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In vitro culture has provided new information on the mechanisms involved in fertilization where two completely different cells fuse together. At the same time, results obtained in vitro have led to new questions. Does the follicle influence the final maturation process of the oocyte and does the oviduct regulate the normal behaviour of spermatozoa? Recent studies indicate a critical influence of both the follicular compartment and the oviduct on the normal fertilization process. Oocytes matured in vivo are more competent to induce pronuclear formation, but are still susceptible to polyspermy. Oocytes matured in vitro can develop fully after fertilization, but require the presence of follicular factors during culture to enhance their developmental competence. Fresh or frozen spermatozoa can penetrate oocytes in vitro, but their conditioning by differential centrifugation or oviductal cells influences the rate of polyspermy. The understanding of these influences is a prerequisite to enhancing in vitro production of pig embryos.

Two concentrations of glycerol in a freezing diluent were tested with respect to the in vitro fertilizing capacity of frozen-thawed boar spermatozoa which, before exposure to oocytes, were subjected to 3 methods of fractionation. These were 1) the upper fraction, 2) the swim-up and 3) percoll gradinet-centrifugation. The highest proportions of motile spermatozoa were obtained by the swim-up procedure, while acrosomal integrity was best preserved by the upper fraction procedure. Raising the glycerol concentration from 2 to 6% (v/v) during freezing decreased the proportion of spermatozoa with a normal apical ridge. Spermatozoa separated by the upper fraction method showed the greatest penetration of oocytes and produced the highest incidence of polyspermy. The glycerol level affected penetration and polyspermy only with spermatozoa separated in a percoll gradient, where the higher level of glycerol increased oocytes penetration and polyspermy. Pronuclei formation was influenced by the separation procedure and by the glycerol concentration in the freezing diluent. The results indicate that frozen boar semen can be used for in vitro fertilization more successfully than fresh semen since penetration by frozen upper fraction spermatozoa was similar to, the degree of polyspermy was lower, and the formation of two pronuclei was greater (P<0.01) than in oocytes exposed to fresh semen.

In a previous study we reported that ovaries from bovine fetuses, which consist mainly of preantral follicles with few antral follicles, are weakly responsive to gonadotropins (FSH and LH). Insulin-like growth factor-I (IGF-I) is known to enhance gonadotropin responsiveness in vitro, but there is a lack of consistent data on the involvement of IGF-I, FSH, and LH during early stages of folliculogenesis in cattle. In the study reported here, we assessed autoradiographically the ontogeny of 125I-gonadotropin and 125I-IGF-I binding activities during preantral and early antral stages in cattle. Follicular growth was initiated around Day 180 of gestation in fetuses. The density of 125I-FSH binding was high in granulosa cells from primary (mean +/- SEM 10.5 +/- 0.7 grains/cell, 0.05-mm diam.) and secondary follicles (10.8 +/- 0.8 to 13.6 +/- 1.2 grains/cell, 0.06-0.15 mm) but increased significantly (p < 0.05) in early antral follicles (18.2 +/- 1.1 grains/cell, 0.16-3.0 mm). Specific 125I-IGF-I binding levels were low in granulosa cells from preantral follicles, averaging 2.5 +/- 0.6-3.1 +/- 0.9 grains/cell. However, after antrum formation, the density of 125I-IGF-I binding increased significantly (p < 0.05) with follicular diameter in granulosa cells and was 5.7 +/- 0.7 and 9.1 +/- 0.6 grains/cell for antral I (0.16-0.5 mm) and antral II (0.6-3.0 mm) follicles, respectively. 125I-FSH and 125I-IGF-I binding densities were low in theca cells from preantral and early antral follicles as well as in the interstitial tissue and granulosa cells from atretic follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

