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L. J. Spicer and S. A. Zinn

Summary. Concentrations of cortisol were determined in pooled fluid of small (< 10 mm) and large (≥ 10 mm) follicles of cyclic cattle (Exp. 1), and in fluid of the largest follicle of 17 post-partum anovulatory cows (Exp. 2). In Exp. 1, concentrations of cortisol in small follicles were greater (P < 0·05) than in large follicles (14·7 versus 13·2 ng/ml), and varied significantly with stages of the cycle; small and large follicles had the highest cortisol concentration during the early luteal phase of the cycle. Large follicles had 2-fold greater concentrations of oestradiol than did small follicles, whereas small follicles had 2-fold greater concentrations of androstenedione than did large follicles. Across pools of follicular fluid, cortisol concentrations were correlated only to androstenedione concentrations (r = 0·65, P = 0·07). In Exp. 2, concentrations of cortisol did not significantly differ between oestrogen-active (oestradiol > progesterone in follicular fluid) and oestrogen-inactive (progesterone > oestradiol) follicles, although oestrogen-active follicles had a 24-fold greater concentration of oestradiol than did oestrogen-inactive follicles. Cortisol concentrations were correlated to hCG binding capacity of thecal cells (r = − 0·35, P = 0·08) and to follicular diameter (r = 0·45, P < 0·05). These results suggest that normally fluctuating concentrations of cortisol in follicular fluid of cattle play little or no active role in follicular differentiation in vivo.

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L. J. Spicer, J. P. Hanrahan, M. T. Zavy, and W. J. Enright

To determine whether concentrations of insulin-like growth factor-1 (IGF-1) in blood of ewes change during the oestrous cycle, oestrus was synchronized for 45 ewe lambs from four genotypes (Finn ewes selected for low ovulation rate (LF), Finn ewes selected for high ovulation rate (HF), unselected control Finn ewes (CF) and Cambridge ewes (CAM)) using progestin sponges and blood samples were taken every day from day 0 (day 0 = day of progestin sponge removal) to day 5, and then every second or third day until 3 days after the next oestrus. Ovulation rates (determined via laparoscopy) following the first oestrus were 1.3, 3.3, 2.0 and 2.1 for LF, HF, CF and CAM groups, respectively. In a second experiment, jugular and utero–ovarian venous blood samples were collected simultaneously from seven Rambouillet crossbred ewes during the mid-luteal phase of an oestrous cycle to determine whether the ovary is a major source of blood IGF-1. In the first experiment, plasma IGF-1 concentrations increased (P < 0.05) between days 0 and 3, and then decreased (P < 0.05) between days 4 and 8 in all groups. IGF-1 concentrations increased again at the subsequent oestrus. There was no significant difference in plasma IGF-1 between HF and LF ewe lambs. Overall, plasma IGF-1 was lowest (P < 0.05) in CAM and highest in CF ewe lambs at all stages. Plasma IGF-1-binding protein activity did not vary with stage of cycle or differ (P > 0.10) among genotypes. Among LF, HF and CF ewe lambs, ovulation rate was not correlated with plasma IGF-1 or IGF-1-binding protein activity. In the second experiment, serum concentrations of IGF-1 in jugular (174 ± 38 ng ml−1) and utero–ovarian (188 ± 43 ng ml−1) venous blood did not differ (P > 0.10) but were highly correlated (r = 0.96). We conclude that (i) plasma concentrations of IGF-1 increase during oestrus in cyclic ewes, (ii) plasma concentrations of IGF-1 are influenced by genotype in sheep but are not genetically associated with ovulation rate, (iii) plasma IGF-1-binding protein activity is not influenced by stage of cycle or genotype in sheep, and (iv) the ovary does not appear to be a major source of blood IGF-1.

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T. D. Hamilton, J. A. Vizcarra, R. P. Wettemann, B. E. Keefer, and L. J. Spicer

