W.J. Enright

Effect of dietary intake on concentrations of insulin-like growth factor-I in plasma and follicular fluid, and ovarian function in heifers☆

The objective of this study was to determine if alterations in dietary intake of heifers can influence IGF-I concentrations in plasma and(or) follicular fluid (FFL), size of follicles, and steroid concentrations in FFL (as an indicator of steroidogenic capacity). Cyclic heifers [n = 23; mean +/- SE body weight (BW) = 373 +/- 7 kg] were individually fed for 10 weeks either: a) 1.8% of BW in dry matter (DM) per d (GAIN; n = 7), b) 1.1% of BW in DM per d (MAINT; n = 8) or c) 0.7% of BW in DM per d (LOSE; n = 8). After 10 wk of treatment, heifers were ovariectomized 36-40 hr after the second injection of prostaglandin F2 alpha analog (2 injections 11 d apart), and plasma and ovaries were collected. Heifers weighed 444 +/- 13,387 +/- 8 and 349 +/- 9 kg in the GAIN, MAINT and LOSE groups, respectively, at time of ovariectomy. Mean diameter of follicles greater than or equal to 10 mm was greater (P less than .05) for GAIN (15.6 mm) than for MAINT (11.0 mm) or LOSE (12.5 mm) heifers. Numbers of follicles and concentrations of IGF-I in plasma and FFL did not differ (P greater than .20) between LOSE, MAINT and GAIN heifers. Progesterone concentrations were greater (P less than .05) in small and medium follicles of GAIN than MAINT or LOSE heifers, but were unaffected by diet in large follicles. Estradiol concentrations in FFL in small, medium and large follicles were unaffected (P greater than .20) by dietary treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of continuous administration of gonadotropin-releasing hormone (GnRH) or a potent GnRH analogue on blood luteinizing hormone and testosterone concentrations in prepubertal bulls

The objective was to determine if continuous administration of gonadotropin-releasing hormone (GnRH) or a potent analogue (GnRH-A) for 28 or 56 d would decrease blood concentrations of luteinizing hormone (LH) and testosterone (T) in 5-month old bull calves. Treatments (5 calves/treatment), using a completely randomized design, were: control (vehicle), 3.3, 10 and 30 micrograms GnRH, and 3.3 and 10 micrograms GnRH-A (Leuprolide) per kg bodyweight/d for 28 d, administered via subcutaneously implanted mini-osmotic pumps. A second pump was implanted on day 28 in controls and bulls receiving 10 micrograms GnRH-A until day 56. Blood samples were taken every second day for plasma LH and T concentrations, and every 15 min for 6 hr on days 1, 14 and 27 (and days 40 and 55 where applicable) for plasma LH concentrations. There was an increase (P < 0.05) in basal plasma LH in response to GnRH and GnRH-A on day 1 (1.6, 2.1, 2.1, 2.5, 2.1 and 1.9 ng/ml for control, 3.3, 10 and 30 micrograms GnRH, and 3.3 and 10 micrograms GnRH-A, respectively; pooled s.e.d. = 0.2), but not on days 14 or 27. The number of LH pulses/6 hr was similar on day 1 for GnRH-treated and control calves, but there was a linear decrease (P < 0.05) in pulse frequency in response to GnRH doses on days 14 (1.8, 1.2, 0.6 and 0.0 for control, 3.3, 10 and 30 micrograms GnRH; s.e.d. = 0.5) and 27 (1.8, 1.0, 0.0 and 0.0; s.e.d. = 0.3). On days 1, 14 and 27, both GnRH-A doses suppressed (P < 0.05) LH pulsatility. GnRH (days 1-14) and GnRH-A (days 1-27) increased (P < 0.05) plasma T in a dose-dependent manner. Mean T was greater (P < 0.05) in 10 micrograms GnRH-A-treated than in control calves during days 29-41 (4.6 and 1.8 ng/ml; s.e.d. = 0.6) and days 43-55 (4.1 and 1.8 ng/ml; s.e.d. = 0.4). In conclusion, continuous administration of GnRH or GnRH-A to 5-month old bulls for 28 or 56 d chronically decreased LH pulse frequency, had no effect on basal LH, but increased testosterone concentrations.

