Induction and synchronization of ovulations of nulliparous and multiparous sows with an injection of gonadotropin-releasing hormone agonist (Receptal).

The objective of this study was to determine if administration of a set dose (10 microg) of a gonadotropin-releasing hormone agonist, buserelin (Receptal; Rc), at set times after altrenogest (Regumate; RU) treatment or after weaning was able to induce and synchronize ovulation in female swine (gilts and sows). The pubertal (n=187) gilts were allocated to four groups, all synchronized with RU. Group 1 (RU) was inseminated twice at detected estrus, Group 2 (RU+Rc120) and Group 4 (RU+Rc104) received 10 microg Rc at 120 or 104 h after the end of RU treatment, respectively, and Group 3 (RU+eCG+Rc104) was treated with 800 IU equine chorionic gonadotropin (eCG) at 24h and Rc 104 h after the end of RU treatment, respectively. Gilts were inseminated twice at predetermined times, namely 144 and 168 h (Group 2), 128 and 144 h (Group 3), and 144 and 152 h (Group 4) after the end of RU treatment, respectively. Pregnant gilts were slaughtered at 30 d. Administration of Rc 104 h after the end of RU feeding synchronized ovulation over a 24-h time window in 97.9% and 100% of the gilts of Groups 3 and 4, respectively, whereas Rc administration at 120 h (Group 2) only successfully synchronized 88.9% of the gilts over 24h. Ovulation rates of gilts of Groups 2 and 4 were similar to that of the control group. Pregnancy rates were numerically higher in Groups 2 and 3 (92% and 96%, respectively) compared with those of Groups 1 and 4 (84% and 81%, respectively). Combination of eCG with Rc administration at 104 h (Group 3) increased ovulation rate (+4 CL) but decreased embryo survival to 62% at Day 30. The weaned sow experiment involved 61 sows of a range of parities (2.7+/-0.9), allocated to two control groups (Control 104 group and Control 94 group) and two treated groups (Rc104 group and Rc94 group), which received 10 microg Rc at 104 and 94 h after weaning, respectively. The females were inseminated at detected estrus. All pregnant sows farrowed. After treatment with Rc 94 h after weaning, 100% of sows ovulated over a 24-h time window versus only 68.7% of controls. Farrowing rate and litter size of the sows treated with Rc at that time were unaffected compared with that of control sows. In contrast, Rc administration at 104 h after weaning may have been too late; only 66.7% of the treated sows ovulated during a 24-h period. This proportion was numerically lower but not significantly different than that for control sows. Farrowing rate and litter size of treated sows were not significantly different than that of controls. Administration of Rc at the dose and times selected in this study tightened synchrony of ovulation in gilts and in sows after weaning. It remains to be established if such a synchrony is suitable to obtain good fertility after a single artificial insemination at a predetermined time.

Pig xenografts to the immunocompetent rat brain: Survival rates using distinct neurotoxic lesions in the nigrostriatal pathway and two rat strains.

Porcine foetal neurons for xenotransplantation in Parkinson's disease (PD) is an alternative source to human fetuses. One of the obstacles facing brain xenotransplantation is the existence of an immune response, which prevents long-term graft survival. Experimental results concerning the survival time of porcine foetal neurons implanted into the brain of immunocompetent rats have been quite different from one study to another, suggesting an effect on graft survival of uncontrolled experimental parameters. To identify such parameters, we have first analyzed the survival of porcine foetal nigral neurons at 5 and 10 weeks after implantation into the striatum of immunocompetent rats having different types of brain lesion affecting cells (quinolinic acid) or projections to the striatum (MPP+, 6-OHDA). In a second experiment, graft survival was analyzed in two strains of recipient rats (female Sprague-Dawley and male Lewis rats) in conditions of ipsilateral dopaminergic denervation using 6-OHDA. The characteristics of surviving grafts were assessed by measuring the graft volume, the number of TH+ neurons, the size of TH+ neurons soma, and CD5+ cell infiltration. Long-term survival (> or = 10 weeks) of porcine neurons could be observed in all experimental models. However, there was no significant difference in graft survival rates and characteristics of the surviving grafts between the lesioned groups, or between Sprague-Dawley and Lewis rats. Altogether, results were highly variable within groups of grafts exposed to similar experimental procedures at both 5 and 10 weeks post-grafting. We conclude that the distinct neurotoxins and host rat strains used in our experimental design are not major factors influencing the rejection time-course of primary neural xenografts.

