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The objectives of the present studies were to determine the effect of insulin, insulin-like growth factor I (IGF-I), testosterone, and FSH on proliferation, progesterone production, and(or) estradiol production of bovine granulosa cells. In addition, existence of IGF-I mRNA in granulosa cells and in vitro IGF-I production by granulosa cells were assessed. Cells from small (1 to 5 mm) and large (> or = 8 mm) follicles were collected from cattle and cultured for either 3 or 4 d. When cells from small follicles were cultured, insulin (.1 to 10 micrograms/mL) and IGF-I (100 to 400 ng/mL) increased (P < .05) cell numbers compared with controls. Insulin alone or IGF-I alone increased (P < .05) progesterone production per cell by severalfold on d 4. In cells from both sizes of follicles, insulin (1 microgram/mL), in the presence of FSH, increased estradiol production per cell. In contrast, IGF-I (100 ng/mL) inhibited estradiol production by cells from small follicles and stimulated estradiol production by cells from large follicles. Insulin-like growth factor II (100 ng/mL) and insulin at higher doses (> or = 5 micrograms/mL) had no effect on estradiol production by cells from small and large follicles. Granulosa cells contained four IGF-I mRNA transcripts and produced IGF-I in vitro. These results support the hypothesis that insulin and IGF-I may have direct local effects on bovine ovarian function, and that these effects are influenced by dose and size of follicle.

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The relationship between ovarian follicular steroidogenesis and insulin-like growth factor binding protein (IGFBP) activity was evaluated during the follicular phase of the bovine estrous cycle. In experiment 1, follicles were collected from cyclic cows (n = 11) slaughtered at 48 h after administration of prostaglandin F2 alpha (PGF; 35 mg i.m.). In experiment 2, cows were injected twice daily with saline (control) or FSH (FSH cows; total dosage = 42 mg) from Day 2 to Day 6 (estrus = Day 0) and with PGF (35 mg i.m.) on Day 7; follicles were collected from control cows (n = 20) slaughtered at 0, 24, 48, or 72 h and from FSH cows (n = 8) at 0 and 48 h after PGF. Follicular fluid was assayed for estradiol (E2), androstenedione (A4), progesterone (P4), and insulin-like growth factor-I (IGF-I) by RIA and for IGFBP activity by ligand blotting and densitometry. Intensities of the 34-kDa (IGFBP-2), 29-27-kDa, and 22-kDa IGFBP bands in follicular fluid were nondetectable or were lower (p < 0.01) in the fluid of large (> or = 8 mm) E-active (E-A; E2 > 50 ng/ml and > P4) follicles than in large E-inactive (E-I), medium (5-7 mm), or small (< 5 mm) follicles. IGFBP-3 (44-40-kDa doublet) was unaffected by follicle stage in experiment 1, but IGFBP-3 was lower (p < 0.01) in follicular fluid of E-A vs. E-I large follicles in experiment 2. Profiles of IGFBP activity were similar in follicular fluid of small, medium, and E-I large follicles. In experiment 2, E2 concentrations in large E-A follicles increased (p < 0.01) from 0 to 48 h after the PGF injection for control cows but decreased (p < 0.01) for FSH cows, whereas follicular fluid IGFBP-2 binding activity decreased from 0 to 48 h after PGF in controls and increased in FSH cows (treatment x time, p < 0.05). IGFBP-3 binding was unaffected by FSH treatment or time after administration of PGF. Profiles of IGFBP activity in homogenates of granulosa or theca cells were similar to follicular fluid profiles except for the absence of IGFBP-3 binding activity. The disappearance of binding activities for IGFBP-2 and smaller-molecular-mass IGFBPs in E-A follicles suggests a possible regulatory role for IGFBPs in follicular maturation and on aromatase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Recent studies have implicated insulin-like growth factor I (IGF-I) as an intraovarian regulator of follicular growth and differentiation. Therefore, we investigated the possibility that cattle selected for twin births may have increased concentrations of IGF-I within the ovarian follicle and(or) in peripheral blood. The estrous cycles of 14 cows with histories of producing twins and 12 control monotocous cows were synchronized with 35 mg of prostaglandin F2 alpha (PGF2 alpha). Blood and follicular fluid were collected 48-50 h post-administration of PGF2 alpha (follicular phase of the estrous cycle). Concentrations of IGF-I were measured by RIA after acid-ethanol treatment of serum or follicular fluid. Twin-producing cows had a greater (p less than 0.05) number of large (greater than or equal to 4 mm) follicles and 47% greater (p less than 0.05) concentrations of IGF-I in peripheral blood than control cows. Cattle selected for high twinning frequency also had greater (p less than 0.05) concentrations of IGF-I (+/- SE) in the two largest follicles than control (unselected) cows (327 +/- 28 vs. 243 +/- 29 ng/ml). IGF-I concentrations in pooled small (1-3.9 mm) follicles were less (p less than 0.05) than in large follicles but did not differ between control and twin-producing cattle. In addition, the percentage of IGF-I concentrations measured in follicular fluid to that of serum was lower (p less than 0.05) in small follicles than in large follicles, and was greater (p less than 0.05) in large follicles of control (93.2 +/- 5.3%) than twin-producing (76.2 +/- 4.4%) cattle.(ABSTRACT TRUNCATED AT 250 WORDS)

