct pattern between fertility in vitro and motility parameters. There was no difference in fertility in vitro between Percoll-separated and un-separated spermatozoa from two of the four boars. However, in vitro fertility of Percoll-separated spermatozoa was higher than that of un-separated spermatozoa from the other two boars.     

Variability in the size of the nucleus in spermatozoa from Houbara bustards, Chlamydotis undulata undulata

Semen collected from 3-year-old male Houbara bustards contained large proportions (6-40%) of spermatozoa with large nuclei. In these spermatozoa, the length of the nucleus was up to twice the mean length of the nucleus in normal spermatozoa. The lengths of the acrosome, midpiece and flagella were all normally distributed, but the length of the nucleus formed a bimodal distribution. The proportion of spermatozoa with large nuclei varied among males, but not among different semen samples collected from the same male throughout the breeding season. The proportion of motile spermatozoa with large nuclei was half that of normal spermatozoa, but their velocity was significantly greater. After insemination into females, spermatozoa with large nuclei were observed in the outer perivitelline layer of eggs laid, indicating that they were stored and transported within the oviduct and reached the egg at about the time of fertilization. Furthermore, there was no difference in the ability to produce viable progeny in females that were mated with males producing greater proportions of spermatozoa with large nuclei compared with those producing 'normal' spermatozoa. Thus, the abnormal spermatozoa did not appear to impede fertility. There were no signs of triploidy in the males that produced spermatozoa with large nuclei, or in their progeny, as demonstrated by the size of erythrocytes. Therefore, it appears that the spermatozoa with large nuclei were the result of aberrant spermatogenesis.     

Cryopreservation of epididymal dog sperm.

A method of cryopreservation was developed for sperm salvaged from the cauda epididymis and vas deferens of domestic dog testes. Four modifications of the glycerol concentration of a buffer used for cryopreservation of dog ejaculates and two freezing rates were assessed for their effect upon post-thaw spermatozoal motility and morphology. There was no statistical difference between the four glycerol concentrations or the two freezing rates and the buffer containing 6% glycerol and the freezing rate provided by 0.5 ml straws was chosen for further study. This method resulted in a significant reduction in the percentage of live spermatozoa detected with Hoechst staining and a reduction in the percentage of capacitated spermatozoa after freeze-thawing. However, there was no difference in the ability of frozen-thawed spermatozoa to penetrate homologous oocytes.This study demonstrates that cryopreservation of epididymal canine sperm can be performed using methods similar to those established for ejaculates of the same species, and that despite some damage, spermatozoa retain their functional ability.     

Plasma membrane and acrosomal integrity in bovine spermatozoa with the knobbed acrosome defect

Previous studies have shown that bovine spermatozoa with the knobbed acrosome defect have a reduced ability to bind to and penetrate the bovine zona pellucida. Cryopreserved spermatozoa from a normal control bull (N) and two bulls with the knobbed acrosome defect (K1 and K2) were subjected to a hypoosmotic swelling test (HOST) to evaluate the functional integrity of the plasma membrane. A capacitation assay and a calcium ionophore challenge test was used to determine the ability of spermatozoa to undergo capacitation and acrosome reaction (AR), respectively. The mean percentage of spermatozoa responding to the HOST was significantly higher for Bull N (68.8 +/- 2.4) than for Bulls K1 (36.1 +/- 4.6) and K2 (40.2 +/- 4.7). The mean percentage of capacitated spermatozoa (54.0 +/- 1.8) was significantly higher for the treatment group (incubation in capacitating medium) for Bull N than that of the negative control group (29.5 +/- 1.8). However, there was no difference between the treatment and the negative control groups of the bulls with the knobbed spermatozoa (36.5 +/- 1.4 and 27.1 +/- 3.0 for Bull K1 and 47.5 +/- 3.8 and 35.2 +/- 6.6 for Bull K2, respectively). Although the mean percentage of acrosome-reacted spermatozoa (60.7 +/- 1.3) was higher for the treatment group (receiving calcium ionophore) for Bull N than that of the negative control (29.5 +/- 1.3), there was no difference between the treatment and the negative control groups for the bulls with the knobbed spermatozoa (47.8 +/- 3.3 and 49.3 +/- 5.0 for Bull K1 and 58.8 +/- 10 and 59.5 +/- 9.7 for Bull K2, respectively). A positive correlation existed between the proportion of spermatozoa that did not respond to the HOST and that undergoing a spontaneous AR. Results suggest that spermatozoa with the knobbed acrosome defect have impaired plasma membrane function which predisposes them to premature capacitation and spontaneous AR on incubation after thawing.     

Selecting a mammalian species for the separation of X- and Y-chromosome-bearing spermatozoa

All (524) male karyotypes in An Atlas of Mammalian Chromosomes (Hsu & Benirschke, 1967-1977) were visually estimated for the chromatin difference between the X- and Y-chromosome-bearing spermatozoa. After more exact measurement of axial chromatid length for 100 karotypes, 24 species were found in which the difference between X and Y chromosomes was greater than 6.2%. It is suggested that such species would be the best for attempts to separate the X- and Y-bearing spermatozoa.     