We have recently reported that although specific 125I-FSH receptors are present in granulosa cells from primary and secondary follicles, gonadotropin responsiveness is very low in ovaries from bovine fetuses, which consist mainly of preantral follicles with few early antral follicles. It is well established that a number of polypeptide growth factors show pronounced mitogenic effects on follicular cells. Therefore, we have compared autoradiographically the ontogeny and cellular localization of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) binding activities to assess their possible involvement in the regulation of early follicular growth in fetuses and neonatal calves. Follicular growth was initiated around Day 180 of gestation in fetuses. 125I-bFGF binding values were high in granulosa cells from preantral follicles (mean +/- SEM, 7.8 +/- 1.1-9.8 +/- 0.7 grains/cell, 0.05-0.15-mm diam.) but decreased in early antral follicles (0.16-3.0 mm) to a constant level (5.7 +/- 1.2 grains/cell). Specific 125I-EGF binding values were low in preantral follicles but showed a 2.5- and 5.0-fold increase in both granulosa cells and the theca interna from antral I (0.16-0.5 mm) and antral II follicles (0.6-3.0 mm), respectively. In atretic follicles, 125I-bFGF specific binding values were high (10.4 +/- 0.8 grains/cell), whereas 125I-EGF binding levels were significantly reduced or absent. None of the radioligands tested bound significantly to primordial follicles. There was no age-related difference in any ligand binding to follicles of comparable size. These results provide novel evidence that bFGF, a potent mitogen, is involved in the regulation of granulosa cell function as early as the preantral stage in cattle.(ABSTRACT TRUNCATED AT 250 WORDS)

In cattle, growing follicles are present in fetal ovaries during the last part of gestation. This study examines the extent of changes in basal and hormone-stimulated adenylyl cyclase (AC) activity in ovaries of the bovine fetus when the first follicles begin to grow. The first growing follicles appeared in fetal ovaries around Day 180 and consisted mainly of primary and secondary follicles; few antral follicles were present before Day 220 of gestation. Basal AC activity in ovarian membranes increased simultaneously with the beginning of follicle growth in the fetus (5.8 +/- 0.9 vs. 9.3 +/- 1.3 pmol cAMP/mg protein/min at 130-180 and 180-210 days of gestation, respectively p less than 0.05). During the same time period, there was a significant increase in both the absolute (16.1 +/- 1.2 to 39.9 +/- 1.4 pmol cAMP/mg protein/min) and the relative (2.8 +/- 0.1 to 4.3 +/- 0.3 times the basal level, p less than 0.05) effects of guanosine triphosphate (GTP). After birth, basal and GTP-stimulated AC activities (pmol cAMP/mg protein/min) increased markedly in ovarian membranes of 1-wk-old calves and then decreased with age; the lowest levels were measured in mature cyclic cows. However, the relative effect of GTP (times the basal level) did not show this age-related variation. Prostaglandin E2 (PGE2) stimulation of AC in ovarian membranes from fetuses was high even on Day 120 (2.1 +/- 0.3 times the control level).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of fetal calf serum (FCS), estrus gilt serum (EGS) BSA, dispersed granulosa cells, hemi-sections of follicular wall, and replacement of medium after 24 hours on in vitro maturation and fertilization of porcine oocytes were studied. The results indicate that the use of BSA for 24 or 48 hours inhibited the expansion of cumulus cells and the maturation of oocytes. An incubation of 24 hours culture in FCS followed by a second 24 hours in BSA containing medium did not decrease the rate of maturation but significantly decreased the polyspermy and mean number of spermatozoa penetrated/oocyte. Renewing the medium with or without removal of cumulus cells during the second incubation increased the maturation rate. Removal of cumulus cells decreased the penetrability, the polyspermy rates of the oocyte and the mean number of spermatozoa/oocyte penetrated. The EGS-supplemented medium, dispersed granulosa cells or hemi-sections of follicular wall did not affect the maturation or fertilization rates. In conclusion, BSA, a protein supplement in maturation medium, inhibited cumulus cell expansion and maturation of porcine oocytes. After resumption of meiosis triggered by FCS, BSA did not influence maturation. The FCS-BSA treatment reduced the incidence of polyspermy and the mean number of spermatozoa penetrated/oocyte without decreasing the rate of maturation and fertilization.

Concentrations of the intracellular Ca(2+)-mediator calmodulin (CaM), were measured by radioimmunoassay during heparin-induced capacitation of bull spermatozoa. Heparin reduced sperm CaM concentrations in a dose-dependent manner corresponding with an increase in in-vitro fertilization rates. Such reductions were observed after heparin treatment for 4-6 h, which is in agreement with the length of the capacitation period in bulls and was concomitant with an increase in CaM concentration in the incubation medium, suggesting translocation of CaM from the spermatozoa to the surrounding milieu. This CaM translocation was inhibited partly by the protease inhibitor benzamidine, suggesting a role for the sperm protease in this process.

