Sperm Head Shape Research Articles

Quantitative changes in sperm head morphology during passage through the male excurrent duct system of the rabbit

A fine adjustment of sperm head size and shape occurs during maturation and storage within the male excurrent duct of the rabbit. This remodelling, as judged by morphometric values of area, perimeter, length, width, and shape factors, takes place mostly in passage from the seminiferous tubules of the testis to the distal caput of the epididymis. The dimensions of sperm heads from the distal corpus of the epididymis break the general tendency toward a reduction in size and more elliptical shapes. A period of transport and storage within the epididymal cauda and vas deferens follows in which there are no further changes in sperm head morphometry. It can be concluded that the period immediately following sperm release from the testis is crucial to the final morphological maturation of spermatozoa. Moreover, the fact that changes are detected in the appearance of sperm heads at successive stages of sperm maturation suggests that the dimensions of a particular epididymal spermatozoon may be taken as an approximate indication of its relative maturity. Mol. Reprod. Dev. 51:203–209, 1998. © 1998 Wiley-Liss, Inc.

Quantitative changes in sperm head morphology during passage through the male excurrent duct system of the rabbit.

A fine adjustment of sperm head size and shape occurs during maturation and storage within the male excurrent duct of the rabbit. This remodelling, as judged by morphometric values of area, perimeter, length, width, and shape factors, takes place mostly in passage from the seminiferous tubules of the testis to the distal caput of the epididymis. The dimensions of sperm heads from the distal corpus of the epididymis break the general tendency toward a reduction in size and more elliptical shapes. A period of transport and storage within the epididymal cauda and vas deferens follows in which there are no further changes in sperm head morphometry. It can be concluded that the period immediately following sperm release from the testis is crucial to the final morphological maturation of spermatozoa. Moreover, the fact that changes are detected in the appearance of sperm heads at successive stages of sperm maturation suggests that the dimensions of a particular epididymal spermatozoon may be taken as an approximate indication of its relative maturity.

Interspecific Variation in Structural Organisation of the Spermatozoon in the Asian Bandicoot Rats, Bandicota Species (family Muridae)

AbstractThe structural organisation of the spermatozoon from two species of bandicoot rats Bandicota bengalensis and Bandicota indica was investigated by light and electron microscopy together with the effect of incubation in Triton‐X 100 and sodium dodecyl sulphate. The sperm head of B. bengalensis is invariably falciform, has a uniform electron‐dense nucleus capped by an acrosome with a posteriolateral equatorial segment, a subacrosomal cytoskeleton with a large rostral perforatorium, and a sperm tail, attached to the lower concave surface of the sperm head, with typical coarse fibres and fibrous sheath. By contrast, the sperm head shapes of B. indica are generally conical or bulbous, the nucleus contains a few large vacuoles, the acrosome lacks an equatorial segment, no recognisable perforatorium occurs, and the sperm tail, which is attached basally, is very short with only modest development of coarse fibres and fibrous sheath. These results indicate that, within the genus Bandicota, huge interspecific differences in morphology of the spermatozoon have evolved. The spermatozoa of B. bengalensis are similar to those of Rattus and many other murids and thus presumably represent the ancestral condition, whereas those of B. indica (and B. savilei) are unlike spermatozoa from any other eutherian mammal so far described. © 1998 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd. All rights reserved

Evolution of the Spermatozoon in Australasian Rodents

The head of the spermatozoon in eutherian mammals contains a nucleus, acrosomal cap and cytoskeleton. It is generally spatulate, paddle-shaped or pear-shaped, but in most murid rodents it is hook-shaped with the anterior region of the nucleus surrounded by an elaborate acrosome and an extension of the subacrosomal cytoskeleton as a perforatorium. This type of spermatozoon is present in Australasian Rattus, together with several other New Guinean genera. However, in most Australasian hydromyine rodents a far greater complexity of structural organisation of the sperm head has evolved in which two further elaborate processes extend from its upper concave surface. These processes contain a huge extension of the cytoskeleton within which filamentous actin is present. By contrast, the form of the sperm head in a few species of Pseudomys, Notomys and Solomys is highly divergent and is either truncated, spatulate or pear- shaped. The evolutionary trends of change in sperm head shape are discussed and it is suggested that the falciform sperm head with the two extra processes in most of the hydromyine rodents is one of the most morphologically complex sperm head types to have evolved in eutherian mammals; it contains a far more extensive development of the cytoskeleton than that of any other mammalian spermatozoon.