The effect of oviductal fluid on protein tyrosine phosphorylation in cryopreserved boar spermatozoa differs with the freezing method

Sperm capacitation takes place in the oviduct and protein tyrosine phosphorylation of sperm proteins is a crucial step in capacitation and acquisition of fertilizing potential. Cryopreserved spermatozoa show altered expression of protein tyrosine phosphorylation in the oviduct. The present study compared two freezing methods (conventional-conventional freezing (CF) and simplified-simplified freezing (SF) methods) for their effect on the ability of boar spermatozoa to undergo protein tyrosine phosphorylation in response to oviductal fluid (ODF). Cryopreserved boar-spermatozoa were incubated with pre- and post-ovulatory ODF for 6 h at 38 °C under 5% CO₂. Aliquots of sperm samples were taken at hourly intervals and analyzed for kinematics and protein tyrosine phosphorylation. Global protein tyrosine phosphorylation in spermatozoa was measured using flow cytometry and different patterns of phosphorylation were assessed using confocal microscopy. Immediately after thawing, no significant difference was observed in post-thaw sperm motility, velocity and global tyrosine phosphorylation between the two methods of freezing although the freezing method significantly (P < 0.05) influenced the effect of oviductal fluid on these parameters during incubation. While spermatozoa frozen by the CF method showed a significantly higher (P < 0.001) proportion of phosphorylation in response to preovulatory ODF during incubation, spermatozoa frozen by the SF method did not elicit such significant response as there was no significant difference in the proportion of tyrosine phosphorylated spermatozoa between treatments at any given time during incubation. If the CF method was used, the proportion of spermatozoa displaying either tail or full sperm phosphorylation increased in response to both preovulatory (EODF) and postovulatory oviductal fluid. However, if the SF method was used, a significant increase in these patterns was noticed only in the EODF treated group. The present study demonstrates that preovulatory isthmic ODF induce tyrosine phosphorylation in a higher proportion of boar spermatozoa compared to the post-ovulatory fluid and that the method of freezing significantly influences the response of post-thaw spermatozoa to porcine ODF.     

Separation of canine epididymal spermatozoa by Percoll gradient centrifugation

The objective was to characterize the separation of canine epididymal spermatozoa on a Percoll gradient. Epididymal spermatozoa were overlaid on a 45 and 90% discontinuous Percoll gradient and centrifuged at 700 x g for 20 min. The Percoll column was separated into six fractions (top to bottom, A-F) after centrifugation. Fractions A-C contained few spermatozoa. Spermatozoa with bent or folded tails and a large amount of granular debris were observed in Fraction B. Fraction D contained many nonmotile spermatozoa, erythrocytes and round epithelial cells. Spermatozoa in Fraction E had significantly lower motility than those in the initial layer. Spermatozoa in Fraction F had motility similar to those before separation. Fraction F contained 40.6% of the motile spermatozoa layered and 67.5% of all motile spermatozoa recovered. There was no significant difference between Fraction F and the initial layer in sperm membrane integrity. In the sperm-oocyte penetration assay, spermatozoa from Fraction F had a significantly higher penetration rate into the immature homologous oocytes than those from Fraction E. Although the recovery rate of the motile spermatozoa was low, the canine epididymal spermatozoa with motility, membrane integrity and penetrating capability could be separated by two-layer discontinuous Percoll gradient centrifugation.     

Fatty acid composition of human spermatozoa and seminal plasma levels of oxidative stress biomarkers in subfertile males

INTRODUCTION: The lipid composition of the sperm membrane has a significant effect on the functional characteristics of spermatozoa. PATIENTS AND METHODS: In the present study, fatty acid composition of spermatozoa and seminal plasma levels of free 15-F(2t)-Isoprostane and catalase were assayed in men with normozoospermia, asthenozoospermia, asthenoteratozoospermia, and oligoasthenoteratozoospermia. RESULTS: In spermatozoa from asthenozoospermic men only oleic acid levels showed a significant difference from normozoospermic men. In spermatozoa from asthenoteratozoospermic and oligoasthenoteratozoospermic samples all of the tested fatty acids were significantly higher than those from normozoospermic samples. Seminal plasma levels of catalase were significantly lower in all patients while levels of free 15-F(2t)-Isoprostane were significantly higher in all patients compared with normozoospermic men. DISCUSSION: Spermatozoa from pathological samples may have higher levels of polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), than spermatozoa from normozoospermic men. Therefore, damage induced by lipid peroxidation would be higher in spermatozoa from pathological samples than those from normozoospermic men.     

Comparative metabolism of spermatozoa from subfertile Delaware and Wyandotte roosters

Sperm metabolism was determined via reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to formazan. When the reaction mixture contained cyanide, which blocks cytochrome oxidase and thus maximizes intermediate electron transfer to INT, and calcium which stimulates fowl sperm motility, the metabolic capacity of spermatozoa from subfertile Delaware and Wyandotte roosters was 90 and 63% of that of spermatozoa from fertile Leghorn roosters. When the assay was performed at 40 degrees C without calcium or cyanide, no difference in metabolism was observed between Delaware and Leghorn spermatozoa (P greater than 0.05). However, the metabolism of Wyandotte spermatozoa was 66% of that observed with Delaware or Leghorn spermatozoa. These results provide further evidence that heritable subfertility in Delaware and Wyandotte roosters is attributable to distinct sperm defects.