Systemic infusion of exogenous ghrelin in male broiler chickens (Gallus gallus domesticus). The effect of pulse frequency, doses, and ghrelin forms on feed intake, average daily gain, corticosterone, and growth hormone concentrations.

There is limited information on the effect of exogenous ghrelin infusion on feed intake (FI) in chickens. Therefore, male broilers were used in 3 factorial experiments to determine the relationships between doses (0, 1, or 4 nM; Dose), frequency (once every two h; 2 h), once every 4th h (4 h) or continuous infusion, and ghrelin forms including acylated-ghrelin (AG) and desacylated-ghrelin (DAG) on FI, ADG, and concentrations of corticosterone and Growth Hormone (GH). Treatments were delivered via a jugular cannula, using programmable pumps for 11 consecutive days. FI and ADG were recorded, and plasma was collected. Data were analyzed using a factorial design. In Experiment 1 the effect of AG pulse frequency and doses were evaluated. There was a linear decrease in FI (P = 0.002) and a linear increase in corticosterone (P = 0.033) and GH (P = 0.011) concentrations when AG was infused. However, ADG decreased with doses (P = 0.011) only when AG was given at 2 h. In Experiment 2 the effect of ghrelin forms and doses given at 2 h was evaluated. There was a linear decrease in FI when AG was infused and a linear increase in FI when DAG was infused (P < 0.05). Birds infused with DAG gained more weight than those infused with AG. There was a linear increase in corticosterone and GH concentrations only when AG was infused (P < 0.01). In Experiment 3 the effect of continuous infusion of 2 doses (0 and 1 nM) of AG and DAG were evaluated. There was a linear decrease in FI and ADG when AG (P < 0.001) was infused and a linear increase in FI and ADG when DAG was infused (P < 0.05). There was an increase in corticosterone concentrations only when AG was infused (P = 0.022). However, GH concentrations were not affected by treatments. We concluded that AG and DAG pulse frequency and doses had a differential effect on FI, ADG, corticosterone, and GH concentrations in broiler chickens.

Capromorelin, a ghrelin receptor agonist, increases feed intake and body weight gain in broiler chickens (Gallus gallus domesticus).

Ghrelin is a hormone that induces orexigenic effects in mammals. However, in avian species, there is scant and conflictive results on the effect of ghrelin on feed intake (FI). Therefore, we evaluated the effect of a ghrelin receptor agonist (capromorelin) on FI, ADG, water intake (WI), animal behavior and concentrations of ghrelin, glucose, growth hormone (GH) and insulin in broiler chickens. One-day-old male broilers were reared as recommended by the industry. At 4 wk of age (experimental day 0; D0), birds were blocked by weight and randomly assigned to 3 treatments in 2 identical trials. Control birds received a vehicle control solution containing 0 mg/kgBW/d of capromorelin. Birds in treatments 2 and 3 received capromorelin at target doses of 6 or 12 mg/kgBW/d of capromorelin (n = 27). FI and WI were measured 3 times a day at 0700 h (Period 1; P1), 1200 h (P2) and 1700 h (P3), while BW was recorded daily. Blood samples were collected on D-1 and D5. Bird behavior (pecking, sitting and standing) was evaluated for 9 h on D2. Data were analyzed using a randomized complete block design with repeated measures over time. Orthogonal polynomial contrasts were used to determine linear and quadratic effects of increasing levels of capromorelin. Polynomial contrasts showed that capromorelin doses linearly increased FI (P = 0.002) and ADG (P = 0.019). There were no treatment, day or treatment x d interactions on glucose, ghrelin and GH concentrations. However, there was a treatment x d interaction (P = 0.041) on insulin concentrations. Concentrations of insulin were higher on D5 for the 0 and 12 mg/kgBW/d treatments as compared with D-1. Polynomial contrasts showed that capromorelin doses linearly increased number of pecks/h (P = 0.018). Per hour FI and WI was higher during P1 (i.e., 0700-1200) as compared to P2 and P3 (P < 0.001). Our observations suggest that capromorelin linearly increases feed intake; thus, the same effect of that reported in mammalian species.