Effects of cimaterol administration on plasma concentrations of various hormones and metabolites in friesian steers

The aim of the experiment was to determine the acute and chronic effects of the beta-agonist, cimaterol, on plasma hormone and metabolite concentrations in steers. Twelve Friesian steers (liveweight = 488 +/- 3 kg) were randomly assigned to receive either 0 (control; n = 6) or .09 mg cimaterol/kg body weight/day (treated; n = 6). Steers were fed grass silage ad libitum. Cimaterol, dissolved in 140 ml of acidified distilled water (pH 4.2), was administered orally at 1400 hr each d. After 13 d of treatment with cimaterol or vehicle (days 1 to 13), all animals were treated with vehicle for a further 7 d (days 14 to 20). On days 1, 13 and 20, blood samples were collected at 20 min-intervals for 4 hr before and 8 hr after cimaterol or vehicle dosing. All samples were assayed for growth hormone (GH) and insulin, while samples taken at -4, -2, 0, +2, +4, +6 and +8 hr relative to dosing were assayed for thyroxine (T4), triiodothyronine (T3), cortisol, urea, glucose and non-esterified fatty acids (NEFA). Samples taken at -3 and +3 hr relative to dosing were assayed for IGF-I only. On day 1, cimaterol acutely reduced (P less than .05) GH and urea concentrations (7.6 vs 2.9 +/- 1.4 ng/ml; and 6.0 vs 4.9 +/- 0.45 mmol/l, respectively; mean control vs mean treated +/- pooled standard error of difference), and increased (P less than .05) NEFA, glucose and insulin concentrations (160 vs 276 +/- 22 mumol/l, 4.1 vs 6.2 +/- 0.15 mmol/l and 29.9 vs 179.7 +/- 13.9 microU/ml, respectively). Plasma IGF-I, T3, T4 and cortisol concentrations were not altered by treatment. On day 13, cimaterol increased (P less than .05) GH and NEFA concentrations (7.7 vs 14.5 +/- 1.4 ng/ml and 202 vs 310 +/- 22 mEq/l, respectively) and reduced (P less than .05) plasma IGF-I concentrations (1296 vs 776 +/- 227 ng/ml). Seven-d withdrawal of cimaterol (day 20) returned hormone and metabolite concentrations to control values. It is concluded that: 1) cimaterol acutely increased insulin, glucose and NEFA and decreased GH and urea concentrations, 2) cimaterol chronically increased GH and NEFA and decreased IGF-I concentrations, and 3) there was no residual effect of cimaterol following a 7-d withdrawal period.

Energy level in winter diets of Fallow deer: effect on plasma levels of insulin-like growth factor-I and sex ratio of their offspring☆

To determine the effect of dietary energy level in the first winter on subsequent puberty onset, pregnancy, growth, and secretion of progesterone and insulin-like growth factor-I (IGF-I) in Fallow deer, prepubertal deer were fed either a high (H) or a low (L) energy diet in a randomised complete block design. June-born female Fallow deer were fed ad libitum either 12.5MJ/kg DM (H, n=29) or 10MJ/kg DM (L, n=29) in pelleted rations once per day during the winter (approximately 4-10 months of age) preceding puberty. Blood samples were collected twice weekly during the peripubertal period. During the winter feeding period, DM intake was similar for both groups but average daily gain was greater (P<0.05) for deer fed the H versus L diet. Onset of puberty was not affected (P>0.10) by dietary treatment. Concentrations of progesterone in plasma did not differ (P>0.10) between dietary treatments before or after puberty, increasing after puberty in both groups, and reaching maximal levels 8-12 days after the onset of puberty. Concentrations of plasma IGF-I increased (P<0.05) before puberty in both groups reaching maximal levels 3-4 days before the onset of puberty but did not differ (P>0.10) between H and L diets before or after puberty. Of the 28 does fed the H diet that calved, 75% of the calves born were male versus 46% in the L diet (P<0.05). In conclusion, increased plasma IGF-I concentrations were associated with the onset of puberty in Fallow deer regardless of the level of dietary energy intake during the preceding winter. Increased dietary energy during winter does not alter pregnancy rates but does alter sex ratio of calves born.