The male:female ratio of developing bovine embryos produced and allowed to develop in vitro and in vivo was determined retrospectively from the cytogenetic analysis of 804 embyos. The overall male:female ratio of the 307 (38%) embryos that could be sexed was 162 (52.8%):145 (47.2%) and did not differ (P>0.05) from the expected 1:1 ratio. Among premorula stage embryos produced in vivo (n = 66) and in vitro (n = 30), the ratios were 1.2:1 and 0.76:1, respectively. Among morulae and blastocysts produced in vivo (n = 74), produced and cultured in vitro (n = 106, and produced in vitro and cultured in vivo (n = 31), the ratios were 1.11:1, 1.3:1 and 0.94:1, respectively, none of which differed significantly from 1:1. There was no difference (P>0.05) in the number of cells or mitotic index between male and female morulae and blastocyst, respectively.

The purpose of this experiment was to determine the ovulation rate after treatment with human chorionic gonadotropin (hCG) in two groups of gilts characterized by different ovarian morphology: grape-type (GT; n = 11) and honeycomb-type (HT; n = 7). At 170 d of age (d 0), gilts were examined by laparoscopy and ovarian type was determined by the distribution of macroscopic follicles present on the ovarian surface. Five to ten minutes after surgery, each gilt received a single injection (i.m.) of 750 IU of hCG. At d 0, GT ovaries had a greater number of large follicles (greater than or equal to 6 mm) than HT ovaries (10.0 +/- .5 vs 2.6 +/- .3; P less than .05), whereas HT ovaries had more small follicles (1 to 3 mm; HT: 42.3 +/- .8 vs GT: 26.7 +/- .9; P less than .05) and total follicles (HT: 59.4 +/- 2.3 vs GT: 52.2 +/- 1.5; P less than .05), although numbers of medium follicles (4 to 5 mm) were similar (GT: 15.6 +/- .8 vs HT: 14.6 +/- 1.7; P greater than .10). Number of induced corpora lutea (CL) per ovary was greater (P less than .05) in gilts with GT ovaries (10.59 +/- 2.9 CL) than in gilts with HT ovaries (5.21 +/- .66 CL). Total weight of luteal tissue (LT) per ovary and serum progesterone concentrations 8 d after induction of ovulation were greater in GT gilts than in HT gilts (GT: 6.37 +/- 1.09 g vs HT: 3.31 +/- .49 g for LT, P less than .05; GT: 21.08 +/- 4.76 ng/ml vs HT: 13.40 +/- 2.05 ng/ml for progesterone, P less than .07).(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study, electroporation was used to test the ability of spermatozoa to carry foreign DNA into the bovine oocytes. Frozen-thawed bovine spermatozoa (10(7)/ml) were electroporated using six different combinations of voltage (500, 1,000, or 1,500 V) and capacitance (1 or 25 microFarads) in the presence of 1 mg/ml of plasmid pRGH527. The portions of plasmids retained by sperm cells after three washings (stable for ten washings) were 4.3, 5.5, 5.1, 6.0, 6.8, and 5.8% for 1 microFarad, 500, 1,000, and 1,500 V and 25 microFarads, 500, 1,000, and 1,500 V, respectively. Nonelectroporated cells have retained only 1% of plasmids. In the same experiment, electroporated spermatozoa were acrosome reacted by treatment with ionophore A23187 to evaluate the fraction of marked plasmids joined at the acrosomal membrane. The results show that 3.5, 5.0, 4.4, 5.0, 6.3, and 4.4% remain tied to the ionophore-treated sperm. Only 0.7% of plasmid was retained after removal of the acrosome of nonelectroporated cells. Acrosome reaction was not significantly induced by the electrical field (EF) (P less than 0.005). EF decrease motility significantly for greater than 100 V in 0.3 M mannitol (M) and mannitol-TALP (MT) (1/1) media and greater than or equal to 500 V (P less than 0.05) in TALP medium. The retained plasmid rate was compared between TALP medium M and MT media and resulted in a percentage of 1.0, 2.5, 6.5 at 1 microFarads, 100 V, and 0.9, 3.8, and 3.8 at 25 microFarads, 100 V in TALP, MT, and M medium, respectively. Sperm cells electroporated at 1 microFarad, 500 or 1,000 V, 25 microFarad, 500 V or 1,000 in TALP medium hold plasmids in proportion of 5.2, 5.4, 7.4, and 6.0%. Electroporation above 100 V in M and MT killed the cells. In a part of this experiment, spermatozoa electroporated in the presence of radiolabeled plasmids have been treated with DNase I and results revealed that 35, 28, 54, 58, and 3% of marked DNA remains in sperm cells following digestion after electroporation in TALP (1,000 V, 1 and 25 microFarads), M medium (100 V, 1 and 25 microFarads), and control, respectively. Using in vitro matured bovine oocytes, the electroporation conditions were correlated with the fertilization rate (85% for control and 55% for electroporated spermatozoa). Autoradiography of embryos following fertilization indicated the presence of plasmids in the cytoplasm and in the zona pellucida.(ABSTRACT TRUNCATED AT 400 WORDS)

