Relationship between variation in conceptus development and differences in estrous cycle duration in ewes

The objective of this study was to examine conceptus development on Day 13 in ewes with estrous cycles of different durations. Ewes (n = 80) were screened according to the length of their estrous cycles. Subsequently, ewes that had either SHORT or LONG cycles were utilized (15.9 +/- 0.1 or 18.6 +/- 0.4 days; mean +/- SEM, p less than 0.01; 10 ewes per group). Jugular blood samples were collected twice daily from Days 0-6 after mating and then once a day until slaughter on Day 13. Concentrations of progesterone in plasma and amounts of ovine trophoblast protein-1 (oTP-1), protein, and prostaglandins (PG) E2 and F2 alpha (PGF2 alpha) in uterine flushings were determined. Concentrations of progesterone were greater (Day by treatment interaction, p less than 0.01) on Days 2-4 for ewes in the SHORT group. On Day 5 and thereafter, progesterone concentrations were not different between groups. More (p less than 0.05) oTP-1 and protein (8.1 +/- 1.3 micrograms and 1.8 +/- 0.3 micrograms versus 2.4 +/- 1.3 micrograms and 0.8 +/- 0.3 mg) were recovered from uterine flushings from ewes in the SHORT versus LONG groups, respectively. The ratio of PGE2:PGF2 alpha was higher (p less than 0.06) in flushings from ewes in the SHORT versus LONG group (1.4 +/- 0.2 versus 0.9 +/- 0.2, respectively). Conceptuses were classified by stage of morphological development. Conceptus development was accelerated (p less than 0.01) in ewes of the SHORT group, as shown by filamentous conceptuses recovered from 78% versus 0% of SHORT versus LONG ewes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of heat stress on conceptus and uterine secretion in the bovine

This study examined the effects of heat stress on conceptus secretion of bovine trophoblastic proteins (bTP) and the uterine secretory environment on Day 17 of pregnancy. After mating to fertile bulls, cows were placed in an environmental chamber at 21 degrees C, 45% RH on Day 7 and were assigned to one of the following treatments on Day 8: Control (21 degrees C, 45% RH), Treatment 1 (37 degrees C, 30% RH) and Treatment 2 (37 degrees C, 40% RH). Cows were slaughtered on Day 17 and each uterine horn was flushed separately with physiological saline to recover the conceptus and uterine contents. The wet weight of the conceptus was recorded and uterine flushings were analyzed for quantitative and qualitative protein changes, prostaglandin F and calcium concentrations. Conceptus tissue was cultured in vitro with 100 muCi of [(3)H]-leucine and the polypeptides released into the medium were analyzed by 2D-PAGE followed by fluorography. Heat stress increased rectal temperature of cows and reduced conceptus wet weight. Uterine content of protein and calcium were increased in the uterine horn ipsilateral to the CL of heat-stressed cows. Although uterine protein increased in heat-stressed cows, no qualitative difference was observed in the polypeptides present in the uterine lumen. Conceptus synthesis and release of bTP were enhanced in treated cows. These responses indicate that heat stress between Days 8 to 17 of pregnancy altered the uterine environment as well as growth and secretory activity of the conceptus.     

Characterization of the interleukin-1beta system during porcine trophoblastic elongation and early placental attachment

Conceptus-uterine communication is established during trophoblastic elongation when the conceptus synthesizes and releases estrogen, the maternal recognition signal in the pig. Interleukin-1beta (IL-1beta) is a differentially expressed gene during rapid trophoblastic elongation in the pig. The current investigation determined conceptus and endometrial changes in gene expression for IL-1beta, IL-1 receptor antagonist (IL-1Rant), IL-1 receptor type 1 (IL-1RT1), and IL-1 receptor accessory protein (IL-1RAP) in developing peri- and postimplantation conceptuses as well as uterine endometrium collected from cyclic and pregnant gilts. Conceptus IL-1beta gene expression was enhanced during the period of rapid trophoblastic elongation compared with earlier spherical conceptuses, followed by a dramatic decrease in elongated Day 15 conceptuses. IL-1RT1 and IL-1RAP gene expression was greater in Day 12 and 15 filamentous conceptuses compared with earlier morphologies while IL-1Rant gene expression was unchanged by conceptus development. The uterine lumenal content of IL-1beta increased during the process of trophoblastic elongation on Day 12. Uterine IL-1beta content declined on Day 15, reaching a nadir by Day 18 of pregnancy. IL-1beta gene expression in porcine conceptuses was temporally associated with an increase in endometrial IL-1RT1 and IL-1RAP gene expression in pregnant gilts. Endometrial IL-1beta and IL-1Rant gene expression were lowest during Days 10-15 of the estrous cycle and pregnancy. The temporal expression of IL-1beta during conceptus development and the initiation of conceptus-uterine communication suggests conceptus IL-1beta synthesis plays an important role in porcine conceptus elongation and the establishment of pregnancy in the pig.     

