Differences in sperm migration through cervical mucus in vitro relates to sperm colonization of the oviduct and fertilizing ability in goats

Sperm migration in estrous cervical mucus can be used to measure the ability of spermatozoa to migrate through the genital tract. The relationship of this test with the sperm colonization of the isthmus, and its impact on fertility has not been evaluated in goats. Our objectives were to determine the differences among spermatozoa of different bucks in their ability to penetrate homologous cervical mucus in vitro and to determine the relationship between sperm displacement through cervical mucus and the ability of spermatozoa to colonize the oviduct and penetrate IVM oocytes, in vivo. Sperm migration in cervical mucus was assessed in flat capillary tubes with a phase contrast microscope. In the first experiment, fresh semen was used to establish differences between males in the ability of their spermatozoa to migrate in cervical mucus. In the second experiment, goats in estrus were inseminated with fresh spermatozoa from males with significant differences in mucus migration ability, and sperm numbers were evaluated at the UTJ. In the third experiment, the fertilization efficiency of IVM oocytes transferred to the oviduct of estrous females inseminated with semen from the same males as earlier, was used to assess the relationship between the mucus migration test and the in vivo fertilization performance of their spermatozoa. Spermatozoa from different males varies significantly in sperm migration efficiency in cervical mucus (15.5a +/- 1.2; 14.9a +/- 1.4; 17.5ab +/- 1.2; 17.0ab +/- 1.5; 19.7b +/- 1.2; 20.1b +/- 1.4 mm; media +/- S.E.M. for males A-F, respectively, P < 0.05). Spermatozoa from males with different mucus migration efficiency values produced different sperm populations at the oviduct reservoir of inseminated females (1,233 +/- 92.3 versus 28.8 +/- 17.0 spermatozoa of males with high and low relative migration efficiency, respectively, P < 0.02). Spermatozoa from males with different mucus migration efficiency values have different fertilization rates of IVM oocytes transferred to oviduct (47/96 (49.0%) versus 25/91 (27.5%) for males with high and low relative migration efficiency, respectively, P < 0.05). Cumulative results suggest that sperm migration in cervical mucus is related to the ability of spermatozoa to colonize the oviduct and to fertilize matured oocytes in vivo.     

Distribution and number of spermatozoa in the oviduct of the golden hamster after natural mating and artificial insemination

A group of female hamsters was mated with males of proven fertility either several hours before or during ovulation. Another group of females was artificially inseminated several hours before ovulation. Females were killed at various times after the onset of mating or artificial insemination, oviducts were fixed and sectioned serially, and spermatozoa were counted individually as to their location in the oviduct. Regardless of the type or time of insemination, the vast majority of spermatozoa that entered the oviduct remained in the lower segments of the isthmus (the intramural and caudal isthmus) without ascending to the ampulla. The lower segments of the oviduct, particularly the caudal isthmus, appeared to be acting as a "sieve" and/or "sperm reservoir." In females mated or artificially inseminated prior to ovulation, virtually no spermatozoa reached the cephalic isthmus or ampulla until the commencement of ovulation. Although a few spermatozoa reached the ampulla by 1 h after the onset of mating, they were the exception rather than the rule. When females were mated during ovulation, spermatozoa spent a minimum of about 3 h in the caudal isthmus before ascending to the ampulla. The number of spermatozoa that entered the oviduct after artificial insemination was considerably lower than in naturally mated animals, but this low number was apparently large enough to ensure complete fertilization.     

A quantitative comparison of the passage of capacitated and uncapacitated hamster spermatozoa through the uterotubal junction.