Effect of sperm survival and CTC staining pattern on in vitro fertilization of porcine oocytes.

Polyspermy in pig oocytes fertilized in vitro remains unacceptably high. In this study, we evaluated the effects of gamete coincubation time, and determined if the proportion of capacitated spermatozoa would be predictive of the fertilizing ability of frozen-thawed semen in vitro. Cumulus-oocyte complexes were collected from slaughterhouse prepubertal gilt ovaries and matured in vitro for 44 h in TCM199, with EGF, FSH, cysteamine and follicular fluid. Fertilization was induced with 2 x 10(5) frozen-thawed spermatozoa/ml in TBM. Penetration of oocytes as well as polyspermic fertilization occurred 2 h after insemination. A strong correlation between penetration and polyspermic fertilization rates has been demonstrated, but there was no correlation between the proportion of capacitated spermatozoa, as assessed by chlortetracycline staining, at the time of insemination and fertilization rates. We also compared the results of IVF in three IVF media: TBM, m199 and TALP. Penetration and polyspermy were very different in these three media: 71 +/- 19% and 25 +/- 13% in TBM, 37 +/- 11% and 6 +/- 2% in m199, 10 +/- 2% and 0% in TALP, respectively. Nevertheless, survival of spermatozoa or modifications of the capacitation status were not different in these media after 6 h incubation. We concluded that survival and capacitation characteristics of the semen used for IVF could not be predictive of the IVF results. It seems necessary to act at the oocyte level to control both variability between replicates and the incidence of polyspermy. Improving the spermatozoa penetration blocking system of the oocytes and reducing the number of sperm-binding sites on the zona pellucida (ZP) are our further objectives.

Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida.

In swine, five to six days post-insemination, morulae and blastocysts are collected together after uterine flushing. The purpose of this study was to vitrify zona pellucida-intact morulae with Open Pulled Straw (OPS) technology and obtain piglets after transfer. Morulae (200) were vitrified after a two-step equilibration in ethylene glycol, dimethyl sulfoxide and sucrose in Hepes-buffered TCM199 + 20% NBCS medium (TCM). 2-6 morulae were loaded into OPS and plunged into liquid nitrogen. At embryo warming, a three-step dilution with decreasing concentrations of sucrose was applied. In each of 10 recipients, 20 morulae were transferred surgically. Day 25, gestation rate and the farrowing rate were 80% and 70%, respectively. The pregnant recipients farrowed from 1 to 8 piglets and the survival of total transferred embryos was 13%. Although survival rates are still compromised, OPS technology is therefore appropriate to cryopreserve porcine morulae with intact zona pellucida.

Effects of cell cycle dependent kinases inhibitor on nuclear and cytoplasmic maturation of porcine oocytes.

The aims of this study were to assess the effectiveness of roscovitine, a potent inhibitor of cell cyclin kinases, to prevent meiotic resumption in porcine oocytes, and to test the subsequent fertilisability and developmental competence of these oocytes. Roscovitine blocked porcine oocytes at the GV stage during 22-44 hr of culture. This effect was dose-dependent, and a concentration of 25 microM was sufficient to prevent meiotic resumption in 92+/-5% of the oocytes after 22 hr in the presence of EGF and FSH. Cumulus expansion was also inhibited under these conditions. The histone H1 kinase activity in oocytes was inhibited in a dose-dependent way, and maintained at a basal level with 25 microM of roscovitine. Synthesis of proteins of 29, 47 and 79 kDa, normally synthesized during maturation, was inhibited too. All these effects were fully reversible. However, the kinetics of maturation were accelerated after roscovitine removal, and the acceleration was more pronounced after 44 hr of inhibition than after 22 hr. Fertilization of oocytes blocked for 22 hr before a 44 hr maturation was decreased compared to control, but was not different from that of oocytes matured for 66 hr. The developmental competence was decreased for the oocytes cultured for 66 hr, including or not an inhibition period, but it was less reduced for oocytes maintained under inhibition for 22 hr. Roscovitine may thus protect oocytes against the aging mechanisms responsible for developmental competence loss, but not against loss of fertilisability. In conclusion, roscovitine provides a useful tool to study the morphological and biochemical basis of porcine oocyte terminal differentiation.

Meiotic and developmental competence of prepubertal and adult swine oocytes.