Effect of intrauterine administration of oestradiol on postpartum uterine bacterial infection in cattle

After parturition fewer first dominant follicles are selected in the ovary ipsilateral to the previously gravid uterine horn in cattle. However, the presence of a large oestradiol-secreting follicle in the ipsilateral ovary is a predictor of fertility, possibly due to a localised effect of oestradiol which increases the rate of elimination of the ubiquitous uterine bacterial contamination that occurs after calving. The present study tested the hypothesis that oestradiol reduces uterine bacterial contamination when administered into the uterine lumen around the expected time for selection of the first postpartum dominant follicle. Animals were infused with saline (n=15) or 10mg oestradiol benzoate (n=15) into the previously gravid uterine horn on Days 7 and 10 postpartum. Peripheral coccygeal blood samples were collected daily and oestradiol concentrations measured by radioimmunoassay (RIA). Uterine lumen swabs were collected 7, 14 and 21 days postpartum for aerobic and anaerobic culture, bacteria were identified and growth scored semi-quantitatively. Plasma oestradiol concentrations were higher for treated animals between Days 7 and 14 (1.4+/-0.1 versus 2.0+/-0.2 pg/ml, P<0.05). Control animals had a similar bacterial growth score on Days 7 and 14, with a lower value on Day 21 (5.7+/-1.0 and 6.1+/-0.7 versus 0.3+/-0.1, P<0.05). However, treated animals had a surprising higher bacterial load on Day 14, than on Days 7 or 21 (7.1+/-0.9 versus 4.0+/-0.6 or 3.6+/-0.6, P<0.05). The increased score was attributable to more pathogens associated with endometritis on Day 14 than Day 7 (5.1+/-1.0 versus 2.5+/-0.5, P<0.05), in particular Prevotella melaninogenicus (1.5+/-0.5 versus 0.7+/-0.2, P<0.05) and Fusobacterium necrophorum (1.5+/-0.4 versus 0.3+/-0.2, P<0.05). In conclusion, administration of oestradiol into the uterine lumen surprisingly increased uterine pathogenic anaerobic bacterial contamination. Thus, it is unlikely that increased fertility associated with a first dominant follicle in the ipsilateral ovary is a consequence of the elimination of bacterial contamination by ovarian oestradiol.     

Insulin-like growth factor-I expression during early conceptus development in the pig

Porcine conceptuses (embryo and associated membranes) in utero undergo developmental morphological transformations coincident with structural and biochemical changes in the uterine endometrium during early gestation. To elucidate a possible role for insulin-like growth factor-I (IGF-I) in these events, porcine endometrial (Days 8, 10, 11, 12, 14, and 30) and conceptus (Days 12, 14, and 16) tissues were characterized for the presence of IGF-I peptide and mRNAs. The corresponding uterine luminal fluids (ULF) at these stages of pregnancy were also analyzed for immunoreactive IGF-I concentration. ULF IGF-I was lowest on Day 8, highest on Day 12, and declined by Day 14. In contrast, endometrial tissue IGF-I content remained constant during this period. Conceptus tissues contained less IGF-I than endometrial tissues; however, conceptus IGF-I values were maximum on Day 12 coincident with peak values for ULF IGF-I. Dot-blot hybridization analyses revealed temporal variation in steady-state levels of IGF-I mRNAs in endometrium. Highest levels of endometrial IGF-I mRNA were detected on Day 12 and were about 4-fold greater than on Day 30 of pregnancy. IGF-I mRNA expression in conceptus tissues on Days 12, 14, and 16 was the same and was significantly less than that in endometrium on Day 12. These results demonstrate the temporal variation of IGF-I mRNA abundance in uterine endometrium and of immunoreactive IGF-I in ULF and in conceptus tissues, with the developmental processes occurring in the conceptuses at early pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential suppression of endometrial prostaglandin F2alpha by the equine conceptus