Effect of short- and long-term feed restriction on ghrelin concentrations in turkeys.

One-day-old broad-breasted white turkeys (Meleagris gallopavo) were reared as recommended by industry standards. In Experiment 1, starting at 5 wk of age (WOA), birds were placed in individual cages with free access to feed and water. Blood samples were taken after 18 h of fasting (FASTING) and at 90 ± 5 min after feeding (1.5 h after feeding). In Experiment 2, birds were weighed, randomly assigned to 2 treatments, and placed in individual cages. In treatment 1 (n = 10), birds were fed ad libitum (FF), while birds in treatment 2 (n = 11) were placed on a restricted diet to allow for an average daily gain of 10.0 g per d from 4 to 11 WOA (RES). In Experiment 1, concentrations of ghrelin (P = 0.012) and glucose (P < 0.001) were increased 1.5 h after feeding compared with concentration during FASTING, whereas concentrations of adrenocorticotropic hormone (ACTH) (P < 0.001) and corticosterone (P = 0.002) were decreased 1.5 h after feeding. Concentration of insulin, free fatty acids, and ketone bodies followed a normal physiological response to fasting and feeding. Similarly, in Experiment 2, concentrations of ghrelin (P < 0.001) and glucose (P = 0.038) were increased in FF birds, whereas concentrations of corticosterone were decreased (P = 0.002) in FF birds. It could be concluded that in turkeys, preprandial (18 h of fasting) and long-term feed restriction is associated with decreased concentration of ghrelin-thus, the opposite effect of that reported in chickens and mammalian species.

The effect of passive immunization against ghrelin on feed and water intake in turkeys.

Five-week-old turkeys were used to evaluate the effect of passive immunization against ghrelin on feed and water intake and animal behavior. In experiment 1, females were reared using normal feeding and lighting management recommended by the industry. At 5 wk of age (d 0 of experiment 1), birds (n = 40) were individually caged (0.65 × 0.4 × 0.4 m) with free access to feed and water. Feed and water intake were measured 3 times a day (0800, 1200, and 1700 h) by recording the weight of feed or water offered minus any unconsumed feed or water remaining. After 3 d of adaptation to the cages (d 3), birds were stratified by BW and feed consumption and randomly assigned to a 2 × 5 factorial arrangement of treatment. Starting on d 3, turkeys were given intravenous (iv) injections (0.5, 1.0, 2.0, 4.0, or 8.0 mL) of pooled undiluted plasma obtained from pigs that were previously actively immunized against ghrelin or iv injections (0.5, 1.0, 2.0, 4.0, or 8.0 mL) of pooled undiluted plasma, obtained from nonimmunized pigs (control). In experiment 2, the 2 highest doses (i.e., 4.0 and 8.0 mL; n = 4/treatment) were repeated in a 2 × 2 factorial arrangement as described in experiment 1. A laptop computer with a built-in color camera and appropriate software was used to record birds for 9 consecutive hours, starting 4 h before treatments were applied. Video clips were saved and a human observer watched and annotated bird behavior associated with feeding, drinking, and standing. Passively immunized birds increased feed consumption (P = 0.04) compared with control animals. Water intake was not affected by treatments. There was a tendency for immunized birds to increase the number of pecks per hour and the amount of time devoted for feeding. Our data suggest that in turkeys, the effect of immunization against ghrelin on feed intake is the opposite of that observed in mammalian species.

Immunization of beef heifers against gonadotropin-releasing hormone prevents luteal activity and pregnancy: Effect of conjugation to different proteins and effectiveness of adjuvants.