Effects of a 1.5-Tesla static magnetic field on spermatogenesis and embryogenesis in mice.

There is a trend toward the use of higher magnetic field strengths in magnetic resonance imaging procedures. Considering this trend and the lack of consensus on the biologic effects of static magnetic fields, it is of considerable interest to examine the biologic effects of a 1.5-tesla (T) static magnetic field on spermatogenesis and embryogenesis in mice. Male and pregnant female Swiss Webster mice were exposed to a 1.5-T static magnetic field for 30 minutes. Effects on spermatogenesis in male mice were investigated by counting testicular spermheads and epididymal spermhead shape-abnormalities as a function of time after exposure. Pregnant female mice were exposed to the field at the two-cell embryo stage, sacrificed immediately, and the ability of these preimplantation embryos to mature into blastocysts was examined in vitro. Exposure to the static 1.5-T magnetic field caused a statistically significant reduction (15%) in testicular sperm on the 16th and 29th days after exposure. However, the increase in spermhead shape abnormalities above normal control values was minimal. A substantial effect was noted on the development of preimplantation embryos with a survival fraction of 0.56 compared with controls. A 30-minute exposure to a 1.5-T static magnetic field appears to cause some deleterious effects on spermatogenesis and embryogenesis in mice.

1501 Acidic fibroblast growth factor and basal forebrain cholinergic neurons in senescence accelerated mice; molecular and immunocytochemical analysis

To test for an association between DNA fragmentation and head shape at high magnification in fresh motile spermatozoa.Observational study.Academic tertiary care center.A total of 60 men in our assisted reproductive program.Quantifying sperm head shape using elliptic Fourier analysis, and detecting DNA fragmentation by use of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.Correlation between percentage of spermatozoa with abnormal head shape and percentage of DNA fragmentation.Elliptic Fourier analysis decomposed sperm head shapes into four quantitative parameters: ellipticity, anteroposterior (AP) symmetry, lateral symmetry, and angularity. The DNA fragmentation was statistically significantly correlated with abnormal angularity, and moderately with abnormal ellipticity but not with abnormal AP symmetry or lateral symmetry. Forward stepwise multiple logistic regression analysis revealed a statistically significantly higher percentage of DNA fragmentation in spermatozoa with abnormal ellipticity and abnormal angularity than in spermatozoa with normal-shaped head (6.1% and 5.4% vs. 2.8%). Spermatozoa with large nuclear vacuoles also correlated with sperm DNA fragmentation, and had a statistically significantly higher percentage of DNA fragmentation (4.7%).Among the morphologic features of the sperm head, abnormal ellipticity, angularity, and large nuclear vacuoles are associated with DNA fragmentation.

1502 Differential vulnerability to aging in the rat serotonergic system

To test for an association between DNA fragmentation and head shape at high magnification in fresh motile spermatozoa.Observational study.Academic tertiary care center.A total of 60 men in our assisted reproductive program.Quantifying sperm head shape using elliptic Fourier analysis, and detecting DNA fragmentation by use of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.Correlation between percentage of spermatozoa with abnormal head shape and percentage of DNA fragmentation.Elliptic Fourier analysis decomposed sperm head shapes into four quantitative parameters: ellipticity, anteroposterior (AP) symmetry, lateral symmetry, and angularity. The DNA fragmentation was statistically significantly correlated with abnormal angularity, and moderately with abnormal ellipticity but not with abnormal AP symmetry or lateral symmetry. Forward stepwise multiple logistic regression analysis revealed a statistically significantly higher percentage of DNA fragmentation in spermatozoa with abnormal ellipticity and abnormal angularity than in spermatozoa with normal-shaped head (6.1% and 5.4% vs. 2.8%). Spermatozoa with large nuclear vacuoles also correlated with sperm DNA fragmentation, and had a statistically significantly higher percentage of DNA fragmentation (4.7%).Among the morphologic features of the sperm head, abnormal ellipticity, angularity, and large nuclear vacuoles are associated with DNA fragmentation.