The effect of different levels of gonadotropin-releasing hormone antibody titres on plasma hormone concentrations, sexual and aggressive behaviour, testis size and performance of bulls

To determine the effect of different levels of gonadotropin-releasing hormone (GnRH) antibody titres (AT) on plasma hormone concentrations, behaviour, testes size and performance, Friesian bull calves (no. = 72) were immunized against either human serum albumin (HSA) conjugated to Cys-Gly-GnRH (no. = 48; immunized) or HSA (no. = 24; controls) at 8 to 10 weeks of age. One booster immunization was administered on either day 28 or 56 (no. = 24 GnRH-immunized and no. = 12 control per booster day). Based on AT 1 week post respective booster, 12 immunized and six control bulls were allocated to one of three AT groups, control (C), medium (M) and high (H) with mean AT of 0·3 (s.e. 0·1), 32 (s.e. 2) and 51 (s.e. 2)% binding at a plasma dilution of 1: 160; respectively. Bulls were blood sampled and weighed every 14 days, and testicular measurements taken every 28 days, from days 0 to 533. Behaviour was observed for 4 h once weekly while bulls were together at pasture and, within AT group, for 20 min 1 week before slaughter. At slaughter, testes and carcass measurements were made. Appropriate data were analysed by ANOVA and correlation coefficients (i) determined. Behavioural data were analysed using x 2 . Mean AT for M and H treatment groups during the experiment were 36·5 and 44·8% binding at a 1: 160 dilution ( P > 0·05, pooled s.e.d. 5·9%), respectively. Level of AT decreased ( P P P P

Interaction between dietary intake and ovariectomy on concentrations of insulin-like growth factor-I, GH and LH in plasma of heifers

The objective of this study was to determine if alterations in dietary intake and(or) ovariectomy influence plasma concentrations of IGF-I, GH and LH in heifers. Cyclic heifers (n = 23) were individually fed for 10 wk either 1) 1.8% of body weight in dry matter per day (GAIN; n = 7) 2) 1.1% of body weight in dry matter per day (MAINT; n = 8); or 3) 0.7% of body weight in dry matter per day (LOSE; n = 8). After 10 wk of dietary treatment, heifers were ovariectomized 36 to 40 h following the second injection of prostaglandin F2alpha analog (2 injections 11 d apart). Heifers weighed 444 +/- 13, 387 +/- 8, and 349 +/- 9 kg in the GAIN, MAINT and LOSE groups, respectively, at the time of ovariectomy; the average daily weight gains during the 10-wk period were 0.96, 0.17 and -0.31 kg, respectively (P < 0.001), for the 3 groups. Blood plasma was collected for 6 h at 15-min intervals 1 d before and 2 wk after ovariectomy. The MAINT group of heifers had greater IGF-I concentrations than either the LOSE or GAIN groups; IGF-I decreased (P < 0.05) by 23 and 35% after ovariectomy in the MAINT and GAIN groups, respectively, but did not change (P > 0.10) in the LOSE groups. Dietary restriction tended to increase (P < 0.10) GH pulse frequency and mean GH. Ovariectomy had no effect (P > 0.10) on mean GH or GH pulse frequency but increased (P < 0.05) GH pulse amplitude in the GAIN groups. Dietary treatment had no effect (P > 0.10) on mean LH, or LH pulse amplitude and frequency. However, across dietary treatments, ovariectomy increased mean LH and LH pulse frequency but did not affect (P > 0.10) LH pulse amplitude. In summary, dietary restriction increased GH secretion while ovariectomy increased LH secretion. There appears to be a dichotomy of response between GH and IGF-I in the way heifers respond to dietary treatment and(or) ovariectomy.