Three experiments were conducted to evaluate methods of immunization against GnRH on antibody titer, luteal activity, and pregnancy in beef heifers. Experiment 1 evaluated the efficacy of adjuvants with 30 heifers. Control heifers were immunized against human serum albumin (HSA) emulsified in Freund's complete adjuvant (FCA). The other 4 treatments contained GnRH conjugated to HSA (HSA-GnRH) emulsified in FCA, Freund's incomplete adjuvant (FIA), DEAE dextran (DD) + mineral oil (MO), or DD+FIA. Treatment was in the mammary gland for all experiments. Titers against GnRH for heifers immunized against HSA-GnRH with FCA, DD+MO, or DD+FIA were greater than titers for HSA-GnRH with FIA or control heifers (P < 0.01). Body weight was reduced (P < 0.05) in control and FCA heifers compared with FIA, DD+MO, and DD+FIA heifers. Heifers immunized with DD+MO and DD+FIA had fewer granulomas in mammary glands than heifers treated with FCA (P < 0.01). In Exp. 2, 36 heifers were used to determine the effect of the protein conjugated to GnRH on titers against GnRH. Heifers (6/treatment) received a primary immunization against GnRH conjugated to HSA (HSA-GnRH), ovalbumin (OA-GnRH), or keyhole limpet hemocyanin (KL-GnRH), or heifers were immunized against each carrier protein. Antigens were emulsified in DD+FIA. Immunization of heifers against OA-GnRH, KL-GnRH, or HSA-GnRH suppressed luteal activity (P < 0.01) for 23, 16, and 12 wk, respectively, and antibody titers against GnRH were greater (P < 0.01) for 19, 5, and 7 wk, respectively, compared with heifers immunized against the carrier proteins. In Exp. 3, 90 heifers were used to determine the effect of immunization against GnRH on ovarian activity and pregnancy rate. Heifers (30/treatment) received a primary and 2 or 3 booster immunizations against GnRH conjugated to OA, and controls received a primary and 2 booster immunizations against OA. All antigens were emulsified in DD+FIA. At 8 wk after primary immunization, heifers were exposed to fertile bulls for 24 wk. Pregnancy rate was less (P < 0.01) for 3-booster heifers (13%) compared with control (83%) and 2-booster (62%) heifers. We conclude that immunization against GnRH, conjugated to OA and emulsified in DD+FIA, does not influence ADG and produces sufficient titers against GnRH to prevent estrous cycles with few mammary granulomas. Immunization against GnRH with 3 booster immunizations prevented luteal activity and pregnancy in most beef heifers for more than 4 mo.

Testis development and gonadotropin secretion in broiler breeder males.

One-day-old chicks were used to evaluate testis development and concentrations of luteinizing hormone, follicle-stimulating hormone (FSH), and testosterone during ontogenesis. Males on a conventional breeder program (control) were reared on a 15L:9D photoperiod and ad libitum food and water intake for 2 wk. On the third week, males were placed on a restricted diet and the photoperiod was reduced to 8L:16D. Males on a pedigree breeder program (broilerized) were reared on a 23L:1D photoperiod and unrestricted food and water intake for 6 wk. At 7 wk, males were placed on a restricted diet and the photoperiod was reduced to 8L:16D. On wk 18, both treatment groups were photostimulated (16L:8D) until the end of the experiment (50 wk). After photostimulation, there was an exponential increase in testis weight (TW), FSH, and testosterone concentrations. At 28 wk, TW from broilerized males were significantly heavier than those from control birds, and concentrations of luteinizing hormone, FSH, and testosterone were maximal at that time. After 28 wk, there was a significant decrease in FSH and testosterone concentrations that were associated with reduced TW. No correlation was observed between BW and TW. However, TW was highly correlated with FSH concentrations and daily sperm production. Our data suggest that management and photoperiod had a profound effect on testicular function that was associated with FSH concentrations in male broiler breeders.

Lamprey GnRH-III releases luteinizing hormone but not follicle stimulating hormone in pigs.