Spermatozoa of murid rodents from Africa: morphological diversity and evolutionary trends

The diversity of the structural organization of the spermatozoa of African murid rodents is described at the light and transmission electron microscopical level of resolution. In most species the sperm head is falciform in shape but it varies somewhat in overall breadth, width, and length. A typical perforatorium is present and the acrosome splits into a large head cap over the convex surface and a smaller ventral segment similar to the sperm head of most Asian and Australasian murids. In a few species, however, the morphology is very different. In Acomys and Uranomys spermatozoa, the apical hook is more bilaterally flattened, has a large apical acrosomal region, and no separate ventral segment. Two species of Aethomys have, in addition to an apical hook, a 4μ long extension of the cytoskeletal material that projects from the concave surface of the sperm head, whereas in Dasymys two large ventral processes extend from the upper concave region which contain nuclear material basally and a huge extension of cytoskeleton apically. In Aethomys chrysophilus type B, the sperm nucleus is unique in form and often has a central region in which threads of chromatin can be seen; it is capped by a massive acrosome whose apical segment is complex and convoluted in structure. Stochomys longicaudatus appears to have a conical sperm head, and in all three Lophuromys species the sperm head is spatulate in shape with the flat, plate‐like nucleus capped by a thin acrosome. The evolutionary trends in changes of sperm head shape and design of these rodents are discussed. It is suggested that some of the differences in morphology may relate to the variation in structural organization of the coats around the egg through which the spermatozoon has to pass in order for fertilization to occur.

Morphometry of stallion spermatozoa by computer-assisted image analysis

Metric measurements of stallion spermatozoal heads were determined for live, unfixed spermatozoa and for Feulgen-stained spermatozoa by videomicroscopy and computerized image analysis. Two ejaculates were collected from each of five stallions of normal fertility. Air-dried semen smears were Feulgen-stained, and live, unfixed spermatozoa were examined as wet-mount preparations. For Feulgen-stained spermatozoa, videoimages (x3850) were captured, and sperm heads were detected via image segmentation and particle analysis. For live, unfixed spermatozoa, phase contrast videoimages (x3850) were measured to determine width and length of the sperm head. For Feulgen-stained spermatozoa, there were significant effects (P < 0.001) of stallion and ejaculate on measured parameters of area, circumference, and the length and width of the sperm head. For live, unfixed spermatozoa, there were significant effects of stallion on length and width and of ejaculate on length of the sperm heads. There was a very poor correlation between length and width of sperm heads between Feulgen-stained and live, unfixed spermatozoa. Two indices of sperm shape (oval factor and aspect ratio) were also determined. Both aspect ratio and oval factor were significantly affected by stallion (P < 0.001); however, oval factor was not affected by ejaculate and therefore may represent a less variable determination of sperm head shape across stallions. Overall, length and width of stallion sperm heads were larger (P < 0.01) for live, unfixed spermatozoa than for Feulgen-stained spermatozoa (length: 6.3 ± 0.4 vs 5.08 ± 0.44; width: 3.08 ± 0.34 vs 2.71 ± 0.28 μm, respectively). Computerized image analysis may be useful as a means to objectively measure sperm head dimensions in the stallion and could be useful in future studies to determine associations with stallion fertility.

Analysis of sperm quality in recombinant inbred mouse strains: correlation of sperm head shape with sperm abnormalities and with the incidence of supplementary spermatozoa in the perivitelline space.

Recombinant inbred strains were developed from reciprocal crosses between two inbred strains of mice (CBA and KE) differing in sperm head shape, proportion of normal sperm heads (CBA, 95%; KE, 78%) and fertilization efficiency (CBA, 100% of fertilized ova; KE, 72%), to determine whether the indices of sperm morphology and function were correlated. The following parameters were analysed in recombinant inbred and progenitor strains: index of sperm head shape (head width in the middle of its length/head length), percentage of abnormal sperm heads, percentage of spermatozoa with progressive movements, efficiency of penetration of hyaluronic acid polymer (Sperm Select) and percentage of fertilized ova after mating males from the tested strains with females from an outbred stock. For each investigated character, recombinant inbred strains, recombinant inbred EXCB and CBXE, could be divided into at least three categories: KE-like, CBA-like and intermediate, suggesting that in each case a minimum of two genes was involved. Recombinant strains derived from the reciprocal crosses of progenitor strains differed only with respect to the proportion of abnormal sperm heads, showing the involvement of the Y chromosome in determining this character. Penetration into Sperm Select was significantly correlated both with fertilization efficiency and sperm motility, while correlation with the proportion of normal spermatozoa did not reach the level of significance. However, there was a significant negative correlation of both sperm abnormalities and the incidence of supplementary spermatozoa in the perivitelline space with the index of sperm head shape.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Spermatogenic Effects of Bromoacetic Acids