Prostaglandin F2alpha secretion by the uterine endometrium between Days 13 and 14 postovulation causes luteal regression in mares. A mechanism involving interruption or suppression of this secretion causes pregnancy to be maintained. The present study was designed to determine the age of the conceptus when maximal suppression of PGF2alpha secretion occurs. Mares were examined daily during estrus with ultrasonography (day 0 = day of ovulation). Conceptus tissues were recovered nonsurgically on Days 9 (n = 7), 12 (n = 5), 13 (n = 5), and 16 (n = 7) and uterine biopsies on Day 14. Both uterine and conceptus tissues were washed in phosphate-buffered saline (PBS) with 100 units penicillin G/ml + 100 microg streptomycin/ml, pH 7.4. Endometrial tissue (approximately 200 mg) plus conceptus tissues were incubated in 15 ml of tissue culture medium 199 (M199) + 10% fetal calf serum and 10 units penicillin G/ml and 10 microg streptomycin/ml at 37 degrees C under 5% CO(2): 5% O(2) : 90% N(2). Samples were taken at 4, 8, and 24 h. Two plates that contained only endometrial tissue and two additional plates with 25 mg flunixin meglumine added along with endometrial tissue were also included in the incubations. Concentrations of PGF2alpha were measured in all samples using radioimmunoassay. There was a trend toward suppression of PGF2alpha secretion by conceptus tissues, regardless of age. However, Day 12 concepti significantly suppressed PGF2alpha secretion compared with that of endometrial tissue incubated alone (P = 0.03).     

Superovulation in heifers given FSH initiated either at day 2 or day 10 of the estrous cycle

The aim of this study was to determine if initiation of superovulation in heifers during the time of development of the first dominant follicle (Days 2 to 6) would give equivalent ovulation and embryo production rates as treatment initiated at mid-cycle. Estrus was synchronized in 60 beef heifers using luprostiol (PG) and they were randomly allocated to treatment with 4.5, 3.5, 2.5 and 1.5 mg of porcine follicle stimulating hormone (FSH) administered twice daily, either on Days 2, 4, 5 and 6 (Day-2 group), respectively, or with similar doses at four consecutive days during mid-cycle (Day-10 group, initiation on Day 9 to 11). All heifers received 500 mug cloprostenol at the fifth FSH injection and 250 mug at the sixth injection. Blood samples for progesterone determination were collected at the time of FSH injections. Heifers were slaughtered 7 d post estrus, and the number of ovulations and large follicles (>/=10mm) were determined on visual inspection of the ovary. Following flushing of the uterine horns the quality of embryos and the fertilization rate were determined. Significant differences between treatments were determined using a two-sided t-test, and frequency distributions were compared using Chi-square tests. The mean number (+/-SEM) of ovulations for heifers in the Day-10 group was 12.9+/-1.0, and 8.5+/-0.9 embryos were recovered. Both the number of ovulations (6.7+/-0.8) and embryos recovered (4.1+/-0.6) were lower (P=0.0001) in heifers in the Day-2 group. Following grading based on a morphological basis, a higher number (P=0.002) of embryos was categorized as Grades 1 and 2 (4.1+/-0.6) and Grade 3 (2.1+/-0.4) in Day-10 heifers than in the Day-2 group (Grade 1 and 2, 1.9+/-0.3; Grade 3, 0.7+/-0.2). The number of Grade 4 and 5 embryos (Day 10, 1.6+/-0.2; Day 2, 1.4+/-0.2) and the number of unfertilized ova (Day 10, 0.7+/-0.4; Day 2, 0.2+/-0.1) did not differ between treatments. Progesterone concentrations were lower (P=0.0001) in Day-2 heifers at FSH treatment prior to prostaglandin, and the decline was more rapid following prostaglandin injection at Day 5 (P=0.02). Results of this study indicate that the number of ovulations and embryos recovered was lower in heifers when FSH treatment was initiated on Day 2 compared with Day 10 of the estrous cycle.     