Female hamsters were artificially inseminated at the time of ovulation with an equal concentration and volume of capacitated sperm suspension in one uterus and uncapacitated sperm suspension in the contralateral uterus. When oviducts were examined 3.5-4.0 h after insemination, a significantly (paired t-test, p less than 0.05) lower number of spermatozoa were found in the oviduct from the side inseminated with capacitated sperm suspension compared to the side inseminated with uncapacitated sperm suspension. The reduction in the number of spermatozoa entering the oviduct on the side inseminated with capacitated sperm suspension was particularly evident when nearly all the spermatozoa in the suspension were hyperactivated. These results suggest that hamster spermatozoa require a progressive linear type of motility pattern to pass efficiently through the uterotubal junction and that under normal conditions in vivo, fertilizing spermatozoa initiate hyperactivated motility after entering the oviduct.     

Impaired transport and fertilization in vivo of calcium-treated spermatozoa from +/+ or congenic tw32/+ mice.

To determine whether calcium alters processes important for fertilization in vivo, mouse (+/+) spermatozoa were incubated in medium with 1.0-1.7 mM calcium prior to artificial insemination (AI) into the cervix of hormonally primed females. Spermatozoa from congenic tw32/+ mice were also tested because their flagella are hypersensitive to calcium. As a control, spermatozoa were incubated in calcium-deficient medium prior to AI. Spermatozoa from mice of both genotypes incubated in calcium-containing medium fertilized significantly fewer eggs after AI than did spermatozoa incubated in calcium-deficient medium. In addition, calcium-treated spermatozoa from tw32/+ mice fertilized significantly fewer eggs than calcium-treated +/+ spermatozoa. Pretreatment with calcium also reduced the number of spermatozoa in the oviducts 0.5-4.5 h after AI, and the oviducts of females inseminated with calcium-treated spermatozoa from tw32/+ mice contained significantly fewer spermatozoa than those of females inseminated with calcium-treated +/+ spermatozoa. These results suggest that preincubation in millimolar levels of calcium changes the physiology of epididymal spermatozoa in such a way as to impair sperm transport to the oviduct and fertilization in vivo.     

Distribution and viability of spermatozoa in the canine female genital tract during post-ovulatory oocyte maturation

ABSTRACT: BACKGROUND: Unlike other domestic mammals, in which metaphase-II oocytes are ovulated, canine ovulation is characterized by the release of primary oocytes, which may take 12 to up to 36 hours. Further 60 hours are needed for maturation to secondary oocytes which then remain fertile for about 48 hours. Oestrus takes 7 to 10 days on average and may start as early as a week before ovulation. This together with the prolonged process of post-ovulatory oocyte maturation requires an according longevity of spermatozoa in the female genital tract in order to provide a population of fertile sperm when oocytes have matured to fertilizability. Therefore the distribution and viability of spermatozoa in the bitch genital tract was examined during post-ovulatory oocyte maturation. METHODS: Thirteen beagle bitches were inseminated on the day of sonographically verified ovulation with pooled semen of two beagle dogs containing one billion progressively motile spermatozoa. Ovariohysterectomy was performed two days later (group 1, n = 6) and four days later (group 2, n = 7). The oviduct and uterine horn of one side were flushed separately and the flushing's were checked for the presence of gametes. The oviducts including the utero-tubal junction and the uterine horns, both the flushed and unflushed, were histologically examined for sperm distribution. RESULTS: The total number of spermatozoa recovered by flushing was low and evaluation of viability was limited. Prophase-I oocytes were collected from oviduct flushing in group 1, whereas unfertilized metaphase-II oocytes were detected in group 2. From day 2 to day 4 after ovulation a significant decrease in the percentage of glands containing sperm (P<0.05) and a marked reduction of the mean sperm number in uterine horn glands were observed. A concomitant diminution of spermatozoa was indicated in the utero-tubal junction accompanied by a slight increase in sperm numbers in the mid oviduct. CONCLUSIONS: Oocyte maturation to metaphase-II stage is accompanied by a continuous sperm detachment and elimination in the uterine horns. Entrance of spermatozoa into the caudal oviduct seems to be steadily controlled by the utero-tubal junction thus providing a selected sperm population to be shifted towards the site of fertilization when oocyte maturation is completed.     