The effect of exogenous administration of lamprey GnRH-III (IGnRH-III) on gonadotropin secretion was evaluated in pigs. Six crossbred barrows (82.4 ± 3.5 kg body weight) were assigned randomly to a replicated 3 × 3 Latin square design to evaluate the effect of 0.1, 1.0 or 10.0 µg/kg body weight of exogenous IGnRH-III on LH and FSH secretion. To facilitate blood collection and infusion of IGnRH-III, barrows were catheterized in the jugular vein 1 day before initiation of experiments. Blood samples were taken at 10-min intervals for 6 h, starting 2 h before treatments were applied. Relative concentrations of LH and FSH were calculated by obtaining the ratio of the average concentration of each hormone 2 h after infusion divided by the average concentration during the 2 h before infusion. Relative concentrations of FSH after IGnRH-III infusion did not influence mean concentration of FSH at any of the doses; yet 10.0 µg/kg body weight had a significant effect on LH secretion (p < 0.01). Relative concentrations of LH averaged 1.2, 1.0 and 3.0 ng/ml (for doses of 0.1, 1.0 and 10.0 µg/kg body weight of IGnRH-III respectively). Only a dose of 10 µg/kg body weight elicited a significant LH increase that was associated with exogenous IGnRH-III infusion. We conclude that IGnRH-III is a weak GnRH agonist and at high doses, IGnRH-III has the ability to release LH but not FSH in barrows.

Active immunization against ghrelin decreases weight gain and alters plasma concentrations of growth hormone in growing pigs.

Ghrelin has been implicated in the control of food intake and in the long-term regulation of body weight. We theorize that preventing the ability of ghrelin to interact with its receptors, would eventually lead to decreased appetite and thereby decrease body weight gain. To test our hypothesis, pigs were actively immunized against ghrelin. Ghrelin((1-10)) was conjugated to BSA and emulsified in Freund's incomplete adjuvant and diethylaminoethyl-dextran. Primary immunization was given at 19 weeks of age (WOA), with booster immunizations given 20 and 40 days after primary immunization. Body weight (BW) and plasma samples were collected weekly beginning at 19 WOA, and feed intake was measured daily. Fourteen days after primary immunization, the percentage of bound (125)I-ghrelin in plasma from immunized pigs was increased compared with control animals (P<0.001). Voluntary feed intake was decreased more than 15% in animals that were actively immunized against ghrelin compared with controls. By the end of the experiment, immunized pigs weighed 10% less than control animals (P<0.1). Concentrations of GH were increased (P<0.05) in immunized pigs. Apoptosis was not observed in post-mortem samples obtained from the fundic region of the stomach. Our observations suggest that immunization against ghrelin induces mild anorexia. This procedure could potentially be used as a treatment to control caloric intake and obesity.

Immunization of pigs against chicken gonadotropin-releasing hormone-II and lamprey gonadotropin-releasing hormone-III: effects on gonadotropin secretion and testicular function.

The objective of this experiment was to evaluate the effects of active immunization against 2 GnRH isoforms on gonadotropin secretion and testicular function in pigs. Synthetic chicken (c) GnRH-II and lamprey (l) GnRH-III peptides, with the common pGlu-His-Trp-Ser sequence at the N-terminal omitted, were conjugated to BSA. Forty-eight male piglets were randomly assigned to 1 of 4 treatments. Pigs on treatment 1 were actively immunized against cGnRH-II, whereas pigs on treatment 2 were actively immunized against lGnRH-III. Control pigs on treatment 3 were actively immunized against the carrier protein (BSA), and pigs on treatment 4 were castrated and actively immunized against BSA. The BSA conjugate was emulsified in Freund's Incomplete Adjuvant and diethylaminoethyldextran. Primary immunization was given at 13 wk of age (WOA) with booster immunizations given at 16 and 19 WOA. Body weight and plasma samples were collected weekly beginning at 11 WOA. Treatments did not affect BW during the experimental period. Antibody titers were increased in animals immunized against cGnRH-II and lGnRH-III (P < 0.001). Cross-reactivity of the antibodies to mammalian GnRH or between cGnRH-II and lGnRH-III was minimal. Concentrations of testosterone were maximal in control boars (treatment 3) and minimal in control barrows (treatment 4) and immunized pigs (treatment x week; P < 0.01). Immunized animals had concentrations of LH (P < 0.001) and FSH (treatment x week; P < 0.03) that were less than control barrows and similar to control boars. At the end of the experiment, intact (noncastrated) pigs were exsanguinated. Testes were removed immediately; Leydig cells were isolated and treated with 0, 1, or 10 ng/mL of LH. There was an LH x GnRH treatment effect on testosterone concentrations (P < 0.03), indicating that Leydig cells were sensitive to the immunization protocol and doses of LH. Taken together, these data suggest that immunization against GnRH isoforms decreased gonadotropin secretion compared with control barrows. Additionally, immunization against cGnRH-II and lGnRH-III reduced the ability of Leydig cells to respond to LH challenges.