Factors related to the time of fixation of the conceptus in mares

The temporal relationships among day of conceptus fixation (cessation of mobility), conceptus diameter, uterine tone, uterine contractility, and myometrial and endometrial thickness of the middle and caudal segments of the uterine horns were assessed in 13 pony mares with fixation in the caudal segment of a uterine horn. The mean day of fixation (14.9 +/- 0.3) was established by 2-h mobility trials. Uterine tone increased (P < 0.0001) gradually over Days 11 to 21, whereas uterine contractility decreased (P < 0.0001) between Days 14 and 18. The diameter of the spherical embryonic vesicle increased (P < 0.0001) between Days 11 and 17. The day of fixation and vesicle diameter on Day 14 were negatively correlated (r = -0.9, P < 0.007); the larger the vesicle, the earlier fixation occurred. Each of 4 uterine-horn diameters (endometrium and endometrium plus myometrium of middle and caudal segments) decreased (P < 0.0001) correspondingly over Days 11 to 21. On the day of fixation conceptus diameter (21.5 +/- 1.0 mm) was similar to endometrial diameter (21.1 +/- 0.4 mm) at the caudal segment. The endometrial diameter represents the distance between the inner opposite walls of the myometrium. The percentage of change between the day before and day of fixation was greater for the conceptus (18.1% increase) than for the endometrial diameter at the caudal segment (1.0% decrease). The results suggest that fixation occurred when the mobile and growing conceptus attained, on the average, a diameter equivalent to the distance between opposite inner myometrial walls at the caudal segment. The uterus became turgid by this time and presumably did not expand adequately to accommodate continued mobility of the expanding conceptus.     

Inhibitory effects of adrenocorticotrophin or corticosterone on progesterone secretion during mid-pregnancy in rats

Conceptus number was reduced to one on Day 7 of pregnancy in rats by aspirating all but a single conceptus (Group E) or left at greater than or equal to 8 conceptuses (Group C). In Group E rats, serum progesterone concentrations remained low from Day 12 until autopsy at Day 21. Hypophysectomy on Day 12 significantly increased serum progesterone values after Day 17 of pregnancy, and these increases were blocked by treatment with ACTH (10 U/day, i.p., Days 12-17). Adrenalectomy on Day 12 also induced slight, but statistically significant, increases in serum progesterone concentration after Day 17, and these were overcome by implantation of a 10 mg capsule of corticosterone. In Group C rats, hypophysectomy or adrenalectomy on Day 12 did not change serum progesterone concentrations, but 40 U ACTH/day inhibited progesterone secretion. We conclude from these results that the pituitary-adrenal system exerts inhibitory effects on progesterone secretion during mid-pregnancy in rats.     

Characterization of the epidermal growth factor receptor in preimplantation pig conceptuses.

Embryos recovered from sows on Days 9-13 of pregnancy (Day 0 = first day of estrus) exhibited saturable and time-dependent specific binding of 125I-epidermal growth factor (EGF). The specific binding (pg/mg protein) was greater (P less than 0.001) for Day 13 elongated conceptuses than for conceptuses of earlier stages. Scatchard analyses showed two classes of binding sites (Kd = 7.0 +/- 2.6 x 10(-11) M, Bmax = 6.2 +/- 1.4 fmol/mg protein and Kd = 3.4 +/- 0.2 x 10(-8) M, Bmax = 420 +/- 80 fmol/mg protein). The EGF receptor in Day 13 conceptus membranes is a 170-kDa protein and was phosphorylated in the presence of EGF and adenosine triphosphate. EGF stimulated protein tyrosine kinase activity about 1.6-fold over basal levels. The results show that the preimplantation pig conceptus possesses EGF-binding sites with the properties of functional EGF-receptors.     

Effect of bovine blastocyst size at embryo transfer on day 7 on conceptus length on day 14: Can supplementary progesterone rescue small embryos?