Immature bovine oocytes are forming an heterogeneous population since only one third of all morphologically normal cumulus-oocyte-complexes will result in a developing embryo after in vitro culture. To increase developmental competence, it is essential to improve the quality of the 2/3 not responding to the in vitro maturation context. To achieve this goal we can address the cytoplasmic domain or the nucleus capacity to control future events leading to normal fertilization and development. To influence for example the quantity and quality of RNA present at meiotic resumption (MR), we need access to uncondensed chromatin before that period. Maintenance of the germinal vesicle for a minimum of 24 h can be achieved with protein synthesis or phosphodiesterase (PI) inhibitors or with cAMP accumulation at a level higher that the one required in the cumulus-granulosa portion to stimulate MR. It is suspected that in vivo, the granulosa layer is responsible for the arresting signal since follicular fluid (BFF) alone is not sufficient for a complete effect in vitro. Low concentrations of granulosa cells in vitro (10(6)/ml) stimulate MR but monolayers with their conditioned medium provide inhibitory conditions (33% GV) to oocytes adhering to the monolayers. To achieve a complete meiotic arrest for 24 h (greater than 75% GV), a high concentration of granulosa cells must be used (10(7)/ml). The embedding of cumulus-enclosed oocytes in agar prior to culture with the cells prevents the significant effect of the cells on MR indicating either a need for contact stimulation or a very labile product.(ABSTRACT TRUNCATED AT 250 WORDS)

The oocytes of cattle are not as sensitive as those of laboratory animals to purines, cAMP, or follicular extracts. To study the resumption of meiosis, a method is needed that is capable of inhibiting meiosis completely for a minimum of 24 h. This study was designed to evaluate interrelationships in granulosa-oocyte-cumulus complexes using fresh granulosa cells aspirated from small follicles (1-5 mm) in which the cumulus is normally firmly attached. Selected oocyte-cumulus complexes obtained from a slaughterhouse (n = 2,236) were co-incubated with one of the following: various concentrations of fresh granulosa cells in tissue cultures medium (TCM) 199 or bovine follicular fluid (BFF) either without or after one washing and/or freezing; resuspended granulosa cells previously cultured for 7 days; blood cells; or medium alone. Additionally, oocyte-cumulus complexes were embedded in agar cylinders before incubation with or without cells. The rate of maintenance of intact germinal vesicles (GV) in oocytes after 24 h ranged from 40-77% when 5-100 x 10(6) unwashed cells/ml BFF were used, compared to only 16% in oocytes cultured in BFF alone. The pattern was the same when washed cells were used (30-77%, using 5-100 x 10(6) cells/ml BFF), but they were not as effective as unwashed cells. With TCM-199 and the same five concentrations of cells (5, 10, 25, 50, and 100 x 10(6)/ml), a similar inhibition was obtained with greater than or equal to 25 but not with 5 (3%) or 10 (5%) x 10(6)/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