This paper reports results from a long-term experiment with a primary objective to increase twinning rate in cattle at the Roman L. Hruska U.S. Meat Animal Research Center. Survival of singles was 13% higher (P < .01) than that of twins at birth, and the difference in survival in favor of singles was of similar magnitude at 72 h (12.9%, P < .01), 150 d (14.8%, P < .01), and 200 d (15.2%, P < .01). Survival of calves with no dystocia was higher than survival of calves with dystocia: 8.6% (P < .01) at birth, 10.8% (P < .01) at 72 h, 12% (P < .01) at 150 d, and 12.2% (P < .01) at 200 d. The effect of dystocia on survival was greater (P < .01) in twins than in singles at birth and at 72 h. Least squares means for dystocia were 20.4% in singles compared with 42.2% in twins. Most of the dystocia in singles resulted from a traction requirement (84.7%) of normal presentations, whereas most of the dystocia in twins (77.8%) resulted from malpresentations, with 59.2% of the malpresentations accompanied with a requirement for traction. Survival in singles ranged from 10.7% to 15.3% greater than in twins at different ages when there was no requirement for assistance in either singles or twins. Calves born as singles were 8.8 kg heavier (P < .01) at birth and 28 kg heavier (P < .01) at 200 d than calves born and reared as twins. Calf weight produced per cow calving was 53.1%, 54.7%, and 58.4% greater (P < .01) at birth, 150 d, and 200 d, respectively, in cows producing twins than in cows producing singles. Cows producing twins had 65.2% more (P < .01) live calves at 200 d than cows producing singles. Single male calves gained 74 g more per day than twin males from birth to 200 d, 45 g more (P < .01) per day from 200 d to slaughter and 57 g more (P < .01) per day from birth to slaughter. Differences between twin and single males in carcass traits were small. A sample of steers from the Twinning Project gained significantly faster and produced significantly more desirable carcasses than a sample of steers from a high performance reference population. Freemartins did not differ (P < .05) from normal females in growth traits, but freemartins had higher (P < .05) scores for marbling with a higher percentage (P < .05) of USDA Choice or better quality grade carcasses and lower estimated percentage retail product.

Constraints to maximal productivity from twinning in beef cattle include increased incidence of dystocia and retained placenta, longer postpartum interval, and lower conception rate. Incidence and cause(s) of the shorter gestation length and of the increased retained placenta and dystocia associated with twinning were evaluated for 3,370 single and 1,014 twin births produced in a population of cattle selected for natural twin births. Gestation length was shorter for twin than for single pregnancies (275.6 vs. 281.3 d, P<.01) and likely contributed to the higher incidence of retained placenta associated with twin births (27.9 vs. 1.9%; P<.01). Incidence of retained placenta was also higher in the spring (March-April) than in the fall (August-September) calving season (18.3 vs. 11.4%; P<.01). The higher incidence of dystocia with twins than with singles (46.9 vs. 20.6%, P<.01) was primarily due to abnormal presentation (37.0 vs. 4.5%, respectively) of one or both twin calves at parturition. First- (40.5%) and second- (22.7%) parity dams with a single birth had more (P<.01) dystocia than older dams (13.4%), whereas dystocia was not affected (P>.10) by parity with twin births. Because of the shorter gestation length and the increased incidence of retained placenta and(or) dystocia, achievement of increased productivity with twinning in cattle necessitates intensive management of twin-producing dams and their calves during the calving season. Management of the increased dystocia can be facilitated by preparturient diagnosis of twin pregnancies, enabling timely administration of obstetrical assistance to facilitate delivery of twin calves and to increase their neonatal survival.

Expression of ovine interferon-tau (oIFNtau), a factor essential for the process of maternal recognition of pregnancy in ruminant ungulates, is restricted to the trophoblast. However, the molecular mechanisms by which oIFNtau expression is restricted to the trophectoderm have not been fully elucidated. The objective of this study was to determine whether oIFNtau gene transcription could be regulated through Cdx2 expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Human choriocarcinoma JEG3 cells were co-transfected with an oIFNtau (-654 base pair, bp)-luciferase reporter (-654-oIFNtau-Luc) construct and several transcription factor expression plasmids. Compared to -654-oIFNtau-Luc alone, transcription of the -654-oIFNtau-Luc increased more than 30 times when this construct was co-transfected with Cdx2, Ets-2, and c-jun. The degree of transcription decreased to 1/4 levels when the upstream region was reduced to -551 bp, and became minimal with further deletions; this was confirmed with the use of the reporter constructs with mutated c-jun, Ets-2, and/or Cdx2 sites. In trophoblast unrelated NIH3T3 cells, which do not support IFNtau gene transcription, the oIFNtau-Luc transcription was enhanced approximately eightfold when the cells were co-transfected with the Cdx2/Ets-2 or Cdx2/Ets-2/c-jun expression plasmids. These findings were confirmed by gel-shift assays examining Cdx binding site on the oIFNtau gene's upstream region, by immunohistochemical study identifying the presence of Cdx2 in day 15 and 17 ovine conceptuses, and by Western blot detecting Cdx2 in day 17 conceptuses. Our results indicate that oIFNtau gene transcription is regulated by Cdx2, and suggest that Cdx2 could be a key molecule in determining oIFNtau gene transcription by the trophectoderm.