Conceptus size on Day 14 after multiple embryo transfer of Day 7 in vitro–produced blastocysts varies greatly within animal. One explanation for this variation may be related to blastocyst cell number at the time of transfer. The aim of this study was to examine the effect of Day 7 blastocyst cell number on Day 14 conceptus size and to examine the effect of progesterone (P4) supplementation on embryo development after the transfer of Day 7 blastocysts containing a low total cell number. The estrous cycles of crossbred beef heifers were synchronized using an 8-day progesterone (P4)–releasing intravaginal device (PRID) with the administration of a prostaglandin F_2α analog on the day before device removal. Only those heifers recorded in standing estrus (Day 0) were used. Heifers were randomly assigned to one of four treatment groups: (1) control: large blastocysts (high total cell number), (2) control: small blastocysts (low total cell number), (3) small blastocysts plus a single intramuscular injection of 3000 IU human chorionic gonadotropin (hCG) on Day 2 after estrus, or (4) small blastocysts plus insertion of a vaginal P4 insert (PRID, 1.55 g P4) between Days 3 and 5 after estrus. In vitro–produced blastocysts were transferred to each heifer on Day 7 (n = 10 blastocysts per heifer), and conceptuses were recovered at slaughter on Day 14. Daily blood samples were collected from Day 0 to 14 to measure serum P4 concentrations. Data were analyzed using the PROC MIXED procedure of SAS. Total cell number on Day 7 was significantly lower in small versus large blastocysts (72.4 ± 3.93 vs. 144.8 ± 3.90, P < 0.05). Conceptus recovery rate was 53.8% overall (140 of 260) and was highest in the large blastocyst group (68.3%, 41 of 60) compared with the other groups (45.7%–55.0%). Concentrations of serum P4 were similar in the two unmanipulated recipient groups but were significantly elevated (P < 0.05) by Day 8 in the hCG-treated heifers and on Days 4 and 5 in the PRID group (P < 0.003). In the absence of supplemental P4, Day 14 conceptuses resulting from the transfer of small blastocysts (2.48 ± 0.54 mm) were smaller than those from large blastocysts (3.32 ± 0.52 mm). Administration of hCG on Day 2 approximately doubled conceptus length on Day 14 (4.94 ± 1.15 mm; P < 0.05), whereas insertion of a PRID from Days 3 to 5 increased conceptus length approximately fivefold (13.09 ± 2.11 mm; P < 0.05) compared with controls. In conclusion, results indicate that supplemental P4 is capable of “rescuing” poor-quality blastocysts, presumably via the now well-described actions on the endometrium and consequent effects on uterine lumen fluid composition.     

Retinol-binding protein: a major secretory product of the pig conceptus

Pig conceptuses and endometrial explants recovered from gilts between Days 10 and 15 of pregnancy were cultured in leucine-deficient or methionine-deficient medium supplemented with 3H-leucine or 35S-methionine, respectively, for 30 h. Conceptus and endometrial tissues from Day 15 of pregnancy were fixed in Bouin's fixative for immunocytochemistry and light microscopy. Conceptus culture medium from Day 15 of pregnancy was pooled, dialyzed, and fractionated by anion exchange and gel filtration chromatography. A family of 3-5 low molecular weight (Mr) acidic (Mr = 19,000-22,000; pI = 5.6-6.5) 3H-leu-labeled proteins were isolated and identified by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), electroblotting, and fluorography. The two major proteins (pCSP-1 and pCSP-2) were excised from a polyvinylidene difluoride transfer membrane, and NH2-terminal amino acids were sequenced. One peptide was sequenced through 33 amino acids and the second, which shared 100% homology, was sequenced through 22 amino acids. Analysis of the larger sequence indicated that it shared 93.9% and 90.9% homology with the first 33 amino acids of human and rabbit plasma retinol-binding protein (RBP), respectively. Analyses of culture medium from pig conceptus incubations by 2D-PAGE and immunoprecipitation with rabbit anti-human RBP serum indicated that immunoreactive RBP was produced between Days 10 and 15 of pregnancy and was present in Day 30 allantoic fluid. Western blotting of enriched fractions of Day 15 conceptus RBP followed by immunostaining indicated that five isoforms of radiolabeled RBP were present. Immunoreactive RBP was detected in trophectoderm and yolk sac of conceptuses and endometrial surface and glandular epithelium at Day 15 of pregnancy. Results from this study demonstrate that pig conceptuses secrete RBP prior to onset of conceptus elongation and throughout the peri-implantation period, which suggests that RBP and associated retinoids influence conceptus development.     