Influence of estradiol, progesterone, and nutrition on concentrations of gonadotropins and GnRH receptors, and abundance of mRNA for GnRH receptors and gonadotropin subunits in pituitary glands of beef cows.

Nutritionally induced anovulatory cows (n = 28) were used to determine the effect of steroids on regulation of synthesis and secretion of gonadotropins. Anovulatory cows were ovariectomized and received intravaginal inserts containing estradiol (E2), progesterone (P4), E2 and P4 (E2P4), or a sham intravaginal insert (C) for 7 d. Concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were quantified in serum and E2 and P4 were quantified in plasma. Cows were exsanguinated within 1 to 2 h after removal of intravaginal inserts and pituitary glands were collected and stored at -80 degrees C until messenger ribonucleic acid (mRNA) for gonadotropin-releasing hormone receptor (GnRH-R) and gonadotropin subunits, pituitary content of GnRH-R, and LH and FSH were quantified. Pituitary glands from five proestrous cows were harvested to compare gonadotropin characteristics between ovariectomized, anovulatory cows and intact cows. Plasma concentrations of E2 were greater (P < 0.05) in E2-treated cows than in sham-treated cows. Concentrations of P4 were greater (P < 0.05) in cows treated with P4 than in sham-treated cows. Mean serum concentrations of LH and FSH were not significantly influenced by steroid treatments. However, frequency of LH pulses of ovariectomized, nutritionally induced anovulatory cows was increased (P < 0.05) by treatment with E2 and amplitude of LH pulses was greater (P < 0.05) in cows treated with E2 or P4 than in cows treated with E2P4 or sham-treated. Quantity of mRNA for LHbeta in the pituitary gland was greater when cows were treated with P4. Concentrations of LH in the pituitary gland were not affected by steroid treatments; however, pituitary concentrations of FSH were less (P < 0.1) in E2 cows than in sham-treated cows. The number of GnRH-R was increased (P < 0.05) in cows treated with E2, but P4 treatment did not influence the number of GnRH-R. Abundance of mRNA for GnRH-R, common alpha-subunit, and FSHbeta were not affected by treatments. Pituitary concentrations of LH were greater (P < 0.05) and concentrations of FSH were less (P < 0.05) in proestrous cows than in ovariectomized, anovulatory cows treated with or without steroids. Abundance of mRNA for GnRH-R, common alpha-subunit, LHbeta and FSHbeta were similar for proestrous and anovulatory cows. We conclude that treatment of nutritionally induced anovulatory cows with progesterone and estradiol may cause pulsatile secretion of LH.

Efficacy of intramuscular treatment of beef cows with oxytetracycline to reduce mastitis and to increase calf growth.