Differing sperm ability to penetrate the oocyte in vivo and in vitro as revealed using colloidal preparations

The penetration ability of boar (Sus scrofa domestica) spermatozoa exposed to viscous preparations under in vivo and in vitro fertilization conditions has been examined. Experiments involving induced ovulation in prepubertal animals and surgical insemination directly into the oviduct isthmus revealed an advantage of colloidal preparations. Based on within-animal comparisons, the incidence of penetration was 100% using both spermatozoa suspended in a viscous preparation of plant extracts and spermatozoa suspended in a control medium. However, percentages of monospermy were 22.2% in 54 oocytes inseminated with the control suspension compared with 62.5% in 48 oocytes inseminated with the colloidal preparation. An in vitro study involving 355 oocytes from slaughterhouse ovaries inseminated with in vitro–capacitated spermatozoa gave similar percentages of penetrated oocytes for both the control and colloidal suspensions. In this case, however, the percentage of monospermy was 32.7% in the control group compared with 10.6% for spermatozoa suspended in the colloidal preparation. Higher mean numbers of sperm inside the oocytes and higher numbers of sperm bound to the zona pellucida were also observed with the colloidal suspensions. In vitro motility and viability for spermatozoa in the colloidal suspensions were enhanced compared with that of the control group. Lower sperm membrane lipid disorder and reactive oxygen species generation were also observed in the viscous solution. These findings suggest that viscous fluids can enhance the ability of sperm to move, bind, and penetrate the oocyte in vitro, although this influence may be masked in vivo due to the already high viscosity in the oviductal fluid close to the time of ovulation.     

Differing sperm ability to penetrate the oocyte in vivo and in vitro as revealed using colloidal preparations

The penetration ability of boar (Sus scrofa domestica) spermatozoa exposed to viscous preparations under in vivo and in vitro fertilization conditions has been examined. Experiments involving induced ovulation in prepubertal animals and surgical insemination directly into the oviduct isthmus revealed an advantage of colloidal preparations. Based on within-animal comparisons, the incidence of penetration was 100% using both spermatozoa suspended in a viscous preparation of plant extracts and spermatozoa suspended in a control medium. However, percentages of monospermy were 22.2% in 54 oocytes inseminated with the control suspension compared with 62.5% in 48 oocytes inseminated with the colloidal preparation. An in vitro study involving 355 oocytes from slaughterhouse ovaries inseminated with in vitro-capacitated spermatozoa gave similar percentages of penetrated oocytes for both the control and colloidal suspensions. In this case, however, the percentage of monospermy was 32.7% in the control group compared with 10.6% for spermatozoa suspended in the colloidal preparation. Higher mean numbers of sperm inside the oocytes and higher numbers of sperm bound to the zona pellucida were also observed with the colloidal suspensions. In vitro motility and viability for spermatozoa in the colloidal suspensions were enhanced compared with that of the control group. Lower sperm membrane lipid disorder and reactive oxygen species generation were also observed in the viscous solution. These findings suggest that viscous fluids can enhance the ability of sperm to move, bind, and penetrate the oocyte in vitro, although this influence may be masked in vivo due to the already high viscosity in the oviductal fluid close to the time of ovulation.     

Sperm entry into zona-free oocytes in the hamster oviduct: Implications for the mechanisms of acrosome reaction induction

The possible importance of the zona pellucida for induction of the acrosome reaction (AR) and establishment of sperm/egg associations in the fallopian tube was investigated by instilling zona-free eggs into the oviductal ampulla of hamsters that had been inseminated with epididymal spermatozoa 6–7 hours previously. The eggs were recovered only 60–90 minutes later because of increasing difficulty with time of collecting zona-free eggs from the oviduct. In the zona-free group, 41 (4%) of 1,101 transferred eggs were recovered, of which 20% contained spermatozoa with decondensing nuclei (mean 4.4/egg). A similar (22%) fertilization rate (mean 3.2 spermatozoa/egg) was found among intact (control) eggs recovered after instillation into the contralateral oviduct. Mammalian spermatozoa are not incorporated even into zona-free eggs before AR occurs. These results thus demonstrate that an AR in functional hamster spermatozoa in vivo and establishment of sperm/egg associations in vivo require no interaction with the zona pellucida nor with other products of ovulation.     