Characterization of the uterine environment during early conceptus expansion in the bovine

Fifty-three Angus and Hereford beef cows were utilized to investigate the effect of the conceptus on uterine environment during the period of pregnancy recognition. Blood samples were collected on Days 10, 12, 14, 16 and 18. Cows were randomly assigned to be either mated on the subsequent oestrus or serve as cyclic controls. Blood samples were then collected daily from Day 10 until slaughter on Day 15, 16 or 17 from the initiation of oestrus. Uteri were flushed with physiological saline and flushings analyzed for quantitative and qualitative protein changes, calcium, oestradiol-17β and prostaglandin F content. Endometrial explants of caruncular and intercaruncular tissue, and conceptus tissue recovered from pregnant cows were cultured with [³H]-leucine to determine quantitative and qualitative polypeptide synthesis and release. Plasma progesterone concentrations were similar between pregnant and cyclic cows from Day 10 through 17. Only the uterine content of prostaglandin F significantly increased in the ipsilateral horn of pregnant cows on Days 16 and 17. This increase in prostaglandin content was related to the increase in conceptus length from 25 to 40–80 mm. Conceptus production of bovine trophoblastic protein-1 was also first clearly detectable in fluorographs of medium from conceptuses measuring 25 mm. The complexity of the polypeptides present in the medium increased with conceptus development. Polypeptide synthesis by the endometrium was similar between tissues and days; however, production of two groups of low molecular weight basic polypeptides continued to be intensified on fluorographs from the pregnant horn on Day 17 compared to cyclic cows.     

Improvement in embryo recovery using double uterine flushing

The objective of the present study was to evaluate the effects of double uterine flushing on the recovery of embryos/ova in cattle. Two hundred and ten embryo recovery procedures were conducted using a double uterine flushing method, and the results were compared with 432 conventional single-flushing procedures. Cyclic Limousin (n = 403) and Guzera (n = 239) donor cows received an intravaginal progesterone releasing device and 2 mg of estradiol benzoate on Day 0. Between Days 5 and 9, donors received decreasing doses of FSH, which ranged from 200 to 300 IU (Bos indicus) and 300 to 500 IU (Bos taurus). On the afternoon of Day 7, donors received an injection of 500 microg cloprostenol and progesterone implants were removed 12 h later (morning of Day 8). Artificial insemination was performed between 14 and 26 h after first detection of behavioral estrus. Cows were randomly assigned to have embryos recovered by a double-flushing method (n = 210) or the conventional single-flushing procedure (n = 432). For the double-flushing procedure, after first flushing the whole uterus with 1L of Dubelco's Phosphate Buffered Saline (DPBS), a Foley catheter was positioned in the uterine body to permit refilling of the uterus with fresh DPBS (80-150 mL). The catheter was closed with the plunger of a disposable 5 mL syringe, and the donors were allowed to rest in a holding area for 30 min. Thereafter, a second flush was performed to recover the solution remaining in the uterus. Animals from the control group were subjected to a single uterine flush. From 210 double-flushing procedures, 1409 viable embryos were recovered. In comparison, from 432 cows receiving the single-flushing procedure, 1993 embryos were recovered. Double flushing increased (P < 0.05) the number of embryos recovered per procedure compared to single flushing (6.7 +/- 0.4 versus 4.6 +/- 0.2, respectively; mean +/- S.E.M.). When double flushing was performed, average recovered embryos/ova increased (P < 0.05) from 8.3 +/- 0.4 to 12.7 +/- 0.7 in Limousin and from 7.9 to 11.5 in Guzera. Also, utilization of double flushing increased (P < 0.05) the number of viable embryos from 4.7 +/- 0.3 to 6.9 +/- 0.5 in Limousin and from 4.5 +/- 0.4 to 6.4 +/- 0.7 in Guzera. Mean total embryos/ova was similar (P > 0.05) between the control group and after the first uterine flushing in the double-flushing group; therefore, both flushings were conducted efficiently. In conclusion, double uterine flushing increased embryo recovery in cattle.     