Effects of growth hormone-releasing factor and vasoactive intestinal peptide on proliferation and steroidogenesis of bovine granulosa cells

Recent studies have suggested that growth hormone-releasing factor (GRF), like vasoactive intestinal peptide (VIP), may enhance follicle-stimulating hormone (FSH)-stimulated steroidogenesis in cultured rat granulosa cells (GC). Because effects of GRF or VIP on GC proliferation have not been reported, we evaluated and compared the effect of GRF to that of VIP using cultured bovine GC. Undifferentiated GC from 1-5 mm bovine follicles were established for 2 days in medium containing 10% fetal calf serum, washed and then cultured in chemically defined medium for an additional 2 days. Two-day treatment with 2.5-1000 ng/ml of VIP had no effect (P greater than 0.05) on proliferation or progesterone production of bovine GC in the presence or absence of 200 ng/ml FSH. In comparison, 100, 250, 500, 1000 or 2000 pg/ml of human [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 analog caused a dose-dependent stimulation (P less than 0.05) of GC proliferation in the absence and presence of 5 micrograms/ml insulin. However, the GRF analog had no effect (P greater than 0.05) on GC progesterone production (expressed as ng/10(5) cells/24 h) in the absence or presence of 5 micrograms/ml insulin. The effects of GRF analog on progesterone production and cell proliferation were not influenced by co-culture with 200 ng/ml FSH. GRF(1-44)-NH2 also stimulated cell proliferation but had no effect on basal or FSH-induced progesterone production. These results suggest that GRF may play a role in GC proliferation during follicular development in the bovine.

Active immunization of post-pubertal heifers against prostaglandin F2α to suppress oestrous behaviour: effect of adjuvant and dose of immunogen

Two experiments were performed to develop an effective prostaglandin F2α immunization protocol to suppress oestrous behaviour in beef heifers. In Experiment 1, a 3 × 2 factorial plan (n=4–5 per treatment) was used to test three doses (3.3, 10 and 30 mg) of a prostaglandin F2α- human serum albumin (PGF-HSA) conjugate as the immunogen and two adjuvants, GNE (proprietary product; Intervet, The Netherlands) and diethylaminoethyl (DEAE)-dextran. Heifers (n=5) in a control group were untreated. Booster immunizations were given on Days 42 and 145 after the primary immunization (Day 0) and data collection for statistical purposes ended on Day 297. After Day 42 the incidence of oestrous behaviour was: (1) greater (P < 0.05) for control than immunized heifers (4.3 and 2.2, respectively), (2) greater (P < 0.05) for heifers immunized using GNE than for heifers immunized using DEAE-dextran (2.6 and 1.9, respectively), and (3) greater for heifers immunized with 30 mg of immunogen than for those immunized with either 3.3 or 10 mg (3.1, 1.7 and 1.9, respectively). Suppression of oestrous behaviour was accompanied by formation of a persistent corpus luteum (CL). Persistent CL were formed in ten of the 28 immunized heifers and the mean (± standard error of the mean) duration of persistence was 397 ± 85 days. In Experiment 2, a 2 × 2 factorial plan (n=6–7 per treatment) was used to test two doses (1 and 10 mg) of the PGF-HSA conjugate as the immunogen and two adjuvants, non-ulcerative Freunds adjuvant (NUFA) and DEAE-dextran. A control group was untreated (n=6). Booster immunization was given on Day 183 after the primary immunization (Day 0) and the experiment finished on Day 384. Antibody titres were higher (P < 0.05) in NUFA-treated heifers than in DEAE-dextran-treated (1 mg) heifers in the 183- to 283-day period. After Day 183, oestrous behaviour was suppressed in 26 out of the 27 immunized heifers. Persistent CL were maintained for longer (P < 0.05) in NUFA-treated heifers (245 days) than in DEAE-dextran-treated heifers (166 days) but there was no difference due to dose of immunogen (208 and 203 days, 1 and 10 mg, respectively). It is concluded that immunization against PGF-HSA results in suppression of oestrous behaviour in heifers due to prolongation of the life-span of the CL; however, efficacy of response is dependent on the immunization regime used.