Acute Spermatogenic Effects of Bromoacetic Acids. Linder, R. E., Klinefelter, G. R., Strader, L. F., Suarez, J. D., and Dyer, C. J. (1994). Fundam. Appl. Toxicol. 22, 422-430.Chlorine and bromine can react with natural organic substances in source waters to form haloacetic acids, major disinfection by-products of water chlorination. Several toxic effects including testicular damage have been attributed to the chloroacetic acids but little information is available on the bromine analogues. In this report we present the results of acute toxicity and acute spermatotoxicity studies of monobromoacetic acid (MBAA) and dibromoacetic acid (DBAA). In adult male rats the acute oral toxicity of MBAA was 10-fold that of DBAA (LD50 177 vs 1737 mg/kg). No reproductive-related endpoints were affected in rats given a single dose of 100 mg MBAA/kg or 14 daily doses of 25 mg MBAA/kg/day. In rats dosed with DBAA, serum testosterone fell to 17% of control 2 days after a single dose of 1250 mg/kg but returned to control levels by Day 14. Marked effects on sperm motion were seen on post-treatment Days 14 and 28. Degenerative flagellar changes in cauda sperm were present on Day 14 while abnormal sperm head shapes and flagellar degeneration were observed in both caput and cauda sperm on Day 28. Histopathology indicated altered spermiation at all timepoints as evidenced by retention of Step 19 spermatids beyond Stage VIII of the cycle of the seminiferous epithelium. Disorganization, distortion, and degeneration of late spermatids were also observed. On Day 14 structures resembling residual bodies were rarely seen in the testis but were numerous in the epididymis. Caput sperm counts were decreased on Day 2 and cauda sperm counts were decreased on Days 14 and 28. The data indicate that DBAA is a testicular toxicant in the rat with late and elongating spermatids being particularly susceptible germinal cells.

Increases in chromosome aberrations and in abnormal sperm morphology in rubber factory workers

Subjects working at a rubber plant in a chemicals warehouse or in calandering and bambury units were analyzed for both sperm parameters and structural chromosome aberrations in peripheral blood lymphocytes. Sperm analysis war performed in a group of 24 workers for comparison with fertile (n = 24) and infertile (n = 24) control groups. The statistical analyses of semen volume, vitality and sperm count did not show significant differences between exposed and fertile groups but significant differences were found from the infertile group. A significantly lower proportion of normal sperm head shapes was found in exposed subjects when compared to the fertile group (40.1 vs. 57.8). Seven exposed workers were re-analyzed 1 year and their sperm parameters did not change. The cytogenetic analysis showed a significant increase (3.90%) in the percentage of cells with aberrations in bambury workers (n = 11). However, no differences were found between calandering workers (n = 8) and control subjects (n = 10). Workplace air samples taken on the day of tissue sampling did not show any increase above the Cuban maximal allowed concentration for benzo[ a]pyrene or toluene.

Genotoxic effect of smoke-dried meat extract in Swiss albino mice using sperm head shape abnormality test

The genotoxic effect of an extract of smokedried meat was assayed by employing in vivo sperm head shape abnormality. A significant dose responsive mutagenic effect was observed using the sperm head shape abnormality test. The result indicates that higher doses i.e., 100 and 200 mg/kg body wt. of smoke meat extract, significantly induced sperm head shape abnormality as compared to lower doses i.e., 20 mg/kg body wt. and control.