The present study was conducted to compare meiotic and cytoplasmic competence of prepubertal and adult porcine oocytes, and the effects of EGF (0 to 100 ng/mL), FSH (0 to 400 ng/mL) and prepubertal pFF (0 to 10%) on nuclear maturation. Prepubertal oocytes were less responsive to FSH and pFF than were adult oocytes in terms of stimulation of nuclear maturation. The best nuclear maturation rates for prepubertal oocytes were obtained with 10 ng/mL EGF and 400 ng/mL FSH, whereas for adult oocytes no additional effect of EGF was seen in the presence of 400 ng/mL FSH. Supplementation with pFF had no additional effect on MII yield over that obtained with EGF plus FSH. After maturation in the presence of EGF, FSH and cysteamine, fertilization rates were not different between adult and prepubertal oocytes, but polyspermy was more frequent in prepubertal oocytes (31 +/- 17% vs. 17 +/- 7% in prepubertal and adult oocytes, respectively, P < 0.05). The addition of pFF to maturation medium decreased oocyte fertilization of adult oocytes and polyspermic fertilization in prepubertal oocytes. Blastocyst yield and developmental competence were significantly reduced in prepubertal oocytes compared to adult oocytes. The mean cell numbers in blastocysts cultured for 7 days ranged from 61 to 74, and did not differ among groups. Finally, the viability of the 2- to 4-cell embryos and blastocysts produced was assessed by embryo transfer experiments. One offspring was obtained after transfer of 2- to 4-cell embryos, and one after transfer of in vitro-produced blastocysts. In conclusion, although prepubertal gilt oocytes appeared less meiotically and developmentally competent than their adult counterparts, they can be used to produce blastocysts able to develop to term.

Relationship between peri-oestrus progesterone levels and time of ovulation by echography in pigs and influence of the interval between ovulation and artificial insemination (AI) on litter size.

Two methods for the determination of ovulation were compared to one ultrasonography performed 5 times a day. Time of ovulation by echography was 40 +/- 5.8 h (mean +/- SD) after the onset of oestrus. Preovulatory LH rise (two blood samples per day) began near the onset of oestrus but, in our conditions, this parameter could not be used to predict ovulation. The basal level of progesterone (two blood samples per day) was determined with a non-linear model, the timing when progesterone rose more than one SD (0.3 ng x mL(-1)) coincided with the timing of ovulation determined by echography (R2 = 0.98). This method was efficient and was used in a field trial to measure the consequences of the variability of the interval between Al and ovulation on litter size. The interval between Al and ovulation had an effect on litter size; litter size decreased by one piglet when this interval increased by 10h.

Piglets born after vitrification of embryos using the open pulled straw method.

Morulae and unhatched blastocysts from Large White hyperprolific (LWh) and Meishan (MS) gilts were selected to test an ultrarapid open pulled straw (OPS) vitrification method with two media. The viability of vitrified/warmed embryos was estimated by the percentage of embryos that developed to the hatched blastocyst stage in vitro or by birth after transfer. In Experiment 1, two cryoprotectant dilution media were compared for cryopreservation of MS and LWh blastocysts: TCM was a standard Hepes-buffered TCM199 + 20% NBCS medium and PBS was a PBS + 20% NBCS medium. After a two-step equilibration in ethylene glycol, dimethyl sulfoxide, and sucrose, 2-5 blastocysts were loaded into OPS and plunged into liquid nitrogen. Embryos were warmed; a four-step dilution with decreasing concentrations of sucrose was applied. In PBS, LWh blastocysts (27%) had a lower viability in vitro than MS blastocysts (67%; P = 0.001). In TCM, no significant difference was observed between genotypes (41% for LWh and 43% for MS blastocysts) and both viability rates were lower than that of the control groups. In Experiment 2, morula-stage LWh and MS embryos were vitrified and warmed using PBS. The viability rate was low and did not differ between LWh (11%) and MS (14%). In Experiment 3, 200 MS and 200 LWh blastocysts were vitrified/warmed as described in Experiment 1 (PBS). In each of 20 MS recipients, 20 embryos were transferred. The farrowing rate was 55% and recipients farrowed four and five piglets (median) for MS and LWh blastocysts, respectively. The OPS method is therefore appropriate for cryopreservation of unhatched porcine blastocysts.

Effect of growth factors, EGF and IGF-I, and estradiol on in vitro maturation of sheep oocytes.

