Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: a study with various mouse strains

Success with in vitro fertilization (IVF) using inbred strains of mice varies considerably and appears to be related to the proportion of motile spermatozoa present in epididymal sperm samples of different strains. In this study, motile spermatozoa were separated from the original samples using a column of Sephadex G25. IVF rates were compared between separated and nonseparated samples of epididymal spermatozoa before and after cryopreservation. Oocytes and spermatozoa were obtained from FVB, DBA/2, C57BL/6J, and BALB/c inbred mice; and from F1 (C57BL/6J;ts DBA/2) hybrid mice, and isogenic gametes were used for IVF. These strains of mice were chosen because of their common use in transgenesis and mutagenesis studies. Dulbecco PBS was used for sperm separation on Sephadex, 18% raffinose, and 3% skim milk for cryopreservation; T6 medium for IVF; and mKSOM(AA) for embryo culture. There was a marked improvement in the rate of fertilization using fresh spermatozoa after motile spermatozoa were separated in C57BL/6J and BALB/c strains (92% vs. 58%, 79% vs. 44%) but no differences were found in fertilization rates between separated and nonseparated spermatozoa in F1, FVB, and DBA/2 strains (99% vs. 83%, 95% vs. 93%, 86% vs. 87%, respectively). After cryopreservation, higher rates of fertilization were obtained with separated motile samples in all strains; the greatest improvements were obtained with spermatozoa from C57BL/6J and BALB/c strains (40% vs. 16% and 51% vs. 14% for separated and nonseparated spermatozoa, respectively). No differences were found between the proportions of 14.5-day fetuses developing from embryos derived from separated and nonseparated spermatozoa with or without cryopreservation (33% to 46%). In conclusion, the fertility of frozen-thawed mouse epididymal spermatozoa improves significantly when highly motile populations of spermatozoa are separated for freezing.     

Analysis of oocyte quality in recombinant inbred mouse strains developed from KE and CBA strains that differ in fertilization efficiency

Recombinant inbred (RI) mouse strains were developed from reciprocal crosses between two inbred strains differing in the proportion of fertilized ova (CBA, 100%; KE, 77%), to analyse the underlying factors. A correlation (r = 0.83, P < 0.01) between fertilization efficiency within 22 RI strains and after mating RI females with KE males proved that oocyte quality was involved. The following oocyte parameters were analysed in RI and progenitor strains: time of meiotic maturation, rapidity of enzymatic removal of egg investments, and proportion of fertilized ova with supplementary spermatozoa in the perivitelline space. Among the RI strains, high incidence of supplementary spermatozoa was correlated with lower efficiency of fertilization (r = -0.58, P < 0.05) and with slow meiotic maturation (r = -64, P < 0.01), suggesting that delayed maturation may affect oocyte ability of being fertilized by the first penetrating spermatozoon. However, significant correlations were also found between characters which coexist within the progenitor strains, but are not likely to be physiologically related; this suggests that RI strains have inherited large blocks of progenitor genomes, not disrupted by recombination. The strain distribution pattern (SDP) of the analysed traits revealed CBA-like, KE-like, and intermediate phenotypes, indicating that they are polygenically determined. No linkages were found between the studied traits and 12 enzymatic markers. However, the SDP for fertilization efficiency showed a preponderance of non-matching strains (15/19) in relation to agouti locus; the known instability of this chromosome region makes it possible that a putative linkage was disrupted by recombination when RI strains were created.     