Spring-calving multiparous Angus x Hereford cows were used to determine the efficacy of intramuscular treatment with oxytetracycline to reduce the incidence of mastitis-causing bacteria, decrease milk somatic cell counts (SCC), and increase calf growth. During 2 yr, milk samples were collected from each quarter from a total of 319 cows at 8 to 14 d after calving and at weaning, to determine the presence of bacteria and SCC. A California mastitis test (CMT) was performed on milk from each quarter of each cow at the initial sample collection. Cows with a CMT score of 1, 2, or 3 in at least one quarter, were randomly assigned to receive either an intramuscular injection of oxytetracycline (n = 63) or the control vehicle (n = 60), and cows with a CMT score of 0 or trace in all four quarters were not treated (n = 196). Calf weights were determined at birth, early lactation, and weaning. The number of somatic cells in milk and the percentage of quarters that were infected increased as CMT score increased (P < 0.01). The presence of mastitis-causing bacteria at calving increased (P < 0.05) the incidence of infection at weaning. The presence of mastitis-causing bacteria at weaning was associated with increased SCC for quarters and average SCC for cows (P < 0.01). Average SCC per cow at weaning increased (P < 0.05) as the number of infected quarters per cow increased. Treatment did not alter (P > 0.10) the percentage of cows or quarters infected with mastitis-causing bacteria or SCC of cows or quarters at weaning. Average SCC per cow was negatively correlated (P < 0.05) with calf weights at early lactation, but not with weaning weights of calves. Treatment did not influence (P > 0.10) calf weights at early lactation or at weaning. Cows with one or more dry quarters after calving had calves that weighed less at early lactation and weaning than cows with four functional quarters (P < 0.01). Intramuscular oxytetracycline treatment of beef cows that had CMT scores of 1 or greater after calving did not reduce intramammary infection rates or increase calf weights at weaning.

Influence of exogenous gonadotropin-releasing hormone on ovarian function in beef cows after short- and long-term nutritionally induced anovulation.

The effect of pulsatile infusion of gonadotropin-releasing hormone (GnRH) on follicular function was evaluated in nutritionally induced anovulatory beef cows. After 4 (short; n = 12) or 18 wk (long; n = 12) of anovulation, cows were randomly assigned within anovulatory group to either 2 microg of GnRH treatment or saline (control; i.v.) every hour for 5 d. Ovarian structures were monitored by daily ultrasonography. Growth rate of the largest follicle (P < 0.01) and maximal size of the largest follicle during treatment were greater (P < 0.01) for GnRH vs control cows. At exsanguination after 5 d of GnRH treatment, the size of the second-largest follicle was greater (P < 0.05) in short (i.e., 4 wk) anovulatory cows than in long (i.e., 18 wk) anovulatory cows and the largest follicle tended (P < 0.10) to be larger in long vs short anovulatory cows. Short anovulatory GnRH-treated cows had more small follicles than short anovulatory control cows or long anovulatory GnRH-treated or control cows (anovulation x GnRH; P < 0.10). Follicular fluid (FFL) concentrations of estradiol (P < 0.01) and androstenedione (P < 0.05) were greater in GnRH vs control cows. Concentrations of insulin-like growth factor-I were greater (P < 0.10) in large vs small follicles in cows that were anovulatory for 4 wk, but not in cows that were anovulatory for 18 wk. The amount of insulin-like growth factor-binding protein (IGFBP)-3 in FFL was greater (P < 0.05) in 4- vs 18-wk anovulatory cows. Amounts of IGFBP-2, -4, and -5 were greater (P < 0.001) in FFL of small (< 5 mm) vs large (> or = 5 mm) follicles regardless of treatment. We conclude that pulsatile treatment with GnRH for 5 d stimulates similar growth of the largest follicles in short- and long-term anovulatory beef cows, and that the duration of anovulation is not a major factor that limits follicular growth w hen anovulatory cowsare treated with GnRH. The primary intrafollicular factors associated with increased follicular size were increased concentrations of estradiol, progesterone, and insulin-like growth factor-I,and decreased concentrations of IGFBP-2, -4, and -5. Increased duration of anovulation was associated with decreased concentrations of IGF-I and IGFBP-3 in FFL.