The objective of these experiments was to determine the effect of exogenous addition of insulin-like growth factor-I (IGF-I, 100 ng/mL), epidermal growth factor (EGF, 10 ng/mL) and estradiol (E2, 100 ng/mL) to the maturation medium of sheep oocytes on their subsequent development in vitro. Addition of IGF-I to the maturation medium did not improve nuclear or cytoplasmic maturation of sheep oocytes at the concentration tested. However, EGF improved significantly the resumption of meiosis (84% oocytes in metaphase II stage after IVM vs. 59% in medium alone). Cleavage rate and blastocyst development rates were improved (P<0.01) after addition of EGF (60% and 29%, respectively), as compared with maturation in TCM 199 alone (39% and 19%, respectively), but remained lower than rates observed after maturation in complete medium containing follicular fluid (FF, 10%) and FSH (81% and 35%, respectively). No additive effect of EGF over FSH was observed during these experiments. Addition of FF to FSH containing maturation medium improved significantly both cleavage (P<0.001) and blastocyst rates (P<0.05). Addition of E2 to the IVM medium is not required when medium already contains FF. However, in defined conditions supplementation of maturation medium with E2 had a positive effect. These results suggest that EGF, FSH and E2 may play an important role in the nuclear and cytoplasmic maturation of sheep oocytes in vitro.

Intrauterine infusion of high doses of pig trophoblast interferons has no antiluteolytic effect in cyclic gilts.

In the pig species, the preimplanting trophoblast is known to synthesize and secrete high amounts of interferon during early development. Previous experiments in cyclic gilts using total conceptus secretory proteins suggested that porcine trophoblastic interferons, unlike those of ruminants, exert no effect on the luteal cycle. In the present experiment, cyclic Meishan gilts were divided into two groups, cannulated on both uterine horns, and given daily injections of either a placebo or increasing doses of a mixture of recombinant interferon-gamma and interferon-delta, on Days 11-14 of the estrous cycle. In treated gilts, the injected doses were much higher than those previously found in uterine perfusates from pregnant gilts. However, no significant differences could be found between the control (n = 4) and the treated (n = 5) group concerning the days of the estrous cycle for mid-decrease of progesterone (control: Day 14.5+/-0.57 [mean+/-SD]; treated: Day 15+/-1.25), the day of estrus (control: Day 19+/-0.96; treated: Day 19.6+/-0.55), and the subsequent ovulation rate (control: 14+/-2.2 corpora lutea; treated: 13.1+/-1.1 corpora lutea). These data confirm that pig trophoblastic interferons, unlike those of ruminants, do not themselves exert an antiluteolytic effect. A possible synergistic effect of embryonic estrogens on the luteal functions of nonpregnant sows remains to be determined.

Growth kinetics of the granulosa cell population in ovarian follicles: an approach by mathematical modelling.

This paper describes, from a mathematical viewpoint, the cellular changes in the granulosa of ovarian follicles during their terminal development. A dynamic model takes into account the processes of (1) cell division, (2) exit from the cell cycle towards differentiation, and (3) apoptotic cell death. Proliferative cells leave the cycle in an irreversible way. The risk of entering apoptosis applies to non-cycling cells. Changes in the cell numbers and in the growth fraction are derived from differential equations. The transitions between the different cell states are ruled by time-dependent rates. Numerical applications of the model concern ovulating and degenerating ovarian follicles in the ewe. The main feature of the ovulating case is the progressive exhaustion of the proliferating compartment for the benefit of the non-cycling cells. From an initial mainly proliferative state the granulosa progressively switches to a highly differentiated state, so that the growth fraction continuously decreases. In the atretic cases, the pattern of changes in the total viable cell number is influenced by the follicular age at the onset of the apoptotic process and by the intensity of the cell death rate. As apoptosis affects the non-cycling cells, the growth fraction is no longer strictly decreasing. The sensitivity of the model to the parameters is studied in a more general framework than the granulosa cell population.

Correlations between chemical parameters, mitogenic activity and embryotrophic activity of bovine oviduct-conditioned medium.