A 1169 base pair fragment of bovine 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) cDNA was used to quantitate 3 beta-HSD messenger RNA (mRNA) levels in the bovine ovary during the estrous cycle. The content of 3 beta-HSD protein was measured by immunoblot analysis using an antiserum developed in rabbits against human 3 beta-HSD, whereas 3 beta-HSD activity was measured using [3H]pregnenolone, [3H] dehydroepiandrosterone, and [3H]androst-5-ene-3 beta,17 beta-diol as substrates. There was a parallel increase in 3 beta-HSD mRNA, protein content, and enzymatic activity levels from days 1-3 after estrus to maximal values at 50-100% above control on days 8-11 after estrus. Thereafter, all values decreased progressively until days 16-17 before a dramatic fall to 5% or less than maximal values on days 18-20 after estrus. Almost superimposable results of enzymatic activity were obtained with the three substrates, thus suggesting a unique 3 beta-HSD or parallel changes in the activity of multiple 3 beta-HSDs. The above-described changes observed during the luteal phase are almost exclusively due to variations in corpora lutea. In fact, 3 beta-HSD activity in ovarian follicles was approximately 10,000 lower than that measured in corpora lutea. The close correlation observed over a wide range of 3 beta-HSD mRNA, protein content, and activity levels suggests that changes of ovarian 3 beta-HSD activity are controlled at the level of 3 beta-HSD gene expression and/or 3 beta-HSD mRNA stability.

This study was designed to investigate the potential use of in vitro matured, in vitro fertilized bovine zygotes for producing transgenic calves by microinjection of foreign DNA. In Experiment 1, the effect of centrifugation (4 min, 15,000 x g, 20 degrees C) on in vitro derived bovine zygotes was evaluated. In vitro development from 2 to 8 cells was not affected (80 vs 78%) when control zygotes (n = 211) were compared with zygotes treated (n = 210) 18 h post insemination. In Experiment 2, the influence of the centrifugation alone on the developmental potential of embryos was evaluated in rabbit oviducts for 120 h. The percentage of control and treated zygotes that developed to 1, 2 to 8, 8 to 32 and more than 32 cells were 7, 54, 10 and 10% vs 7, 40, 11 and 10%, respectively. In Experiment 3, the effect of pronuclear injection with plasmid containing CRF (corticotropin releasing factor) gene or pOCAT 330 Delta 1 plasmids; 2 microg/ml in Tris 10 mM, EDTA 0.2 mM, 18 to 20 h post insemination was evaluated by in vivo development in the rabbit oviduct. The embryos submitted only to centrifugation and vortexing resulted in a morula-blastocyst (> 32 cells) rate of 25% (n = 226) compared with the injected zygotes of which only 5% (n = 206) achieved the same stage. We conclude that in vitro produced bovine zygotes have a reduced developmental potential following microinjection, and this effect is not due to the centrifugation process.

This study was undertaken to create an in-vitro model using granulosa cell monolayers to replace the role of the follicle in the maturation of bovine oocytes. Cumulus-oocyte complexes were co-incubated with fresh or 7-day granulosa cell cultures (with new or conditioned medium) or with conditioned medium alone, in the presence or absence of IBMX (isobutylmethylxanthine), adenosine or heparin. Progression to the metaphase-II stage was significantly affected by the co-culture of oocytes with bovine granulosa cell monolayers and to a lesser degree when cultured with supernatant alone (conditioned medium). The oocytes attached rapidly to the monolayer, suggesting that the intimate contact between the granulosa cells and the cumulus-oocyte complexes is an important signal for the maintenance of meiotic arrest. Heparin did not prevent maturation itself, but prevented attachment of cumulus-oocyte complexes to monolayers, thereby reducing their inhibitory effect. Adenosine prevented cumulus expansion and reduced maturation and IBMX was an effective inhibitor only in the presence of additional granulosa cells.