Effect of in vitro and in vivo culture on embryo development from prepubertal goat IVM-IVF oocytes

The aim of this study was to analyze different culture systems on embryo development of prepubertal goat oocytes. We compare (i) the effect of the age of donor (goat) of oocytes on in vitro maturation, fertilization and subsequent embryo development, (ii) the effect of the origin of oviduct cells from coculture of prepubertal goat embryo development, and (iii) the effect of in vivo culture in rabbit oviducts for 1, 2 and 3 days on the development of prepubertal goat embryos produced in vitro. In Experiment 1, at 24 h post-insemination (hpi), oocytes from adult goats were allocated in TCM199 with oviduct cells from adult goats, and oocytes from prepubertal goats were randomly placed in drops with oviduct epithelial cells from adult (aOEC) or prepubertal (pOEC) goats. Cleavage rate and embryo development were evaluated at 48 hpi and after 7 days coculture, respectively. In Experiment 2, at 24 hpi, prepubertal oocytes were allocated in TCM 199 with pOEC. At 40-42 hpi, a group of embryos remained in the coculture (control group), and the rest were transferred to rabbit oviducts (three rabbits for replicate) for culturing in vivo for 24, 48 and 72 h. After these in vivo cultures, embryos were recovered, evaluated and placed in TCM199 with pOEC until Day 8 post-insemination. The maturation, fertilization and blastocyst rates did not differ significantly between oocytes obtained from adult and prepubertal goats. The percentage of blastocysts obtained from prepubertal goat embryos cocultured with aOEC or pOEC was also similar (12.1% versus 12.2%). The transfer of prepubertal goat embryos to rabbit oviducts for 1, 2 and 3 days did not improve the blastocyst rate compared to the control group (9.7, 10.9, 4.1 and 11.5%, respectively). In conclusion, in our conditions, there were no significant differences in embryo development between oocytes obtained from prepubertal and adult goats, and the embryo development from prepubertal goat oocytes were similar in the different culture systems compared.     

Capacitation status of hamster spermatozoa in the oviduct at various times after mating

Female hamsters were mated shortly after the onset of oestrus or immediately after ovulation. At various times after mating, spermatozoa were flushed from the isthmus of the oviduct using a modified Tyrode's medium supplemented with 20% hamster serum. Cumulus oophorus-free eggs were introduced into the suspensions of isthmic spermatozoa. Some eggs were removed every 30 min and examined for evidence of fertilization. For females mated shortly after the onset of oestrus, spermatozoa recovered from the oviducts 8 h after mating (about 1.5 h after ovulation) could penetrate eggs within 30 min and were considered fully capacitated. When spermatozoa were recovered at earlier times (1, 2, 4 and 6 h after mating) they required additional time (2, 1.5, 1 and 1 h respectively) in vitro before penetrating eggs. Therefore, when mating occurs shortly after the onset of oestrus, spermatozoa in the oviduct do not appear to become fully capacitated until about the time of ovulation. For females mated immediately after ovulation, spermatozoa recovered from the oviducts at 4 h after mating could penetrate eggs within 30 min. Spermatozoa recovered at 1 and 3 h after mating required 2 and 1 h respectively in vitro before penetrating eggs. These results suggest that sperm capacitation proceeds at a faster rate when mating occurs after ovulation.     