Phenotype and fertilizing capacity of spermatozoa of chimaeric mice produced from two strains that differ in sperm quality

Spermatozoa of males from the inbred mouse strains, KE (albino) and CBA (agouti), are distinguishable by head shape and differ in quality: CBA spermatozoa show a lower percentage of abnormal heads and higher efficiency of fertilization. Aggregation chimaeras were produced to investigate whether these differences are intrinsic or extrinsic to spermatogenic cells. Among 24 overt chimaeras, 14 were males: 1 was sterile, 8 produced either KE or CBA spermatozoa, as recognized by shape and by progeny testing, and 5 (21%) were germ-line chimaeras. Both the level of abnormal sperm heads and the efficiency of fertilization of CBA and KE spermatozoa produced by chimaeric males (except two subfertile ones) were within the range characteristic for the respective strain. All 5 germ-line chimaeras, irrespective of their coat colour composition, produced about 98% KE and only 2% CBA spermatozoa, which indicated strong selection against CBA germ cells. However, mature CBA spermatozoa showed high competitive ability, because the proportion of agouti progeny (from the CBA component) sired by those males was significantly higher than the proportion of CBA spermatozoa, estimated from vaginal plug preparations after every mating. The fact that this difference was particularly striking for one chimaera with a preponderance of CBA somatic component may suggest some influences extrinsic to spermatogenic cells. We conclude from this study that sperm head shape, the level of sperm abnormalities and fertilizing capacity are determined largely autonomously by genes acting in the germ cells. The internal environment created by foreign somatic cells exerts only minor modifications, unless there has been deterioration beyond the range of 'normality'.

Quantitative evaluation of radiation‐induced changes in sperm morphology and chromatin distribution

Sperm head cytometry provides a useful assay for the detection of radiation-induced damage in mouse germ cells. Exposure of the gonads to radiation is known to lead to an increase of diploid and higher polyploid sperm and of sperm with head shape abnormalities. In the pilot studies reported here quantitative analysis of the total DNA content, the morphology, and the chromatin distribution of mouse sperm was performed. The goal was to evaluate the discriminative power of features derived by high resolution image cytometry in distinguishing sperm of control and irradiated mice. Our results suggest that besides the induction of the above mentioned variations in DNA content and shape of sperm head, changes of the nonhomogeneous chromatin distribution within the sperm may also be used to quantify the radiation effect on sperm cells. Whereas the chromatin distribution features show larger variations for sperm 21 days after exposure (dpr), the shape parameters seem to be more important to discriminate sperm 35 dpr. This may be explained by differentiation processes, which take place in different stages during mouse spermatogenesis.

Experimentally Induced Testicular Alterations in Boars: Sperm Morphology Changes in Mature and Peripubertal Boars

Seven mature boars and 8 peripubertal boars were used to study the effects of elevated testicular temperature. The scrotum of the boars was covered by a textile-aluminium foil insulation device for 100 hours. Insulation of the scrotum in the peripubertal boars took place at an age of 100 days. Pronounced changes in sperm characteristics, e.g. decreased sperm motility and high numbers of proximal cytoplasmic droplets, abnormal sperm head shapes and abnormal acrosomes, were seen during week 3 to 5 after scrotal insulation in the mature boars. An "immature" semen picture, i.e. low sperm motility, low total sperm count and high numbers of proximal cytoplasmic droplets and abnormal sperm head shapes, was seen in the first ejaculates in the peripubertal experimental boars. The semen picture was normal in the control boars. A maturation of the semen picture over time was seen in the experimental boars. However, remaining abnormalities in the sperm morphology together with the testicular histology indicate lasting pathological changes in the testes.

Semen quality in workers producing reinforced plastic

A comparison of semen parameters from 25 workers at a reinforced plastic production plant and from 46 age-matched male fertility patients was carried out. For a period of more than three months before the semen analyses, the 25 workers had been exposed to styrene and acetone in concentrations exceeding the Danish threshold limit values. Serum concentrations of follicle stimulating and luteinizing hormones, semen sample volume, and concentration of sperm showed no differences between the two groups. Significant differences between the two groups were found in the percentage of live sperm, immotile sperm, and normal sperm. The reinforced plastic plant workers showed, in terms of semen quality, better values in the percentages of live sperm and immotile sperm, whereas they had a reduced percentage of normally shaped sperm. It was found that the reinforced-plastic workers had increased percentages of the amorphous and the pyriform sperm head shapes as compared to the fertility clientele. In sperm midpiece and sperm tail defects, the two groups showed similar values. From the data it could be concluded that work with production of reinforced plastic might result in an increase in the number of abnormal sperm heads.