This experiment was designed to analyze the effect of adenylate cyclase stimulation on cumulus-enclosed immature oocytes. More than 1400 selected (complete and unexpanded cumulus) oocytes from follicles 1 to 5 mm in diameter were recovered from ovaries obtained at slaughter and cultured for 24 h in TCM-199+10% fetal calf serum (FCS), with or without the adenylate cyclase stimulator, and in the presence or absence of bovine follicular fluid (BFF, 50%), or in complete BFF. In a second set of experiments, oocytes treated for 24 h were further cultured for a second 24 h with TCM-FCS alone. Oocytes were classified as germinal vesicle (G); intermediate (I; up to Metaphase I); matured (M; Anaphase I to Metaphase II); or degenerated (D), and cumulus expansion was evaluated. Products used were sodium fluoride (NaF), isobutylmethylxanthine (IBMX), adenosine (ADE) and forskolin (FK), all known to stimulate accumulation of cAMP in cells without the involvement of a hormone receptor except for adenosine, which acts as a substrate or as an agonist. The results indicate that NaF (0.01 M), IBMX (0.2 mM), FK (0.1 mM) and complete BFF can significantly reduce the proportion of oocytes reaching the mature state. Combination of NaF or FK to BFF (50%) are also effective at the significant level. Cumulus expansion was always limited when meiotic progress was affected or when adenosine was present in the culture media. When oocytes were cultured for a second 24 h in the control media, only NaF had a significant residual effect, but many oocytes were showing degenerative changes after the second incubation period. This method provides a new means to block oocyte nuclear maturation.

The 125I-calmodulin gel overlay procedure was used to evaluate the effect of a heparin treatment on the calmodulin-binding proteins of bull spermatozoa. At concentrations that increase the in vitro fertilization rate of in vitro-matured oocytes, heparin induced a decrease in the binding to calmodulin (CaM) in 3 sperm proteins of 28, 30, and 49 kDa. The binding of these proteins to CaM was higher when Ca2+ was absent from the overlay procedure, and this binding was negatively correlated to the fertilization rate. These results suggest that sperm capacitation is associated with a decrease in the binding of CaM to the 28, 30, and 49 kDa sperm CaM-binding proteins. Implications of such a decrease are discussed.

Bovine follicular oocytes (n = 454), obtained after laparoscopy, were used to study in vitro capacitation, fertilization, and embryo development. Capacitation was accomplished by treating bovine spermatozoa with high ionic strength medium. Maturation, fertilization, and development studies were carried out in Brackett's defined medium or in Ham's F-10. In vitro fertilization rates, ranging from 14% to 55%, were found to be influenced by individual variations among males. Brackett's defined medium was found to be superior to Ham's F-10 for oocyte maturation, fertilization, and growth, these media giving cleavage rates of 60% and 32%, respectively. Oocytes with expanded cumuli at the time of recovery cleaved at a rate of 43%, which is significantly different from oocytes recovered without granulosa cells (22%) or oocytes with compact cumuli and corona cells (5%). The in vitro development pattern of the in vitro-fertilized embryos was found to be similar to that observed in vivo. Embryos were observed at the 2-cell stage 44.5 +/- 6.3 h after in vitro insemination, 4-cell after 59.0 +/- 9.4 h, 8-cell after 74.8 +/- 12.7 h, and 16-cell after 96.2 +/- 13.9 h (observations at 12-h intervals). The procedures described here resulted in cleavage rates of up to 60% using follicular oocytes embedded in expanded cumuli cells and semen samples from selected males.

Bovine cumulus-oocyte complexes (COC) (n = 4230) were used in this study to assess the effects of culture method, hormonal supplementation, and cumulus cell concentration on maturation, fertilization and development of resulting embryos. Five treatments were evaluated. 1) 10 COC/50-microliter drops under oil in TCM 199 supplemented with 10% heat-treated fetal calf serum, follicle-stimulating hormone (0.5 microgram/ml), luteinizing hormone (5 micrograms/ml), and estradiol-17 beta (1 microgram/ml); 2) as in 1 without hormones; 3) as in 1 but in 3 ml TCM-199 in petri dishes without paraffin oil; 4) as in 2 but only 1 COC/50-microliter drop; and 5) as in 1 but with denuded oocytes. After 24 h maturation, the frequencies of oocytes reaching metaphase II were 98, 84, 92, 93, and 87%, respectively, for the five treatments. In the same order, percentages of normal fertilization were 73, 70, 62, 81, and 62%, and the frequencies of embryos containing two or more blastomeres at 65 h postinsemination were 69, 82, 66, 51, and 43%. The same five treatments were used in a second study in which 3,199 oocytes were fertilized, allowed to cleave in vitro to the 2- to 3-cell stage (42 h postinsemination), and transferred to oviducts of sheep (one treatment/oviduct) for 4 days. The frequencies of morulae or blastocytes obtained were 28, 18, 23, 24, and 11% for the five treatments, respectively. After nonsurgical transfer to bovine recipients (n = 8) using fresh or frozen-thawed embryos, three pregnancies past 50 days were obtained. Only one went to term with the birth of a live heifer calf.