Embryo development rates after transfer of oocytes matured in vivo, in vitro, or within oviducts of mares

Objectives of the present study were to use oocyte transfer: 1) to compare the developmental ability of oocytes collected from ovaries of live mares with those collected from slaughterhouse ovaries; and 2) to compare the viability of oocytes matured in vivo, in vitro, or within the oviduct. Oocytes were collected by transvaginal, ultrasound-guided follicular aspiration (TVA) from live mares or from slicing slaughterhouse ovaries. Four groups of oocytes were transferred into the oviducts of recipients that were inseminated: 1) oocytes matured in vivo and collected by TVA from preovulatory follicles of estrous mares 32 to 36 h after administration of hCG; 2) immature oocytes collected from diestrous mares between 5 and 10 d after aspiration/ovulation by TVA and matured in vitro for 36 to 38 h; 3) immature oocytes collected from diestrous mares between 5 and 10 d after aspiration/ovulation by TVA and transferred into a recipient's oviduct <1 h after collection; and 4) im mature oocytes collected from slaughterhouse ovaries containing a corpus luteum and matured in vitro for 36 to 38 hours. Embryo development rates were higher (P < 0.001) for oocytes matured in vivo (82%) than for oocytes matured in vitro (9%) or within the oviduct (0%). However, neither the method of maturation nor the source of oocytes affected (P > 0.1) embryo development rates after the transfer of immature oocytes.     

Optimization of in vitro bovine embryo production: effect of duration of maturation,length of gamete co-incubation,sperm concentration and sire

The aim of these experiments was to investigate the effect of duration of IVM, duration of gamete co-incubation, and of sperm dose on the development of bovine embryos in vitro. In addition, the speed of sperm penetration of six bulls of known differing in vivo and in vitro fertility was examined. In Experiment 1, following IVM for 16, 20, 24, 28 or 32 h, cumulus oocyte complexes (COCs) were inseminated with 1 x 10(6) spermatozoa/ml. After 24 h co-incubation, presumptive zygotes were denuded and placed in droplets of synthetic oviduct fluid (SOF). In Experiment 2, following IVM and IVF, presumptive zygotes were removed from fertilization wells at 1, 5, 10, 15 or 20 h post insemination and placed in culture as described above. In Experiment 3, following IVM, COCs were inseminated with sperm doses ranging from 0.01 x 10(6) to 1 x 10(6) spermatozoa/ml. Following co-incubation for 24 h, presumptive zygotes were placed in culture as described above. In Experiment 4, following IVM, oocytes were inseminated with sperm from six bulls of known differing field fertility. To assess the rate of sperm penetration, oocytes were subsequently fixed every 3 h (up to 18 h) following IVF. Based on the results of Experiment 4, in Experiment 5, following IVM for 12, 18 or 24 h, COCs were inseminated with sperm from two sires with markedly different penetration speeds. After 24 h co-incubation, presumptive zygotes were denuded and placed in culture. The main findings from this study are that (1) the optimal duration of maturation of bovine oocytes in vitro to maximize blastocyst yield is 24 h, (2) sperm-oocyte co-incubation for 10 h is sufficient to ensure maximal blastocyst yields, (3) sperm concentrations of 0.25 x 10(6) and 0.5 x 10(6) spermatozoa/ml yielded significantly more blastocysts than any other concentration within the range of 0.01 x 10(6) 1 x 10(6) spermatozoa/ml, (4) there are marked differences in the kinetics of sperm penetration between sires and this may be a useful predictor of field fertility, and (5) the inferior development associated with slower penetration rates may in part be overcome by carrying out IVF at a time when the actual penetration is most likely to coincide with the completion of maturation.     