Effects of supplemental zinc and manganese on ruminal fermentation, forage intake, and digestion by cattle fed prairie hay and urea.

One in vitro and one in vivo metabolism experiment were conducted to examine the effects of supplemental Zn on ruminal parameters, digestion, and DMI by heifers fed low-quality prairie hay supplemented with urea. In Exp. 1, prairie hay was incubated in vitro for 24 h with five different concentrations of supplemental Zn (0, 5, 10, 15, and 20 ppm) and two concentrations of supplemental Mn (0 and 100 ppm), both provided as chloride salts. Added Mn increased (P < 0.02) IVDMD, but added Zn linearly decreased (P < 0.03) IVDMD. Added Zn tended to increase the amount of residual urea linearly (P < 0.06) at 120 min and quadratically (P < 0.02) at 180 min of incubation, although added Mn counteracted these effects of added Zn. Six 363-kg heifers in two simultaneous 3 x 3 Latin squares were fed prairie hay and dosed once daily via ruminal cannulas with urea (45 or 90 g/d) and with Zn chloride to provide the equivalent of an additional 30 (the dietary requirement), 250, or 470 ppm of dietary Zn. After a 7-d adaptation period, ruminal contents were sampled 2, 4, 6, 12, 18, 21, and 24 h after the supplement was dosed. Supplemental Zn did not alter prairie hay DMI (mean = 4.9 kg/d) or digestibility, although 470 ppm added Zn tended to decrease (P < 0.06) intake of digestible DM, primarily due to a trend for reduced digestibility with 470 ppm supplemental Zn. Zinc x time interactions were detected for both pH (P = 0.06) and NH3 (P = 0.06). At 2 h after dosing, ruminal pH and ruminal ammonia were linearly decreased (P < 0.05; P < 0.01) by added Zn. At 5 h after feeding, ruminal pH was linearly increased (P < 0.05) by added Zn, suggesting that added Zn delayed ammonia release from urea. The molar proportion of propionate in ruminal fluid was linearly and quadratically increased (P < 0.02; P < 0.01) whereas the acetate:propionate ratio was linearly and quadratically decreased (P = 0.02; P < 0.05) by added Zn. Through retarding ammonia release from urea and increasing the proportion of propionate in ruminal VFA, Zn supplementation at a concentration of 250 ppm may decrease the likelihood of urea toxicity and increase energetic efficiency of ruminal fermentation.

Nutritionally induced anovulation in beef heifers: ovarian and endocrine function preceding cessation of ovulation.

Angus x Hereford heifers were used to determine endocrine and ovarian function preceding nutritionally induced anovulation. Six heifers were fed to maintain body condition score (M), and 12 heifers were fed a restricted diet (R) until they became anovulatory. Starting on d 13 of an estrous cycle, heifers were given PGF2alpha every 16 d thereafter to synchronize and maintain 16 d estrous cycles. Ovarian structures of M and R heifers were monitored by ultrasonography daily from d 8 to ovulation (d 1 of the subsequent cycle) until R heifers became anovulatory. Concentrations of LH and FSH were quantified in serum samples collected every 10 min for 8 h on d 2 and 15 (48 h after PGF2alpha), and estradiol and IGF-I were quantified in daily plasma samples from d 8 to 16 during the last ovulatory cycle (Cycle -2) and the subsequent anovulatory cycle (Cycle -1). During the last two cycles before anovulation, M heifers had 50% larger (P < .0001) ovulatory follicles than R heifers and 61% greater (P < .0001) growth rate of the ovulatory follicles. There was a treatment x cycle x day effect (P < .001) for concentrations of estradiol. The preovulatory increase in estradiol occurred in the R and M heifers during Cycle -2 but only in M heifers during Cycle -1. A treatment x cycle x day effect (P < .05) influenced LH concentrations. During Cycle -2, LH concentrations were similar for M and R heifers, but during Cycle -1, M heifers had greater LH concentrations than did R heifers. Concentrations of FSH were greater (P < .05) in R than M heifers after induced luteolysis when R heifers failed to ovulate. There was a treatment x cycle interaction (P < .05) for IGF-I concentrations, and M heifers had 4.7- and 8.6-fold greater IGF-I concentrations than did R heifers during Cycle -2 and -1, respectively. We conclude that growth rate and diameter of the ovulatory follicle, and concentrations of LH, estradiol, and IGF-I are reduced before the onset of nutritionally induced anovulation in beef heifers.