Bovine follicular oocytes (N = 5991) were exposed to an analog of cyclic adenosine 3',5'-monophosphate (cAMP), dibutyryl cyclic AMP (db-cAMP) (2.5, 5, and 10 mM), the phosphodiesterase inhibitor isobutyl methyl xanthine (IBMX, 0.2 mM), or the purine, hypoxanthine (0.5, 1.0, 2.0 mM), and the nucleoside, adenosine (0.05, 0.1, 0.2 mM), for 6 or 21 h to assess their effects on oocyte nuclear maturation. Potential effects of bovine follicular fluid (BFF) were also evaluated after different preculture washing procedures. In a separate experiment, denuded oocytes were used to study the effect of cumulus removal on meiotic inhibition. Db-cAMP decreased the frequency of germinal vesicle breakdown (GVBD) at 6 h (88% for control and 51%, 45%, and 32% for 2.5, 5, and 10 mM concentrations, respectively). IBMX had a comparable effect with only 41% of the oocytes resuming meiosis. Hypoxanthine and adenosine alone or in combinations also decreased the number of oocytes undergoing GVBD at 6 h. Only 22% GVBD occurred when the combined highest concentration of both substances was used compared to 88% in controls. If oocytes were incubated in 50% BFF after a wash in control medium during processing, 56% would resume meiosis at 6 h vs. 35% if the washing procedure included inhibitors (db-cAMP + IBMX). Total BFF (100%) during washing and maturation prevented 72% of the oocytes from resuming meiosis. Db-cAMP and IMBX combined or BFF also inhibited meiotic resumption of denuded oocytes. At 21 h, the inhibitory effects were less pronounced, with most oocytes only delayed in completing the first reduction division.(ABSTRACT TRUNCATED AT 250 WORDS)

A 125I-labelled calmodulin gel overlay procedure in the presence and the absence of Ca2+ was used to evaluate bull spermatozoa calmodulin-binding proteins. Frozen spermatozoa were thawed, washed and incubated for 6 h before being processed for SDS polyacrylamide gel electrophoresis and the 125I-labelled calmodulin gel overlay procedure. In non-incubated spermatozoa, up to 14 binding proteins were detected. Some exhibited greater calmodulin binding in the presence of Ca2+ while others exhibited greater binding when Ca2+ was absent. When heparin (2 micrograms/ml) was present in the incubation medium, a decrease in the calmodulin binding to the proteins of Mr 28,000 and 30,000 was detected in the presence of Ca2+ and EGTA. This effect of heparin was time- and dose-dependent and was increased by the presence of the acrosin inhibitor benzamidine. Sperm capacitation could thus be related to a decrease in the binding of calmodulin to these proteins.