In vitro fertilization of sheep oocytes matured in vivo

Incubating washed ram spermatozoa in a modified Brackett's defined medium buffered with Hepes (DM-H) containing 20% of heat-inactivated sheep serum appears to be a reliable method of capacitating sperm for in vitro fertilization. Raising the Ca(++) concentration in the fertilization medium (DM-H-SS) to 10 mM stabilized the fertilization rate of various rams (2). This study was designed to determine if the developmental competence of the oocytes fertilized under such conditions was normal. Thirty-seven ewes, treated with progestagen sponges, were superovulated with porcine follicle stimulating hormone (pFSH: 16 mg). An intramuscular injection of gonadotropin releasing hormone (GnRH: 100 mug); given 24 to 26 h after sponge removal, induced the synchronization of ovulations 24 h later. Ovulated oocytes (n = 229) recovered with flushing of the oviducts were inseminated in vitro and 17 h later either fixed in acetic/alcohol (n = 115) to evaluate fertilization or transferred (n = 114) into 38 synchronized recipients (three oocytes/recipient) to evaluate their developmental competence. Of the fixed oocytes, 82.6% were fertilized and 61.7% were monospermic. Nineteen of the recipient ewes (50%) were pregnant at Day 18, and 16 ewes produced a total of 26 live young (mean: 1.63/ewe). The results showed a high efficiency of in vitro fertilization of ovulated oocytes in sheep following a pFSH-GnRH treatment and the in vivo developmental competence of oocytes fertilized in the presence of elevated Ca(++) concentration.     

Sperm Localization in the Oviducts of Artificially Inseminated Dairy Cattle

Eight animals, 3 heifers and 5 primiparous cows, were artificially inseminated by intrauterine deposition of frozen-thawed semen. The insemination dose comprised 20×106 or 200 × 106 spermatozoa, frozen in French mini straws. Four animals were inseminated at fixed time interval (72 or 84 h) after cloprostenol injection. The remaining 4 animals were inseminated in spontaneous oestrus. Slaughter took place 2 or 12 h after insemination. After fixation the oviducts were cut into segments, which were serial-sectioned and stained. Six sections per segment were examined under the microscope for sperm recovery. The number of spermatozoa recovered from the oviducts varied considerably among animals. Recovery was poor (less than 50 spermatozoa) in 4 animals. Recovery was low when insemination took place in induced oestrus and with the lower sperm number (20×106). In animals in which more than 50 spermatozoa were found the distribution varied both between animals and between oviducts within the same animal. Overall, more spermatozoa were found in the lower (UTJ, isthmus and AIJ) than in the upper (ampulla) parts of the oviducts. In 3 out of 4 animals more spermatozoa were recovered from the left than from the right oviduct. Only in 1 animal were the majority of spermatozoa found in the oviduct ipsilateral to the follicle-bearing ovary.     

The influence of pre- and post-ovulatory insemination on sperm distribution in the oviduct,accessory sperm to the zona pellucida,fertilisation rate and embryo development in sows

The aim of present study was to investigate the influence of pre-compared with post-ovulatory insemination, on the distribution of spermatozoa in the oviduct, the accessory sperm counts on the zona pellucida and early embryonic development. Thirty-six crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire) were artificially inseminated once either at 20-15 h before (group AIB) or at 15-20 h after (group AIA) ovulation by using a pooled semen of two boars. Thereafter, they were randomly allocated to one of five groups: slaughter at 5-6h after AI (group I-AIB), at 20-25 h after ovulation (groups II-AIB and II-AIA), at 70 h after ovulation (groups III-AIB and III-AIA), on day 11 (groups IV-AIB and IV-AIA, first day of standing oestrus=day 1) and on day 19 (groups V-AIB and V-AIA).The plasma levels of oestradiol-17beta and progesterone differed significantly (P相似文献   

Influence of hardening of the zona pellucida on in vitro fertilization of bovine oocytes

The influence of hardening of the zona pellucida of in vivo matured bovine oocytes on fertilizability was investigated. For the study, 163 preovulatory and 73 postovulatory oocytes recovered from superovulated heifers were used. The preovulatory oocytes, before they were used for in vitro fertilization, consisted of: 1) those cultured in vitro for 4 to 6 h to permit final maturation and 2) those incubated in the rabbit oviduct for 4 to 5 h to permit final maturation and induce hardening of the zona pellucida. A few oocytes served as a control of nuclear maturity and the zona pellucida solubility. Preovulatory and postovulatory oocytes were both inseminated in vitro using frozen-thawed, heparin treated and swim-up separated spermatozoa. Significant differences (P<0.01) were established between fertilization rates of cultured preovulatory oocytes (68.8%) and those incubated in the rabbit oviducts (42.9%), or those recovered from bovine oviducts (40.7%). It can be concluded that hardening of the zona pellucida distinctly influences the fertilizability of oocytes. This factor should be taken into account when considering the source of oocytes or the kind of treatment to be used for in vitro fertilization.     