Germ cells and fertilization differences among Japanese flounder Paralichthys olivaceus, summer flounder Paralichthys dentatus and their first and second generations

The morphology of gametes and the fertilization biology of Japanese flounder Paralichthys olivaceus (Po), summer flounder Paralichthys dentatus (Pd) and their hybrids were examined. Multiple generations (two hybrids: Po♀× Pd♂ (F1) and Pd♀× Po♂; two backcrosses: F1♀× Po♂ and F1♀× Pd♂) were obtained by artificial insemination. Comparison of egg ultrastructure among Po, Pd and F1 showed the morphology of micropyle region and the distribution density of pores were species specific. There were c. 100-200 accessory openings around the micropyle in Po, but not in Pd and F1. The zona radiata thickness and number of parallel bands were similar between F1 and Po, which were different from Pd. Comparison of spermatozoa ultrastructure revealed a close relationship between Po and Pd. Cytologically, the six crosses obeyed normal fertilization and cleavage processes, and only one male pronucleus was observed in a fertilized egg, indicating a monospermic fertilization pattern. Analysis of the time distribution from fertilization to first cleavage revealed an obvious delay at pronucleus fusion in the Pd × Po cross. The delay might indicate some cytoplasmic-nuclear incompatibility during the process of fertilization.     

Fertilization of cumulus-free,zona-intact mouse ova in vitro at high and low sperm concentrations

The effect of varying the sperm concentration between 2 × 10⁵ sperm/ml and 8 × 10⁶ sperm/ml on fertilization of cumulus-free, zona-intact F1 (CBA × C57BL) mouse ova by QS and F1 (CBA × C57BL) mouse spermatozoa was studied. The spermatozoa from both strains of mice exhibited optimal fertilization rates at 2 × 106 sperm/ml. However, at sperm concentrations greater than 4 × 106 sperm/ml and less than 1 × 106 sperm/ml, fertilization rates were significantly reduced. F1 spermatozoa were more susceptible to dilution than QS spermatozoa. A significant interaction between strain and sperm concentration indicated that the two strains produced different fertilization rates at different sperm densities. Extracts of epididymal fluid, medium from capacitated spermatozoa, or ampulla fluid did not improve the fertilization rate at 2 × 105 sperm/ml, but retaining the cumulus oophorus did. The decrease in fertilization rate at 8 × 106 sperm/ml can in part be attributed to a nondialysable inhibitor from the neat sperm preparation that appeared to be of epididymal origin.     

Tissue culture‐induced genomic alteration in maize (Zea mays) inbred lines and F1 hybrids

Genomic alteration is a common phenomenon associated with plant tissue culture, which often encompasses genetic changes and epigenetic modifications (e.g. cytosine methylation). Here, we studied genomic alteration in maize by assessing calli and regenerated plants derived from three inbred lines (M17, J7 and JC) and two pairs of reciprocal F1 hybrids (pair I: M17/J7 and J7/M17 and pair II: M17/JC and JC/M17). By employing two molecular markers, the amplified fragment length polymorphism and methylation‐sensitive amplified polymorphism, we found that both types of genomic alterations occurred in calli and regenerated plants of all the studied maize inbred lines and F1 hybrids, but the extent and pattern of changes varied substantially across the genotypes. Among the three inbred lines, M17 showed markedly higher frequencies of both genetic (from 2.1% to 3.8%) and methylation alterations (from 6.5% to 9.9%, by adding up the various patterns) than the other two lines which showed similar frequencies for both types of alterations (genetic: 0.5–1.8%, methylation: 2.1–3.7%). Of the two F1 hybrid pairs, while pair I showed genetic variation frequencies similar to that of the inbred parent with lower changing frequency and pair II was intermediate of those of the parents, both pairs showed frequencies of methylation alteration more or less intermediate of those of their inbred parental lines. Parent‐of‐origin effects in both genetic and methylation changes were detected in only one of the hybrid pairs (primarily pair II) for a given changing pattern. Statistical testing confirmed the genotypic difference in both genetic and methylation (hypomethylation) alterations among the regenerants. Taken together, it could be concluded that the frequency and pattern of both genetic and cytosine methylation alterations in maize tissue culture were largely genetic context‐dependent traits, but stochasticity also played an important part. F1 hybrids were not significantly more stable than their inbred parental lines under tissue culture conditions.     