In cows, protein synthesis is required for germinal vesicle breakdown (GVBD). This study examines more closely the need for protein synthesis and the nuclear changes in the bovine oocyte during 24 h of culture. Bovine oocytes with compact and complete cumulus were washed and incubated in groups of 10 for up to 24 h in 50-microliters drops of TCM-199 supplemented with follicle-stimulating hormone (NIAMADD, 0.5 micrograms/ml), luteinizing hormone (LH) NIAMADD, 5 micrograms/ml), estradiol-17 beta (1 microgram/ml), pyruvate (20 microM), and 10% heat-treated fetal calf serum. Medium was overlaid with paraffin oil. Oocytes (n = 891) were fixed at the end of each 3-h interval from 0 to 24 h of culture, or at 24 h after addition of cycloheximide (10 micrograms/ml at 10 different times during maturation (0, 1, 2, 3, 6, 9, 12, 15, 18, 21 h; n = 175). At each time point, the chromosomal status of oocytes was evaluated, frequencies were computed, and the time spent on each step was determined. The germinal vesicle (GV) was present from 0 to 6.6 h, GVBD at 6.6 to 8.0 h, chromatin condensation at 8.0 to 10.3 h, metaphase I at 10.3 to 15.4 h, anaphase I at 15.4 to 16.6, telophase I at 16.6 to 18.0 h, and metaphase II at 18.0 to 24 h. Cycloheximide blocked oocyte maturation at GVBD, if added from 0 to 3 h; at chromatin condensation, if present from 6 to 24 h; and at metaphase I, when present from 9 to 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Cow embryos fertilized in vitro (1-8-cell) (n = 113) were transferred surgically to the ligated oviduct of pseudopregnant female rabbits (31 +/- 4 h after 75 i.u. hCG). Rabbits were killed 99 +/- 5 h later and 67 embryos were recovered: 45 (67%) had cleaved at least once, 15 had reached the morula stage and 2 blastocysts were obtained. Transfer of cleaved embryos (2-8-cell) led to a high recovery rate (84%) compared to 39% for one-cell embryos. Of the embryos incubated for more than 99 h in the rabbit oviduct, 41% were at the morula stage. Embryos incubated in vivo (n = 21) (8-cell to blastocyst) were transferred to the uterus of 14 synchronized recipient heifers by a surgical (N = 5) or a non-surgical (N = 9) procedure: 6 pregnancies resulted (4 from the non-surgical procedures). In addition, 27 (2-8-cell) cow embryos developed in vitro were transferred to the oviduct of synchronized heifers by a paralumbar surgical approach on a standing animal, but no pregnancies resulted. It is therefore concluded that (1) the rabbit oviduct can be used to obtain cow eggs at an embryonic stage sufficiently advanced to permit transfer to the uterus of a synchronized recipient; and (2) the pregnancy rate after in-vitro fertilization and incubation in the rabbit oviduct are similar to results with fertilization in vivo.

Three experiments were performed to evaluate the potential for parthenogenetic activation of bovine oocytes in in vitro fertilization systems and to determine the chromosome complement of the resulting parthenogenotes. In the first experiment, immature oocytes from slaughtered cattles were matured in vitro in Defined Medium (DM) for 24 h to simulate in vitro fertilization conditions. Subsequently, a portion was fixed, and the remainder were transferred to rabbit oviducts. Oocytes were then cultured for 6-8 h or for 24 h with Colcemid present during the last 6 to 8 h and fixed on slides and examined. In the second experiment, mature oocytes were collected from the preovulatory follicles, and the oocytes were subjected to the same culture as in experiment I. In the third experiment, oocytes were treated as in experiment II, except that instead of transfer to rabbit oviducts, they were cultured an additional 48 h in vitro. In experiment I, 131 oocytes were fixed after culture in DM. Of the 79 oocytes analyzed in the pre-rabbit group, 71 (90%) were at the second meiotic metaphase (MII), and 8 (10%) were at pre-MII stage; none were activated. After transfer to rabbits, 291 were fixed. Of these, 80 were analyzed; 37 (46.3%) were MII, 7 (8.6%) were pre-MII, and 36 (45%) were activated. Of the 36 activated oocytes, 26 (72.2%) were haploid, 4 (11.1%) were diploid, 1 (12.8%) was tetraploid, and 5 (13.8%) were in the process of endoreduplication. In experiment II, 51 oocytes were fixed after culture in DM of which 36 (70.6%) could be analyzed; 30 (83.3%) were MII, and 6 (16.7%) were pre-MII. After culture in the rabbit, 68 were fixed of which 27 (39.7%) could be analyzed. Of these 27, 20 (74.1%) were MII, and 7 (25.9%) were activated; 6 were haploid, and 1 was endoreduplicating. In experiment III, 30 oocytes were fixed at the end of the culture period; only 10 could be analyzed of which 8 (80%) were MII and 2 (20%) were pre-MII. In all, 46% of in vitro and 26% of in vivo matured oocytes were activated, based on chromosomal analysis. Of those activated, the majority (74.4%) were haploid, suggesting that activation occurs at or after completion of MII. Endoreduplication appears to be one of the mechanisms leading to the formation of diploid and polyploid parthenogenotes.

The infertility of many cows could be treated by in vitro fertilisation. In the present study laparoscopy was utilised to recover the in vivo matured oocytes from the ovary of a standing donor. After the capacitation of fresh semen with high ionic strength medium and in vitro fertilisation, a rabbit oviduct was employed as an incubator for four to five days, in order to obtain sufficiently aged embryos to be transferred to the uterus of a recipient. Using surgical or non-surgical transfer six calves were obtained.