To establish parameters predicting the quality of bovine oviduct epithelial cell-conditioned media, we compared media conditioned by oviduct cells from cows at Day 2 (n = 3) and Day 15 (n = 3) of the estrous cycle. In addition, we tested the influence of time of conditioning. Media were evaluated for their embryotrophic activity using a cumulus cell co-culture system as a control. The same media were tested for their mitogenic activity on NIH 3T3 cells and for chemical parameters, including total protein, and de novo synthesized protein as well as for concentrations of glucose, lactate and ammonium. Analysis of variance did not reveal a significant effect by stage of the estrous cycle on the embryotrophic activity of conditioned media. However, there was a significant effect by time of conditioning on the proportion of 5- to 8-cell embryos (P < 0.01) and of blastocysts and hatched blastocysts (P < 0.05). None of the conditioned media (19 to 31% blastocysts) was superior to the cumulus cell co-culture system (32% blastocysts). In the conditioned media, the proportion of 5- to 8-cell embryos correlated positively with mitogenic activity on 3T3 cells (r = 0.64; P < 0.05), whereas the proportion of blastocysts was not significantly correlated with this parameter. In summary, our results provide evidence for an effect of time of conditioning on embryotrophic activity of oviduct epithelial cell-conditioned media. The fact that mitogens for NIH 3T3 cells affect the proportion of 5- to 8-cell embryos but not of blastocysts suggests different culture requirements for early and late preimplantation stage development of bovine embryos.

Follicular growth and maturation in hyperprolific and large white sows.

The mechanisms whereby hyperprolific sows achieve their increased ovulation rate (+5 oocytes on average) compared with normal Large White sows were explored in this study. The following specific questions were asked. Is increased ovulation rate related to 1) increased follicular populations within the ovaries or 2) alterations in terminal follicular growth and maturation? The population of antral follicles in six sows of each genotype was studied using histological techniques on ovaries obtained at the preovulatory stage. No difference between the total number of antral follicles, number of healthy or atretic follicles in specific size classes, and in size of the preovulatory follicles could be detected. The number of granulosa cells contained in preovulatory follicles was also similar between genotypes. Terminal follicular growth and maturation were studied in 15 sows of each genotype killed at d 1, 3, or 5 (n = 5.d-1.genotype-1) after the end of Regumate administration (d 0). Small (< or = 3.5 mm) follicles were counted, and follicles > 3.5 mm were dissected, measured, and incubated in vitro. Steroid concentrations (estradiol and testosterone) in culture medium were then measured. The two genotypes differed in the patterns of growth of their ovulatory follicles, because at d 3 all ovulatory follicles were obvious in Large White sows. In contrast, between d 3 and 5, seven additional ovulatory follicles grew in hyperprolific sows. Differences in follicular maturation between genotypes were also detected. Whereas testosterone concentrations in culture medium were similar in the two genotypes, estradiol concentrations were markedly (P < .01) increased in hyperprolific follicles. This indicates that these follicles may have an increased aromatizing ability. How this generates the altered pattern of follicular growth and the increased ovulation rate of hyperprolific sows remains to be established.

Effects of oxygen, CO2/pH and medium on the in vitro development of individually cultured porcine one- and two-cell embryos.

The gas atmosphere and medium composition are critical factors in the in vitro development of one- and two-cell embryos of several species. The present study evaluated the effect of different O2/CO2 concentrations (2/5, 2/10, 5/2.5, 5/5, 5/10, 10/10 and 21/5) on pig one- and two-cell embryo development. The embryos were individually cultured, for 6 days at 39 degrees C in a medium rich in bicarbonate and glutamine and containing pyruvate and lactate but lacking glucose. When the CO2 levels increased from 2.5% to 10%, the pH of the medium decreased from 8.2 to 7.5 and the development of the embryos was affected, but this depended mainly on the O2 levels. Pig embryo development was inhibited by 2 and 21% O2 levels. The optimum level for pig embryo development was 5% O2 and 5% CO2, whatever the criteria used to evaluate embryo development. At these optimal levels, the mean number of cells per embryo was 26 +/- 1.7 (ls mean +/- SE), and 50% of the one- and two-cell embryos developed to blastocysts. The substitution of 0.5% bovine serum albumin (BSA) in the medium by 0.3% polyvinyl-pyrrolidone (PVP) significantly decreased the one- and two-cell embryo development. When the calcium and chloride contents of the medium with PVP were reduced, however, the embryo development was similar to that observed in the medium containing BSA. Pig embryo development in vitro was found to be optimal under an atmosphere of 5% O2 and 5% CO2 and PVP could replace BSA as the high molecular weight supplement.

Failure to produce transgenic offspring by intra-tubal insemination of gilts with DNA-treated sperm.