An increased level of sperm abnormalities in mice with a partial deletion of the Y chromosome

Two congenic lines of mice, one with a partial deletion of the Y chromosome, differ in the percentage of spermatozoa with abnormal heads: B10.BR/SgSn males give 22.6% and B10.BR-Ydel/Ms males give 64.2% abnormal sperm. The F1s resulting from crosses of B10.BR/SgSn males with females of five common inbred strains exhibited significantly lower levels of abnormal sperm than the parental strains, as opposed to F1 hybrids sired by B10.BR-Ydel/Ms mutant males, where very high levels of abnormal spermatozoa were found. About 30% of abnormal spermatozoa, produced by males with deletion on the Y chromosome, were characterized by a flat acrosomal cap. This class of abnormality was never observed in non-mutant males, suggesting a mutant-specific defect. These results demonstrate the important role of the Y chromosome in spermatogenesis.     

The minimum effective spermatozoa : egg ratio for artificial insemination and the effects of mercury on sperm motility and fertilization ability in Clarias gariepinus

The optimal ratio of spermatozoa : egg (15 000 : 1) for artificial insemination of African catfish Clarias gariepinus gave fertilization and hatching rates of 80 and 67%, respectively. Below a sperm : ova of 3000 : 1 fertilization success decreased significantly. Excessive sperm (>15 000 : 1) partly inhibited fertilization success. Sperm motility was decreased significantly by 0·001 mg 1⁻¹ Hg²⁺ as HgCl₂, but its effect on fertilization was dependent on the sperm : ova ratio, since excess sperm masked the effect of the pollutant. The most sensitive sperm : ova ratio for monitoring pollutant effects on fertilization success was 1500 : 1, which corresponds to half the minimal amount that yields a high fertilization rate in artificial insemination. There was a good correlation between fertilization and hatching rates ( r =0·83; P<0·05). Although both fertilization and hatching rates provide equally good indicators of fertilization success, the more rapid fertilization rate test is recommended since it requires only 12 h.     

Herpes simplex virus infection of human spermatozoa correlates with decreased frequency of blastocyst formation and frequency of embryo implantation during in vitro fertilization

We have conducted a comparative analysis of developing human embryos in the course of in vitro fertilization (IVF) as a method of sterility treatment of two groups of patients: herpes simplex virus (HSV) was detected in the fraction of motile sperm of male partners in group I (n = 28) and no HSV was found in sperm in group II (n = 103). We assessed number of fertilized ova, embryos during cleavage, and blastocysts as well as such parameters as frequency of implantation and frequency of pregnancy in IVF cycles. It was established that the presence of HSV in spermatozoa did not affect the efficiency of fertilization or cleavage of zygotes. At the same time, in cases of virus-infected male gametes, the frequency of blastocyst formation was two times less (p = 0.015), and the frequency of embryo implantation and pregnancy was, on average, five times lower (p = 0.01 and p = 0.016, respectively). Based on the obtained results, a conclusion was made about the negative influence of HSV-virus in male gametes at the early stages of embryo development and the frequency of achievement of pregnancy in respect to the methods of assisted reproductive technologies (ART).     

Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa

Efficient and dependable mouse cryopreservation methods are urgently needed because the production of mice with transgenes and disrupted and mutant genes is now commonplace. Preservation of these unique genomes provides an essential safeguard for future research. Unfortunately, mouse spermatozoa appear more vulnerable to freezing than other species, e.g., bovine and human. In this study, we examined the efficiency of intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) in generating embryos from mouse spermatozoa frozen with 18% raffinose and 3% skim milk for cryoprotection. A comparison was made between the inbred strain C57BL/6J, commonly used in mutagenic and transgenic studies, and a hybrid strain B6D2F1 (C57BL/6J x DBA/2J). C57BL/6J spermatozoa are known to be more sensitive to freezing than B6D2F1. Fertilization of oocytes after IVF was significantly lower with C57BL/6J spermatozoa when compared with B6D2F1 spermatozoa for both fresh and frozen spermatozoa (fresh, 89 vs. 55%; frozen, 56 vs. 9%). Freezing also reduced the fertility of B6D2F1 spermatozoa (89 vs. 56%). Fertilization improved dramatically after ICSI with fresh and frozen C57BL/6J spermatozoa (90 and 85%) and also with frozen B6D2F1 spermatozoa (87%). The development of two-cell embryos to the blastocyst stage was lower for C57BL/6J than B6D2F1 (42-61% and 84-98%) in all treatments but similar for embryos within each strain. The normality of chromosomes from fresh and frozen spermatozoa was assessed in oocytes prior to first cleavage. The majority of oocytes had normal chromosomes after IVF (98-100%) and ICSI (87-95%), indicating that chromosomal abnormalities were not responsible for the poorer development in vitro of C57BL/6J embryos. In conclusion, our data show that ICSI is a more efficient and effective technique than IVF for generating embryos from frozen spermatozoa. More important, ICSI is especially valuable for strains where IVF with fresh spermatozoa produces few or no embryos.     

Meiotic behavior as a selection tool in silage corn breeding

In breeding programs, commercial hybrids are frequently used as a source of inbred lines to obtain new hybrids. Considering that maize production is dependent on viable gametes, the selection of populations to obtain inbred lines with high meiotic stability could contribute to the formation of new silage corn hybrids adapted to specific region. We evaluated the meiotic stability of five commercial hybrids of silage corn used in southern Brazil with conventional squashing methods. All of them showed meiotic abnormalities. Some abnormalities, such as abnormal chromosome segregation and absence of cytokinesis, occurred in all the genotypes, while others, including cytomixis and abnormal spindle orientation, were found only in some genotypes. The hybrid SG6010 had the lowest mean frequency of abnormal cells (21.27%); the highest frequency was found in the hybrid P30K64 (44.43%). However, the frequency of abnormal meiotic products was much lower in most genotypes, ranging from 7.63% in the hybrid CD304 to 43.86% in Garra. Taking into account the percentage of abnormal meiotic products and, hence, meiotic stability, only the hybrids CD304, P30K64, SG6010, and P30F53 are recommended to be retained in the breeding program to obtain inbred lines to create new hybrids.     

Effects of platelet activating factor on the motility and acrosome reaction of bovine spermatozoa

The effects of platelet activating factor (PAF) on motility and the acrosome reaction of ejaculated bull spermatozoa were evaluated. Washed spermatozoa (30 x 10(6)/ml) were incubated (39 degrees C) for up to 2 h with 10 to 200 muM PAF in a modified Tyrode's solution (pH 7.4) containing 3 mg/ml bovine serum albumin. Sperm motility was evaluated subjectively and by computer-assisted semen analysis. Percent acrosome-reacted spermatozoa was quantified microscopically from fixed smears following Giemsa staining. Percent fertilization by PAF-treated spermatozoa was determined using in vitro-matured bovine ova. Percent sperm motility decreased with >/= 50 muM PAF, while the rate of motility loss increased with PAF concentration (P<0.001). Percent acrosome reactions increased with PAF concentration during incubation (P<0.001). Acrosomal loss was rapid and complete with 200 muM PAF. At concentrations between 80 to 120 muM PAF, bull spermatozoa underwent acrosome reactions without a rapid loss of motility and penetrated in vitro-matured bovine ova at a rate comparable to that of heparin-capacitated spermatozoa (68 versus 54%, respectively). Incubation of bull spermatozoa with 10 to 50 muM PAF for 45 min had no effect on percent progressive motility, sperm velocity or other motility parameters. These results indicate that PAF can be used to induce acrosome reactions in bull spermatozoa and to promote in vitro fertilization of bovine ova. Under the conditions used in this study, PAF did not stimulate bovine sperm motility.     