Ovarian function of nutritionally induced anoestrus cows was evaluated in vivo (Expt 1) and in vitro (Expt 2). In Expt 1, 32 nutritionally induced anoestrous beef cows were divided into four treatment groups receiving: (1) saline infusions at one pulse every 4 h for 13 days (control); (2) 2 μg GnRH at one pulse every 4 h (2 μg infused in 1.8 ml saline over 5 min) for 13 days (GnRH-4); (3) 2 μg GnRH at one pulse every 1 h for 13 days (GnRH-1); and (4) continuous infusion of 2 μg GnRH (a total of 2 μg in 34 ml h−1) for 13 days (GnRH-C). On the last day of treatment, cows were killed, ovaries were removed and follicular fluid samples (n = 149) were collected. The percentage of cows with luteal activity on day 13 was significantly different (P < 0.01) among treatments (0, 25, 75 and 25% for control, GnRH-4, GnRH-1 and GnRH-C cows, respectively). Owing to the large percentage of ovulatory cows in the GnRH-1 group (n = 6), anovulatory cows (n = 2) were removed from this treatment group for statistical analysis, as were cows with luteal tissue from the GnRH-4 (n = 2) and GnRH-C (n = 2) groups. The numbers of small (1.0–4.9 mm) and medium plus large (≥ 5 mm) follicles were not affected (P > 0.10) by treatment. However, GnRH-4 cows (n = 6) had greater (P < 0.05) concentrations of oestradiol in follicular fluid than did control (n = 8) but not GnRH-1 (n = 6) or GnRH-C (n = 6) cows. Concentrations of insulin-like growth factor I were greater (P < 0.05) in the follicular fluid of GnRH-1 cows than in all other treatment groups. Concentrations of androstenedione and progesterone in follicular fluid were not affected (P > 0.10) by treatment or follicle size. The binding activity of insulin-like growth factor binding proteins was not affected by GnRH treatment. However, the binding activity of insulin-like growth factor binding protein 2, 29–32 kDa and 22 kDa insulin-like growth factor binding proteins were greater (P < 0.05) in small versus medium plus large follicles. In Expt 2, granulosa cells were collected from nutritionally anoestrous cows to determine whether ovarian cells from anoestrous cows have the capacity to respond to insulin-like growth factor I or insulin in vitro. Both insulin-like growth factor I (20 and 200 ng ml−1) and insulin (10, 100 and 1000 ng ml−1) increased (P < 0.05) granulosa cell proliferation and progesterone production. In conclusion, pulsatile infusion of 2 μg GnRH (every 1 or 4 h) for 13 days into nutritionally induced anoestrous cows results in increased intrafollicular oestradiol and insulin-like growth factor I concentrations and can stimulate ovulation without markedly affecting concentrations of androstenedione or progesterone, or the binding activity of insulin-like growth factor binding proteins, in follicular fluid. In addition, granulosa cells from nutritionally induced anoestrous cows have the capacity to respond to insulin-like growth factor I and insulin in vitro, indicating that the decrease in trophic factors observed with restricted feeding does not reduce the response of the ovary to insulin-like growth factor I and insulin.

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K. Leung, V. Padmanabhan, L. J. Spicer, H. A. Tucker, R. E. Short, and E. M. Convey

Summary. Thirty primiparous suckling beef cows were slaughtered on Day 7, 14, 28,42 or 56 after parturition. Some had resumed oestrous cyclicity by the time they were slaughtered on Days 42 and 56. Amongst acyclic cows between Days 7 and 42, pituitary LH concentrations and basal and GnRH-induced release of LH from pituitary explants doubled. Pituitary FSH concentration and basal release in FSH increased only by 15–20%, while GnRH-induced release of FSH in vitro was unchanged. During post-partum anoestrus, overall mean concentrations of serum FSH did not change, whereas overall mean concentrations and pulse amplitudes of serum LH increased. Numbers and affinity constants of GnRH-binding sites in pituitary glands remained constant during the post-partum period studied. We conclude that, under these experimental conditions, numbers and affinity constants of GnRH-binding sites in the pituitary gland of post-partum beef cows do not limit the ability of the anterior pituitary gland to release gonadotrophins.

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M. G. Murphy, W. J. Enright, M. A. Crowe, K. McConnell, L. J. Spicer, M. P. Boland, and J. F. Roche

Summary. Friesian × Hereford heifers (n = 19; mean ± s.e.m. body weight (BW) = 375 ± 5 kg) were used in a randomized incomplete block design. Heifers were fed 0·7 (n = 7; L), 1·1 (n = 7; M) or 1·8% (n = 5; G) of BW in dry matter (DM)/day for 10 weeks. Ovaries were examined by ultrasound, for one oestrous cycle, from week 5 of treatment. Maximum diameter of dominant follicles was smaller (P < 0·05) in L (11·8 ± 0·1 mm) than in M (13·7 ± 0·2 mm) or G (13·2 ± 0·3 mm) heifers. Growth rate (mm/day) of dominant follicles during the oestrous cycle was not affected (P > 0·05) by dietary intake. Persistence of dominant follicles was shorter (P < 0·05) in L (9·8 ± 0·2 days) than in M (11·9 ± 0·3 days) or G (12·7 ± 0·4 days) heifers. Three dominant follicles were identified during the oestrous cycle of 5 of 7 L, 3 of 7 M and 1 of 5 G heifers (P < 0·10); 2 dominant follicles were identified in the remaining heifers (n = 2 of 7, 4 of 7 and 4 of 5, respectively). Length of the luteal phase and luteal-phase concentrations of progesterone were not affected (P > 0·05) by treatment. Low dietary intake reduced the diameter and persistence of dominant follicles during the oestrous cycle of beef heifers and tended to increase the proportion of oestrous cycles with 3 dominant follicles.