The reproducibility of the use of sperm cells as vectors of foreign DNA in the genome of pigs was verified in the present study and the effectiveness of four different procedures for sperm treatment was assessed. For each gilt, approximately 6 x 10(6) ejaculated boar spermatozoa were incubated for 30 min in 1 mL TALP medium containing 3 micrograms of linearized pSV2CAT plasmid DNA. Before incubation, spermatozoa were treated in four experimental groups: (1) cells were stored at 16 degrees C for 24 h and then washed three times in TALP; (2) cells from the fresh, undiluted sperm-rich fraction of an ejaculate were used immediately after collection, following the same procedure as (1); (3) cells were treated as in (2) with an extra wash; and (4) incubation with DNA was performed in TALP medium supplemented with 0.5 mg mL(-1) poly-L-lysine hydrobromide. As determined by immunolocalization, plasmid DNA molecules were found to be associated with 12-17.1% spermatozoa, depending on sperm treatment. Of 35 inseminated gilts, 20 gave birth to a total of 126 piglets. None of the piglets showed sign of exogenous DNA incorporation in any of the tissues tested, as assessed by the polymerase chain reaction and Southern blot. The potential of modifying the pig genome through "transformed' spermatozoa was not confirmed by these experiments.

Analysis of [3H]estradiol-17beta metabolites in calf perirenal fat.

Residues of estradiol-17 beta (E2 beta) in the kidney fat of one milk-fed calf were studies using radiometric methodologies. A three-month old Friesian male veal calf was injected intramuscularly daily for 3 d with 333 mg of [6,7(n)-3H]-E2 beta (specific activity: 7.55 MBq mmol-1) dissolved in 2 ml of propylene glycol and slaughtered 3 h after the last administration. Total estrogens were about 280 ng g-1 in perirenal fat. After a de-lipidation step, the relatively polar metabolites that were extractable with dichloromethane represented the main fraction of the metabolites, which accounts for almost 50% of the total radioactivity of the tissue, of which E2 beta was the major metabolite (19.7%) and E1 and E2 alpha represented only 7.7 and 3.2%, respectively. Conjugated estrogens accounted for only 15.2% of the total estrogen content. Non-polar estrogens (about 25% of total estrogens) were removed specifically with isooctane during the de-lipidation step and were further purified on silica and alumina columns before being chromatographed by normal-phase HPLC. The radioactive metabolites were eluted as estrogen-17 esters. The HPLC analysis of the estrogens released following hydrolysis of the esters indicated that E2 beta was the main estrogen acylated by long-chain fatty acids in the fraction of lipoidal estrogens. The presence of such a class of estrogens in fat could be of interest for the detection of estrogens a considerable time after estradiol administration.

Localization and quantification of insulin-like growth factor-I (IGF-I) and IGF-II/mannose-6-phosphate (IGF-II/M6P) receptors in pig embryos during early pregnancy.

To assess a potential role of insulin-like growth factor-I (IGF-I) and -II (IGF-II) in early embryonic development, the presence of their receptors was investigated by both immunohistochemistry and autoradiography experiments on whole embryos at Days 4 and 6 of pregnancy, on embryo sections at Days 8 and 10, and on placenta at Day 20 of pregnancy. Immunohistochemistry experiments were performed by using specific polyclonal antibodies raised against human IGF-I and IGF-II/mannose-6-phosphate (IGF-II/M6P) receptors. By autoradiography, specificity of [125I]-IGF-I and [125I]-IGF-II binding on embryonic cells was assessed by competition with unlabeled IGF-I and IGF-II, and quantification of the autoradiographic signal was performed by image analysis. The presence of IGF-I receptors on porcine trophectoderm cells was detected neither by immunohistochemistry nor by autoradioradiography on whole embryos or embryo sections. IGF-I receptors were present in the placenta at Day 20 of pregnancy, but only on endometrial cells. In contrast, IGF-II/M6P receptors were detected on porcine trophectoderm cells by both immunohistochemistry and autoradiography on whole embryos, on embryo sections at Day 8 and Day 10 of pregnancy, and on fetal and maternal compartments of the placenta at Day 20. The number of IGF-II/M6P receptors on trophectoderm cells was greatly heterogeneous between embryos within the same litters. There was no relationship between the number of IGF-II/M6P receptors on trophectoderm cells and the age or size of embryos between Day 8 and Day 10 of pregnancy. The involvement of the IGF-II/M6P receptor in early embryonic development in the pig remains to be determined.