Improved in vitro fertilization and development by use of modified human tubal fluid and applicability of pronucleate embryos for cryopreservation by rapid freezing in inbred mice

We examined in vitro fertilizability and development of 10 inbred mouse strains (C57BL/6J, C57BL/10, C57BL/10.D2/newSn, C57BL/10-Thy1.1, C57BL/10.Br/Sn, C3H/He, RFM/Ms, STS/A, BALB/c-nu and C.B-17/Icr), and the viability of frozen-thawed in vitro fertilized (IVF) embryos after embryo transfer (ET). In seven strains, fertilizability was significantly greater in modified human tubal fluid (mHTF) compared with modified Krebs-Ringer's bicarbonate solution (TYH medium). The TYH medium supported almost no fertilization in four strains. More than 80% of IVF embryos developed to the blastocyst stage by 120 h in potassium-enhanced simplex optimization medium (KSOM). Reciprocal fertilization between C57BL/6J and BALB/c-nu gametes in TYH medium yielded poor fertilization o f BALB/c-nu due to spermatozoal deficiencies. Increased concentrations of bovine serum albumin and spermatozoa during capacitation and Percoll washing did not drastically affect fertilization. The mHTF, but not TYH medium, supported BALB/c-nu spermatozoa penetration into the zona pellucida irrespective of capacitation media. In vitro fertilized embryos frozen-thawed rapidly were transferred to surrogate mothers at the two-cell stage. Compared with that of unfrozen controls, rapid freezing had no significant effect on fetus development except in C57BL/10.D2/newSn mice. These results suggest that mHTF medium is superior with respect to IVF of inbred mice, and that KSOM adequately supports in vitro fertilized embryo development in inbred mice. The data also indicate that rapid freezing of pronucleate embryos following IVF is suitable for cryopreservation and embryo banking of inbred mice and for the production of genetically modified mice.     

Genetic aspects of experimental tuberculosis in mice

Susceptibility of inbred mouse strains, their F1 hybrids, offsprings from BC1 and RI strains from CXB pannel to tuberculosis infection was studied. Mice were infected with virulent strain of M. tuberculosis H37Rv. All F1 hybrids, with the exception of (BRSUNT X I/St)F1, showed superresistance, due apparently to heterosis effect; (BRSUNT X I/St)F1 demonstrated an intermediate level of resistance, as compared with parental strains. Analysis of [(BALB/c X B6)F1 X B6]BC1, [(BALB/c X B6)F1 X BALB/c]BC1 offsprings and RI strains indicated that strains BALB/c and B6 have different alleles of at least two genetic loci mediating susceptibility to tuberculosis.     

Oocyte and zygote zona pellucida lectin binding in BALB/cBy and C57BL/6By mice and their F1 hybrids

Zona pellucida intact oocytes and zygotes from two inbred strains of mice (BALB/cByEss and C57BL/6ByEss) and their F1 hybrids were reacted with a lectin panel (ConA, WGA, sWGA, PNA, UEA I, LTA, BSB4, DBA, PHA-P, LPA, and LFA). No major differences were observed between groups of mice for the majority of the lectin binding patterns. However, oocytes from BALB/cByEss and the F1 (C57BL/6ByxBALB/cBy) gave identical binding patterns for PNA. Following fertilization BALB/cByEss and the F1 (C57BL/6ByxBALB/cBy) bound UEAI and LTA more strongly, but the other two groups of mice demonstrated identical weaker binding of UEAI and LTA. These results indicate the possible influence of the paternal genotype on zona pellucida formation.     