Ultrasonographic evaluation of the conceptus from days 10 to 60 of pregnancy in jennies

Daily ultrasound examinations were conducted from Days 10 to 60 (ovulation = Day 0) of pregnancy to monitor the conceptus in jennies (n = 12). The embryonic vesicle was first detected on Day 11.5 +/- 0.9 (mean +/- SD; range 10 to 13 d) and was mobile until movement ceased (fixation) on Day 15.5 +/- 1.4 (range, 13 to 18 d). The vesicle was spherical from Days 10 to 18 (mean growth rate, 3.2 mm/d), non spherical (irregular) with a reduced growth rate (0.5 mm/d) from Days 19 to 29, and then grew at a moderate rate (1.6 mm/d) up to Day 46. On average, detection of the embryo proper (consistently located on the ventral aspect of the yolk sac) and embryonic heartbeat were Days 20.7 +/- 1.2 and 23.5 +/- 1.3, respectively. Formation of the allantoic sac was first detected on Day 24.4 +/- 1.7 and was complete on Day 36.8 +/- 1.6. Descent of the fetus (and formation of the umbilical cord) began on Day 37.9 +/- 1.7 and was complete on Day 44.1 +/- 2.1. Crown-rump length averaged 3.7, 15.4, 22.7, 37.5 and 59.6 mm on Days 20, 30, 40, 50 and 60, respectively. In general, morphologic features and dates of occurrence were similar to those reported previously in the mare.     

Intrauterine infusion of highly enriched bovine trophoblast protein-1 complex exerts an antiluteolytic effect to extend corpus luteum lifespan in cyclic cattle

Intrauterine infusion of enriched bovine trophoblast protein-1 complex (bTP-1) resulted in extension of interoestrous intervals and corpus luteum function in cyclic cattle. Conceptus proteins were obtained by culture of Day 17-18 conceptuses for 72 h. Media from the first (n = 28), second (n = 26) and third (n = 19) 24 h of conceptus incubations were utilized. A highly enriched preparation of bTP-1 was obtained by a combination of ammonium sulphate precipitation, ion-exchange chromatography, and h.p.l.c. gel filtration. Degree of purity of the final preparation was confirmed by gel electrophoresis and immunoblotting with antiserum to ovine trophoblast protein-1. Jersey cattle (3 per group) received intrauterine infusions, twice daily from Day 15.5 to 21.0, of bovine serum albumin, the entire array of bovine conceptus secretory proteins (bCSP) from the 3 days of conceptus culture, or bTP-1. Infusions were via a catheter into the uterine horn ipsilateral to the corpus luteum. Oestrous cycle length in bTP-1-treated cows (26.1 +/- 1.3 days) was greater than for cows given BSA (19.5 +/- 1.3 days) or bCSP (21.5 +/- 1.3 days). Similarly, progesterone concentrations in serum remained elevated for a longer period of time for bTP-1-treated cows than for cows treated with BSA or bCSP. Residual variance associated with vena cava concentrations of PGF-2 alpha at Days 19-21 after oestrus (which included the variance between 15-min periods within cows) was reduced in cows treated with bTP-1 as compared to other groups. Lack of a bCSP effect may have been due to low amounts of bTP-1 in conceptus-conditioned medium from cultures of greater than 24 h. None the less, purified bTP-1 was effective in extending luteal function and appears to be the antiluteolytic agent of early pregnancy.     

Effect of recombinant alpha interferons on fertility and interestrous interval in sheep

In Experiment 1, 12 unmated cyclic ewes received twice-daily intrauterine injections on Days 12 to 14 of one of the following treatments: 1) ovine conceptus secretory proteins (oCSP) containing 25 mug of ovine trophoblast protein-1 (oTP-1) as determined by RIA; 2) 25 or 50 mug recombinant human interferon alpha1 (rhlFN); or 3) 1500 ug of serum proteins (oSP) from a Day-16 pregnant ewe (estrus = Day 0) per uterine horn. Ewes receiving oCSP had longer interestrous intervals (27 +/- 2 days; P<0.05) than ewes receiving oSP (17 +/- 2 days). Ewes receiving either dose of rhlFN had an interestrous interval of 16 +/- 2 days which did not differ (P>0.10) from that of oSP-treated ewes. In Experiment 2, 59 normally cycling ewes, mated on Day 0, received twice-daily intramuscular injections of either 2 mg recombinant bovine interferon alpha1 (rblFN) or placebo on Days 12 to 15 post estrus. On Day 16, pregnancy was confirmed by flushing a morphologically normal conceptus from the uterus. Pregnancy rates for rblFN-treated (80%) and placebo-treated (62%) ewes were not different (P>0.10). Uterine flushings and conceptus-conditioned medium were assayed for oTP-1. Total oTP-1 in conceptus-conditioned culture medium was higher (P<0.02) when conceptuses were from placebo-treated (104 +/- 14 mug/conceptus) than from rblFN-treated (56 +/- 12 mug/conceptus) ewes; while total oTP-1 in uterine flushings was similar (P>0.10) for placebo-treated (132 +/- 15 mug/conceptus) and rblFN-treated (147 +/- 17 mug/conceptus) ewes. The interval from mating to subsequent estrus following conceptus removal was 31 +/- 1 and 28 +/- 1 days for pregnant ewes treated with rblFN and placebo, respectively. Interestrous intervals for nonpregnant ewes were longer (P<0.02) for rblFN-treated (27 +/- 3 days) than for placebo-treated (18 +/- 2 days) ewes.     