Presence of leukaemia inhibitory factor and interleukin 6 in porcine uterine secretions prior to conceptus attachment.

Leukaemia inhibitory factor (LIF) plays an important role in embryo development and implantation. We detected peak LIF activity in porcine uterine luminal fluids (ULF) at day 12 of gestation and during day 7 and 13 of the oestrous cycle. A radio-receptor competition assay showed the presence of a molecule in ULF specifically binding to human LIF receptor (LIF-R). LIF activity was partially neutralized by anti-human LIF antibody. Interleukin-6 (IL-6) activity was detected in ULF throughout the oestrous cycle and pre-implantation period. An anti-murine alpha chain (gp80) of IL-6 receptor (IL-6R) specifically neutralized this activity. LIF and IL-6 mRNA were only detected in day 11 endometrium. The presence of LIF or IL-6 in the uterine cavity has not been previously reported. Our results extend LIF production by endometrium during the oestrous cycle and pre-implantation period to another mammalian species other than mouse.

Effect of passive immunization against testosterone on reproductive hormone secretion and ovarian function in dairy cows and pubertal beef heifers.

Multiparous dairy cows were divided in 3 groups from Day 5 up to Day 56 post partum: high energy level (Group H, n=10), low energy level (Group L, n=10) and low energy level plus anti-testosterone bovine immunoglobulins (Group LI, n= 10). Undernutrition decreased body weight, body condition score, milk yield and energy balance in Groups L and LI compared to Group H (P<0.05), but had no effect on secretory pattern of LH. Passive immunization against testosterone increased LH secretion in Group LI (P<0.05). Follicular score and the presence of follicles >/= 10mm on the ovary were not affected by underfeeding but were higher in Group LI than in Group L after immunization (P<0.01). The duration of the first luteal phase was shorter in Group H than in Groups L and LI and maximum progesterone levels reached were higher in Group LI than in Group H (P<0.01). Reproductive performance was not depressed by underfeeding and immunization. In the pubertal beef heifers maintained in anestrus by undernutrition had very low LH secretion. After passive immunization against testosterone, the increase of LH pulses number became almost significant (P=0.07). Following injection of exogenous LH, the number of follicles >/= 9mm was higher in immunized (Group I, n=8) than in control heifers (Group C, n=7). Group I developed a dominant follicle sooner and of greater size than Group C. Passive immunization against testosterone increased LH secretion and follicular development.

Differential expression, during the estrous cycle and pre- and postimplantation conceptus development, of messenger ribonucleic acids encoding components of the pig uterine insulin-like growth factor system.

The temporal patterns of endometrial expression for mRNAs encoding insulin-like growth factor-I (IGF-I), IGF-II, IGF-binding protein-2 (IGFBP-2), and the type I IGF receptor (IGF-IR) were elucidated in cyclic and pregnant pigs. Peak levels of IGF-I mRNAs occurred on day 12 in cyclic and early pregnant gilts, while IGFBP-2 mRNA levels were lowest on day 10. Pregnant gilt endometrium had higher levels of both RNA classes than the corresponding cyclic endometrium. IGF-II and IGF-IR mRNAs remained low during this period. In pregnant pig endometrium and rat uterus, levels of IGF-I mRNA decreased, while those of IGF-II and IGFBP-2 mRNAs increased with stage of pregnancy. Decreased endometrial production of IGF-I mRNA during pregnancy paralleled that in the myometrium. IGF-II mRNA tissue abundance was placenta greater than endometrium greater than myometrium. In contrast, IGFBP-2 mRNA levels were higher in endometrium than in placenta and myometrium. Endometrial expression of IGF-II mRNAs was limited to surface and glandular epithelial cells; epithelial and stromal cells expressed IGFBP-2 mRNAs at comparable levels. Expression of IGF-IR mRNAs was low and did not change with pregnancy. The endometria of two breeds of pigs that exhibit different levels of prolificacy were also examined for IGF mRNAs. On day 12, endometrium from the Large White breed with high conceptus mortality had higher levels of IGF-II and IGFBP-2 mRNAs than did endometrium from the Meishan breed with low conceptus mortality. Expression of IGF-I mRNAs was higher in endometria of Meishan than Large White gilts on day 12. The differential expression of IGF mRNAs with stage of gestation and the correlation of relative ratios of IGF mRNAs with prolificacy during the critical period of maternal recognition of pregnancy suggest an important role(s) for IGFs in conceptus and fetal development.