Effect of albumin on fertilization of mouse ova in vitro

Serum albumin is an obligatory component of the incubation medium for the fertilization of mouse ova. Normal, untreated bovine serum albumin supports high rates of fertilization of cumulus-free ova both with and without their zonae pellucidae. Heat-treated or trichloroacetic acid-extracted bovine serum albumin is unable to support the fertilization of a majority of zona-intact ova but fertilization of zona-free ova is unimpaired. Spermatozoa incubated in medium containing heated bovine serum albumin fertilize zona-intact ova when 2 mM caffeine is present but the progress of sperm head decondensation is delayed when compared to normal controls. Trichloroacetic acid extracted BSA preferentially and irreversibly inhibits zona penetration by spermatozoa, but this effect is not mediated by an inhibition of spermatozoal motility or zona-binding ability. This effect occurs after only a 10-min preincubation of the spermatozoa in the extracted BSA or when the medium contains only a 10% (v/v) proportion of this albumin. It is estimated that mouse spermatozoa under the conditions used take 2 hr to penetrate the zonae pellucidae of 50% of ova and effect fertilization.     

Linkage between sperm abnormality level and major urinary protein phenotype in mice

Segregation of sperm abnormality level and the pattern of major urinary proteins (MUPs) were investigated in F2 and B1 hybrid males obtained from crosses involving two contrasting inbred strains of mice: CBA/Kw (Mup-1a1a, 3.3% abnormal sperm) and C57BL/Kw (Mup-1b1b, 21.9% abnormal sperm). In the progeny of both crosses mean levels of abnormal spermatozoa were significantly higher for males typed as Mup-1b1b than for heterozygous Mup-1a1b males. Moreover, all F2 hybrid males showing very high percentages of abnormal sperm were Mup-1b1b homozygotes. Similarly, among B1 males with a high level of deformed spermatozoa, a statistically significant majority were Mup-1b1b genotypes. Our results suggest that at least two genes which influence sperm abnormality level are segregating in these crosses. Both appear to be recessive for high sperm abnormality level, and one shows weak linkage to Mup-1 on chromosome 4.     

Effect of sperm penetration in vitro on completion of first meiosis by bovine oocytes arrested at various stages in culture.

Bovine oocytes cultured for 12-20 h in TC-199 were incubated for 24 h in fertilization medium, Brackett and Oliphant medium with bovine serum albumin (10 mg ml-1), caffeine (5 mmoll-1) and heparin (10 micrograms ml-1), with or without frozen-thawed spermatozoa. High penetration rates (93-96%) and significantly (P < 0.001) higher maturation rates were obtained in oocytes incubated with (93-100%) than without (62-72%) spermatozoa. However, when oocytes at the germinal vesicle stage were cultured for 44 h fertilization medium, maturation of oocytes to metaphase II was reduced. However, all oocytes that were first cultured for 20 h and further for 24 h with spermatozoa were penetrated and 40% of the penetrated oocytes reached metaphase II. All of the remaining oocytes that did not mature arrested at the stages of condensed germinal vesicle (39%) or prometaphase I (22%). These results indicate that oocytes at metaphase I at and after sperm penetration are stimulated by sperm penetration to complete maturation.     

4-way bred rats as experimental animals

The present study is an attempt to utilize hybrids among several inbred strains of rats as useful animals for the studies of effectiveness and toxicology on drugs., Four-way crosses were made among the LEW, WM, F344 and DRY strains of rats, and their characteristics were examined. From the breeding data of diallel crosses among these four strains and reciprocal crosses among their F1 hybrids, the mating type indicating the highest reproductivity was (LEW X WM) F1 X (F344 X DRY) F1. These four-way crosses were designated as LWFD. The reproductivity of this mating type was exceedingly higher than those of four strains. In order to examine the susceptibility to thiamine hydrochloride, the acute toxicity test was practiced in inbred strains, F1 hybrids and four-way crosses. As a result, in spite of highly heterogeneous population, the LWFD did not show a peculiar response in comparison with four strains and their F1 hybrids. Furthermore, hematological and clinico-biochemical values of the LWFD did not show a large variability as presumed. From these results, it is suggested that hybrids such as four-way crosses among inbred strains can be used as useful animals for the studies of effectiveness and toxicology on drugs.     