Equine fetal kinetics: Allantoic-fluid shifts and uterine-horn closures

Uterine filling by the conceptus and uterine constrictions and allantoic-fluid shifts after filling were studied by daily ultrasound examinations on Days 40 to 74 in 10 mares. Allantoic fluid was first detected at the extremities of the uterus on the following days: 1) tip of umbilical-cord horn, Day 60.0 +/-1.9 (mean +/-SEM); 2) tip of noncord horn, Day 64.8 +/-1.3; and 3) near cervix, Day 48.5 +/-1.3. Transient uterine constrictions with expulsion of allantoic fluid were detected in 37% of 271 examinations. Other end points were studied by weekly ultrasound examinations from Day 40 to term. Height of cross-sections of the fetal body increased (P<0.01) progressively over Monthes 2 to 8, whereas the height of placental fluid minus fetal body decreased (P<0.05) after Month 3. An increasing (P<0.05) frequency of examinations with a reclosed uterine horn occurred between Months 3 and 4. The frequency of reclosed noncord horns continued to increase (P<0.05) after Month 4 and reached 96% at Month 7. The frequency of reclosure of cord horns plateaued (no significant differences) at 54% over Months 4 to 7 and then rapidly decreased (P<0.05). Both uterine horns were closed during the examination that preceded the permanent entry of the fetal hind limbs into the cord horn in all of 9 mares in which this temporal relationship was studied. After entry of the fetal hind limbs, the cord horn remained constricted upon the limbs until at least Month 10. The results provide rationale for the following hypotheses: 1) The gradual decrease in fetal mobility (location and presentation changes) previously reported for Months 4 to 8 is associated with growth of the fetus and a concomitant decrease in volume of allantoic fluid and number of open uterine segments; 2) Closure of both uterine horns near mid-pregnancy plays a role in final selection of cranial fetal presentation followed by entry of the fetal hind limbs into the cord horn.     

Onset of secretion of proteins with antiviral activity by pig conceptuses

In Exp. 1, antiviral activity was detected in Day-15 pregnant uterine flushings (6222 +/- 2167 units/ml) and in conceptus culture medium collected at 0, 1, 2, 4, 8, 16, 24, 32, 40, and 48 h (95, 375, 650, 1216, 1600, 2100, 2017, 2083, 3500 and 5000 units/ml, respectively; R2 = 0.81, P less than 0.01; y = 190.0 + 252.7x - 11.2x2 + 0.2x3. In Exp. 2, antiviral activity of Day-15 conceptus culture medium was reduced 99% after boiling for 20 min (P less than 0.01) and, after 18 h dialysis (6000-8000 Mr cut-off), 100% of the activity was in the retentate. In Exp. 3, antiviral activity was not detected in cultures of conceptuses from Days 10 and 11 and activity was maximal for Day 14 and Day 15 conceptuses (2100 and 2083 units/ml, respectively). Effects of day were best described by a quadratic regression equation (y = 17,652 - 3263x + 150x2; R2 = 0.55, P less than 0.01). In Exp. 4, changes in antiviral activity detected in uterine flushings from pregnant gilts on Days 8, 10, 11, 12, 14 and 15 (1.3, 0, 6.7, 63.3, 580 and 1663 units/ml, respectively) were described by the equation y = -20,743 + 6189x - 606x2 + 20x3 (R2 = 0.85, P less than 0.01). In Exp. 5, low antiviral activities (5-30 units/ml) were detected in all plasma samples collected from the uterine artery and uterine vein of pregnant and cyclic gilts, but values were not significantly influenced by pregnancy status, day or site of collection.(ABSTRACT TRUNCATED AT 250 WORDS)