An evaluation of the mouse sperm morphology test and other sperm tests in nonhuman mammals: A report of the U.S. environmental protection agency Gene-Tox program

The literature on the mouse sperm morphology test and on other sperm tests in nonhuman mammals was reviewed (a) to evaluate the relationship of these tests to chemically induced spermatogenic dysfunction, germ-cell mutagenicity, and carcinogenicity, and (b) to make an interspecies comparison of responses to chemicals. A total of 71 papers were reviewed. The mouse sperm morphology test was used to assess the effects of 154 of the 182 chemical agents covered. 4 other murine sperm tests were also used: the induction of acrosomal abnormalities (4 agents), reduction in sperm counts (6 agents), motility (5 agents), and F 1 sperm morphology (7 agents). In addition, sperm tests for the spermatogenic effects of 35 agents were done in 9 nonmurine mammalian species; these included analyses for sperm count, motility, and morphology, using a large variety of study designs. For the mouse sperm morphology test, 41 agents were judged by the reviewing committee to be positive inducers of sperm-head shape abnormalities, 103 were negative, and 10 were inconclusive. To evaluate the relationship between changes in sperm morphology and germ cell mutagenicity, the effects of 41 agents on mouse sperm shape were compared to available data from 3 different mammalian germ-cell mutational tests (specific locus, heritable translocation, and dominant lethal). The mouse sperm morphology test was found to be highly sensitive to germ-cell mutagens; 100% of the known mutagens were correctly identified as positives in the sperm morphology test. Data are insufficient at present to access the rate of false positives. Although it is biologically unclear why one might expect changes in sperm morphology to be related to carcinogenesis, we found that (a) a positive response in the mouse sperm morphology test is highly specific for carcinogenic potential (100% for the agents surveyed), but (b) overall, only 50% of carcinogens were positive in the test (i.e., sensitivity ⋍ 50%). Since many carcinogens do not produce abnormally shaped sperm even at lethal doses, negative findings with the sperm test cannot be used to classify agents as noncarcinogens. We conclude that the mouse sperm morphology test has potential use for identifying chemicals that induce spermatogenic dysfunction and perhaps heritable mutations. Positive test results may also be useful in assessing carcinogenic potential. Insufficient numbers of chemicals agents have been studied by the other sperm tests to permit similar comparisons. A comparison of 25 chemicals tested with sperm counts, motility, and morphology in at least 2 species (including man, mouse and 9 other mammals) demonstrated good agreement in response among species. With further study, interspecies comparisons of chemically induced sperm changes may be useful for predicting and evaluating human effects.

Studies on Dimensional Characteristics of Spermatozoa in Pure and Strain Crosses of White Leghorn Cockerels

Sperm head dimensional characteristics were evaluated in three pure strains (M, N, and P) and three strain crosses (N × P, M × N, and P × M) of White Leghorn cockerels. A total of 120 collections from 24 cockerels were taken. The average sperm head length recorded was 14.58 ± .09 μ, with an average maximum value of 15.29 ± .19 μ for spermatozoa from the P × M strain cockerels. The average sperm breadth was found to be .722 ± .005 μ. Maximum values were found for spermatozoa from the M × N strain cockerels that averaged .735 ± .004 μ. There was a significant difference (P<.01) between strains for sperm head length. The P × M strain had a significantly longer sperm head length than the other strains. This may be associated with heterosis. There were no significant differences among strains for sperm head breadth.The mean sperm head area was recorded to be 10.61 ± .08 μ2. Maximum sperm head area was observed in semen from P × M strain cockerels (11.03 ± .15 μ2). The average sperm head shape was found to be 20.14 ± .22. There were significant differences (P<.01) among strains for sperm head area and sperm head shape.The repeatability estimates for all the dimensional characteristics showed that they are highly repeatable traits, indicating that additive gene action is playing an important role in the expression of these traits.

Chemically-induced transmissible abnormalities in sperm-head shape.

The characteristics controlling sperm-head shape are carried on the autosomes. The effects on the progeny of alterations in this genome can be measured. Chemically-induced transmissible genetic damage in mice has been measured by assessment of the incidence of abnormal sperm among the progeny of CBA mice (of either sex) that have been treated with mutagens prior to mating with BALB/C mice.