Brief coincubation of gametes in porcine in vitro fertilization: role of sperm:oocyte ratio and post-coincubation medium

In this study, a short coincubation time of 10 min was used to determine the effect of different sperm:oocyte ratios during in vitro fertilization (IVF), and different periods of post-coincubation in a medium that is not appropriate for IVF, on fertilization parameters. In the first experiment, a total of 1624 in vitro matured oocytes, from 4 replicates, were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000, 1500, 1000 and 500 sperm:oocyte) and coincubated for 10 min or 6 h. The oocytes from 10 min of coincubation were washed in IVF medium to remove spermatozoa not bound to the zona pellucida and transferred to another droplet of the same medium (containing no spermatozoa) for 6h. The oocytes from the other group remained with the spermatozoa for 6h. Oocytes from both groups were then cultured in embryo culture medium (IVC) for 12h to assess fertilization parameters. In the second experiment, 1872 in vitro matured oocytes, in 3 replicates were inseminated with frozen-thawed spermatozoa using the same sperm:oocyte ratios as in the first experiment. The oocytes were coincubated for 10 min and transferred directly to IVC medium for 18 h (group A), to IVF medium (containing no sperm) only for 2h and then to IVC medium for 16 h (group B), or to IVF medium (containing no sperm) for 6h and then to IVC medium for 12 h (group C or control). There was an effect of sperm:oocyte ratio on all fertilization parameters in experiment 1. The efficiency of IVF (number of monospermic oocytes/total number inseminated) was higher (P<0.05) for oocytes coincubated with spermatozoa for 10 min and inseminated with 1500 and 1000 sperm:oocyte (35.8+/-3 and 37.6+/-2.7%, respectively) and for those coincubated for 6h with 500 spermatozoa per oocyte (37.2+/-3.1%). In experiment 2, the penetration and efficiency rates obtained in group A were poor (between 3 and 15%) irrespective of the sperm:oocyte ratio. However, in group B the fertilization parameters were similar to the controls and were also affected by the sperm:oocyte ratio. These results demonstrate that coincubation time may be reduced to 10 min to increase the efficiency of fertilization depending on the sperm:oocyte ratio, and that the spermatozoa bound to the zona pellucida require a maximum of 2h in an appropriate medium to penetrate the oocytes.     

Birth of pouch young after artificial insemination in the tammar wallaby (Macropus eugenii)

Timing of artificial insemination (AI) in marsupials is critical because fertilization must occur before mucin coats the oocyte during passage through the oviduct. In this study, timing and the site of insemination were examined to develop AI in the tammar wallaby (Macropus eugenii). Birth and postpartum (p.p.) estrus was synchronized in 46 females. Epididymal spermatozoa (n=4) or semen collected by electroejaculation (n=42) were inseminated early (4-21 h p.p.) into the urogenital sinus (n=7), the anterior vaginal culs de sac (n=7), the uterus by transcervical catheter (n=5), or the uterus by injection (intrauterine artificial insemination, IUAI) (n=5). A further 16 females were inseminated late (19-48 h p.p.) by IUAI. All females were monitored for birth. A third group of six females was inseminated late (21-54 h p.p.) by IUAI and 0.4-6.6 h later, sperm had reached the oviduct in all animals. In total, an oocyte to which spermatozoa were attached was recovered and two young were born after IUAI using epididymal (n=1) or electroejaculated (n=2) spermatozoa, but no young resulted from insemination at other sites. Two females were successfully inseminated at 43 and 47 h p.p., later than most other animals, and the third was inseminated much earlier (18 h p.p.) but with highly motile spermatozoa. These young represent the first macropodids born by AI and the first marsupials conceived using epididymal spermatozoa.     