The effect of immunization of heifers against α 1–26 inhibin conjugate on the superovulatory response to pFSH

A total of 121 heifers was blocked by time and diet and then randomly assigned, within block, to an inhibin-immunized (I) or a control (C) group. Immunized heifers (n = 61) received a primary immunization (Day 0) with 0.33 mg of an alpha 1-26 bovine inhibin fragment-human serum albumin (HSA) conjugate injected with non-ulcerative Freunds and DEAE-dextran adjuvants. Booster injections were given on Days 28 and 56. Control heifers (n = 60) received HSA and adjuvants. On Days 56 and 83 the ovaries of heifers were examined by ultrasound to determine the ovulation rate, and blood samples were collected for antibody titer determination. On Day 84, 61 heifers (C, n = 30; I, n = 31) received a total of 24 mg of porcine follicle stimulating hormone (pFSH), while 60 heifers (C, n = 30; I, n = 30) received 12 mg im pFSH, which was administered twice daily for 4 d in decreasing doses during the mid-luteal phase of the estrous cycle. Luteolysis was induced with prostaglandin F(2alpha) analog. The heifers were artificially inseminated and were slaughtered 7 d after estrus. Embryos were recovered and morphologically graded on a scale of 1 to 5 (1 = excellent; 5 = degenerated). Antibody titers (percentage binding at 1:125 serum dilution) differed (P < 0.01) between Group C and I heifers at Days 56 (0.1 vs 30%) and 83 (0.2 vs 37%), and 26% of Group I and 1% of Group C heifers (P < 0.01) had twin ovulations on Day 83. The mean number of embryos recovered was reduced (P = 0.02) in Group I heifers (8.9 +/- 1.2) compared with C heifers (12.1 +/- 1.1); however, the mean number of freezable embryos (Grades 1 and 2) was not affected (P = 0.61) by immunization, and there was no interaction with pFSH (P = 0.36). Ovulation rate as well as embryo yield and quality were not different (P > 0.10) between Group C and I heifers when 12 mg pFSH were administered; however, immunization decreased the superovulatory response to 24 mg of pFSH.

Follicular growth and serum follicle-stimulating hormone (FSH) responses to recombinant bovine FSH in GnRH-immunized anoestrous heifers

The effect of recombinant bovine FSH (rbFSH) administration on serum FSH and follicular growth was determined in 16 GnRH-immunized heifers (primary day 0 and booster day 28 using 0·4 mg human serum albumin (HSA) – Cys-Gly-GnRH in DEAE-dextran). All heifers produced GnRH antibody titres (mean 58·8 (s.e. 2·8)% binding at 1: 640 serum dilution on day 42), follicular growth ceased (follicles ≤ 4 mm) between days 33 and 49, progesterone concentrations were ≤ 0·5 ng/ml in all heifers by day 53, and pulsatile LH secretion was markedly reduced (v. luteal phase control heifers) on day 63. Heifers were blocked by antibody titre and LH concentrations on day 63 and assigned (no. = five or six per treatment) on day 78 to: (1) control (saline given i.m. four times per day for 4 days), (2) 0·5 mg equivalent (USDA bFSH BP 1) of rbFSH (Granada Biosciences, TX) given i.m. four times per day for 6 days (12 mg in total) or 3) 1·5 mg rbFSH given i.m. four times per day for 4 days (24 mg in total). Ovaries of heifers were examined by ultrasound and blood samples were collected at 3- to 6-h intervals from days –1 to 10 and then twice a day until follicles ≥ 5 mm were no longer detectable. The increase in serum FSH concentrations was different ( P v . 3·3 (s.e. 0·3) days) or LF (5·3 (s.e. 0·3) v . 4·8 (s.e. 0·8) days) or in the duration of the presence of MF and/or LF (13·2 (s.e. 4·5) v . 16·6 (s.e. 2·6) days). Increasing dose of rbFSH increased ( P