Cytogenetic studies on two F1 hybrids of autotetraploid rice varieties showing extremely high level of heterosis

Mechanisms of two F1 hybrids (D46A × DTP-4 and D46A × Dminghui63) of autotetraploid rice (2n = 4x = 48) showing extremely high pollen fertility 87.40% and 85.97%, respectively, seed set 82.00% and 79.00%, respectively and extremely high level of heterosis were analyzed cytologically. The chromosome pairing of D46A × DTP-4 and D46A × Dminghui63 was normal at metaphase I(MI), and had almost no I or III, with an average of 0.020I +14.36 II 6.44rod+7.91ring) +0.01III + 4.80 IV + 0.01VIII and 0.06 I + 17.67 II (11.01rod + 6.67ring)] + 0.06 III +3.10IV+0.01VI, respectively. The most frequent chromosome configurations were 10II+7IV and 12II+bIV. The bivalent frequency was less frequent in hybrids than that in restoring parents, and the same results were gained from univalents, trivalent and multivalents. However, the quadrivalent frequency was significantly higher in hybrids than that in restoring parents at MI. The other meiotic phases progressed normally, except for low percentages of PMCs with lagging chromosomes at AI and low percentages of PMCs with micronuclei at telophaseI (TI) and telophaseII (TII). PMCs with lagging chromosomes at AI and PMCs with micronuclei at TI and TII showed negative correlation between pollen fertility and seed set. Above 90% of the PMCs could form normal microspores, which resulted in the production of viable pollen grains, abnormal microspores were observed including penta-fission and hexa-fission. Based on these results we suggest that the two F1 hybrids had better behaviors of chromosome pairing and genetic stability than autotetraploid rice and other autotetraploid plants ever studied.     

Effect of time of ovulation on fertilization after intrabursal transfer of spermatozoa (ITS): improvement of a new method for artificial insemination in mice

The timing of AI in relation to ovulation was examined to improve intrabursal transfer of spermatozoa (ITS) in mice, a new method of AI that involves transfer of spermatozoa into a space near the infundibulum. Two microliters of fresh epididymal B6C3F1 spermatozoa (containing 2 x 10(5) spermatozoa) were inseminated 1, 7, 12, or 17 h after hCG administration. At 1.7 days after ITS, normal cleaving embryos were recovered at rates ranging from 6 to 50% (21.5 +/- 15.8%; mean +/- S.D.), 40-100% (75.2 +/- 20.2%), 33-100% (60.1 +/- 19.3%), and 6-47% (22.7 +/- 13.3%), respectively. The rate obtained by ITS 7h after hCG administration was comparable (P > 0.05) to that (90.5 +/- 6.3%) for embryos obtained after natural mating (control), but rates at all other times were significantly less than control. To examine whether in vivo fertilization rate differs when spermatozoa from various mouse strains are used, B6C3F1 females were inseminated with spermatozoa from ICR, C57BL/6N and C3H/HeN mice 7 h after hCG administration. There were strain differences (P < 0.01 for ICR and B6C3F1 versus C57BL/6N and C3H/HeN) for in vivo fertilization rates (83.9 +/- 10.3%, 75.2 +/- 20.2%, 33.6 +/- 24.5% and 25.6 +/- 16.1% for ICR, B6C3F1, C57BL/6N and C3H/HeN, respectively). Similar rates (72.9 +/- 7.3% and 27.5 +/- 46.2% for ICR and C57BL/6N, respectively) were also obtained when oocytes were inseminated with spermatozoa of the same strain. In addition, females (B6C3F1) inseminated by ITS of fresh B6C3F1 spermatozoa 7 h after hCG administration yielded normal mid-gestational fetuses with an average litter size of 7.0 +/- 4.9, which seemed much higher than the previously reported litter size of 3.2. In conclusion, the timing of AI was considered a key factor affecting in vivo fertilization efficiency.