Acrosome reaction of bovine spermatozoa in vivo: sites and effects of stages of the estrous cycle

Thirty-two cows were inseminated near the uterotubal junction at various stages of the estrous cycle and slaughtered 16 h later to determine the effects of stage of the estrous cycle and tubal site of sperm recovery on the frequency of acrosome-reacted bull spermatozoa. Slaughter times were 46, 70, 144, or 168 h after each cow was injected with prostaglandin (PG) F 2 alpha or during the luteal phase of the estrous cycle. Sperm were recovered from the upper uterus and the isthmus and ampulla of the oviducts and stained for both viability and acrosome reaction. The highest frequency of acrosome-reacted sperm was found in the ampulla ipsilateral to a dominant follicle (largest follicle present) or recent ovulation and primarily at 70 h after PGF2 alpha (P less than 0.05). Also, fewer sperm were acrosome reacted prior to (46 h post-PGF2 alpha) and well after (168 h post-PGF2 alpha) estrus than during or immediately postestrus (70, 90, and 144 h post-PGF2 alpha; P less than 0.05). Except for two cows, one at 46 h and one at 70 h, all cows with more than 50% acrosome-reacted sperm in the ampulla had ovulated before slaughter. These data suggest that capacitated sperm become localized in the ampulla of the oviduct of the ovulatory side around the time of ovulation.     

Sperm binding properties and secretory activity of the bovine oviduct immediately before and after ovulation

The possibility that differences in hormonal regimes between the two oviducts in the cow around ovulation affects secretory activity of the oviduct epithelial cells and/or sperm-oviduct binding was studied. Oviducts were collected immediately after slaughter at 6 hr before to 5 hr after timed ovulation of 14 normally cyclic cows that had been inseminated (n = 6) or not (n = 8) and material obtained from the same cows was processed in three ways. First, in vivo, after artificial insemination of the cows, low numbers of sperm cells (approx. 15 per oviduct) were found within the entire oviducts as observed by scanning electron microscopy (SEM). Almost all sperm were located in the isthmus and then only on ciliated cells and showed without exception fully matured, intact morphology. Secretory activity of noninseminated oviduct epithelia was induced after ovulation which was most predominant in the pockets of the ipsi-lateral ampulla compared to the contra-lateral ampulla (P < 0.01). Second, ex vivo, explants dissected from oviducts of the noniseminated cows were incubated with sperm. In all cases, the sperm bound to the explants in a similar pattern as observed in vivo and this binding was strictly fucose-dependent. The main difference with in vivo experiments was the high numbers of sperm bound at any site of the oviduct ( approximately 3,000 cells per mm(2)) indicating the high sperm binding capacity of the oviduct epithelia. Ovulation induced a striking drop in sperm binding capacity in the oviducts and was most pronounced in the isthmus ( approximately 1,300 cells per mm(2); P < 0.001) and to a lesser extent in the ampulla ( approximately 2,000 cells per mm(2), P < 0.01). Third, in vitro, pieces of tissue dissected from oviducts of the noninseminated cows were cultured to mono-layers. Culturing epithelial cells resulted in loss of their normal morphological appearance. In all cases, the sperm binding capacity in monolayers was very low (<50 cells per mm(2)) when compared to corresponding explants (P < 0.0001). Sperm binding to monolayers originating from the isthmus (<25 cells per mm(2)) was lower than in those from the ampulla (40-50 cells per mm(2); P < 0.01) and remained similar after ovulation. In all three approaches, no significant differences were found in sperm-oviduct binding characteristics and sperm-distribution in the ipsi- versus contra-lateral oviducts. This indicates, that systemic endocrine changes around ovulation rather than specific oviduct changes at the ipsi-lateral oviduct induce secretion in oviduct epithelial cells, and thus induce sperm release.