Keywords: follicle; heifer; ultrasound; nutrition

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M D Ashworth, J W Ross, D R Stein, D T Allen, L J Spicer, and R D Geisert

Early exposure of pregnant gilts to oestrogen, prior to the normal period of porcine conceptus oestrogen secretion, disrupts the uterine environment resulting in complete embryonic mortality during the period of placental attachment to the uterine surface. The current study evaluates the uterine insulin-like growth factor (IGF) system following endocrine disruption of early pregnancy in gilts through exposure to exogenous oestrogen on Days 9 and 10 of gestation. Endometrial IGF gene and protein expression, IGF-I receptor (IGF-IR) gene expression, and uterine lumenal content of IGF binding proteins (IGFBPs) were evaluated in control and oestrogen-treated gilts on Days 10, 12, 13, 15 and 17 of gestation. Oestrogen treatment altered endometrial IGF-I and IGF-IR gene expression on Days 12 and 13 of gestation. Uterine content of IGF-I and IGF-II in control gilts was greatest on Days 10, 12, and 13 followed by a four- to sixfold decrease on Day 15 of gestation. Oestrogen treatment caused a premature proteolysis of IGFBPs within the pregnant pig uterus on Day 10 of gestation, and an earlier decline in uterine lumenal IGF-I content. Results demonstrate that early exposure of pregnant gilts to oestrogen causes premature loss of uterine IGFs during the period of conceptus elongation. Timing for the release of uterine IGFs during early porcine conceptus development may play an important function in the ability of the conceptus to attach and survive during the establishment of pregnancy.

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R. B. Simpson, C. C. Chase, L. J. Spicer, R. K. Vernon, A. C. Hammond, and D. O. Rae

Angus (n = 14) and Brahman (n = 14) cows were used to evaluate the effects of insulin administered concomitantly with FSH in a superovulation regimen. Cows were allotted to four pen replicates by treatment and breed, and received FSH (i.m.) twice a day for 5 consecutive days (first day of injections = day 0 of study) plus concomitant administration of either saline (control) or long-acting bovine insulin (0.25 iu kg−1 body mass; s.c.). Blood samples were collected at intervals of 6 h during the injection period and analysed for plasma insulin, glucose, insulin-like growth factor I (IGF-I) and IGF-I binding protein (IGFBP) activity. Cows were ovariectomized on day 5. The number and diameter of follicles were recorded. Follicular fluid was aspirated for determination of IGF-I, IGFBP activity, oestradiol and progesterone. Mean plasma concentration of glucose was lower in insulin-treated than in control cows averaged over days 1–5 (56 ± 3 versus 82 ± 3 mg dl−1; P< 0.01). Plasma concentration of IGF-I and IGFBP activity were not affected (P>0.10) by treatment, but were higher in Brahman than in Angus cows (IGF-I: 41 ± 6 versus 19 ± 6 ng ml−1, P < 0.05; IGFBP activity: 17.5 ± 0.4 versus 15.8 ± 0.04% (10 μl)−1; P < 0.03). Insulin treatment did not affect the number of small (1.0–3.9 mm), medium (4.0–7.9 mm) or large (> 8.0 mm) follicles. Brahman cows had a greater (P<0.01) number of medium and total follicles (19.4 ± 2.5 and 60.5 ± 5.5, respectively) than did Angus cows (7.5 ± 2.6 and 30.5 ± 5.6, respectively). Diameter of large follicles was greater in insulin-treated than in control cows (11.4 ± 0.2 versus 10.6 ± 0.1 mm; P < 0.05). Follicular fluid IGF-I concentration in large follicles was higher in insulin-treated Brahman cows (60 ± 2 ng ml−1) than in control Brahman cows (37 ± 2 ng ml−1), but was lower in insulin-treated Angus cows (31±3 ng ml−1) than in control Angus cows (38 ± 2 ng ml−1; treatment × breed interaction, P< 0.01). IGFBP activity in fluid from large follicles was not affected by insulin treatment in Brahman cows but was reduced (P < 0.05) by insulin treatment in Angus cows. In large follicles, concentration of oestradiol in follicular fluid was higher in insulin-treated than in control cows (144 ± 36 versus 29 ± 28 ng ml−1; P< 0.10), while progesterone concentration was lower in insulin-treated than in control cows (71 ± 24 versus 178±19 ng ml−1; P< 0.05). The percentage of large follicles that were oestrogen-active was higher (P < 0.05) in insulin-treated cows (54 ± 4.9%) than in control cows (25 ± 4.5%). In summary, insulin treatment resulted in increased follicle diameter, higher oestradiol and lower progesterone concentrations in the fluid of large follicles, but did not increase the number of follicles. Brahman cows had higher plasma and follicular fluid concentrations of IGF-I, lower oestradiol concentrations and a greater number of follicles than did Angus cows.