Influence of dose, frequency, and duration of infused gonadotropin-releasing hormone on secretion of luteinizing hormone and follicle-stimulating hormone in nutritionally anestrous beef cows.

Nutritionally induced anovulatory cows were ovariectomized and used to determine the relationships between dose, frequency, and duration of exogenous gonadotropin-releasing hormone (GnRH) pulses and amplitude, frequency, and concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum. In Experiment 1, cows were given pulses of saline (control) or 2 micrograms of GnRH infused i.v. during a 0.1-, 1.25-, 5-, 10-, or 20-min period. Concentrations of LH and FSH during 35 min after GnRH infusion were greater than in control cows (P < 0.01), and FSH concentrations were greater when GnRH infusions were for 10 min or less compared with 20 min. In Experiment 2, the effect of GnRH pulse frequency and dose on LH and FSH concentrations, pulse frequency, and pulse amplitude were determined. Exogenous GnRH (0, 2, or 4 micrograms) was infused in 5 min at frequencies of once every hour or once every 4th hr for 3 d. There was a dose of GnRH x frequency x day effect on LH and FSH concentrations (P < 0.01), indicating that gonadotropes are sensitive to changes in pulse frequency, dose, and time of exposure to GnRH. There were more LH pulses when GnRH was infused every hour, compared with an infusion every 4th hr (P < 0.04). Amplitudes of LH pulses were greater with increased GnRH dose (P < 0.05), and there was a frequency x dose x day effect on FSH pulse amplitude (P < 0.0006). We conclude that LH and FSH secretion in the bovine is differentially regulated by frequency and dose of GnRH infusions.

Ovarian function in nutritionally induced anoestrous cows: effect of exogenous gonadotrophin-releasing hormone in vivo and effect of insulin and insulin-like growth factor I in vitro.

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 micrograms GnRH at one pulse every 4 h (2 micrograms infused in 1.8 ml saline over 5 min) for 13 days (GnRH-4); (3) 2 micrograms GnRH at one pulse every 1 h for 13 days (GnRH-1); and (4) continuous infusion of 2 micrograms GnRH (a total of 2 micrograms 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 (> or = 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 micrograms 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.

Body condition at parturition and postpartum weight gain influence luteal activity and concentrations of glucose, insulin, and nonesterified fatty acids in plasma of primiparous beef cows.

Effects of body condition score (BCS) at parturition and postpartum weight gain on luteal activity and concentrations of glucose, insulin, and NEFA in plasma were evaluated during the breeding season in 242 primiparous beef cows over 3 yr (Y) at three locations (L). At approximately 90 d prepartum, cows were blocked by breed, expected calving date, and BCS and randomly assigned to diets so that cows would calve in BCS of 4, 5, or 6. At calving, cows were blocked by breed, calving date, and BCS and randomly allotted to gain .45 (M) or .90 (H) kg/d, from parturition to the start of breeding (postpartum nutrition; PPN). During the 60-d breeding season, weekly blood samples were obtained from cows, and progesterone, insulin, glucose, and NEFA were quantified. Progesterone concentrations greater than 1 ng/mL for more than 1 wk indicated luteal activity. To determine the possible value of blood constituents as predictors of luteal activity, categorical data analyses were performed. Cows with greater BCS at parturition had greater concentrations of glucose during breeding (P < .07). Similarly, PPN influenced glucose at the beginning of breeding, but the differences were minimal after d 28 (PPN x day; P <.001). Cows with greater BCS at parturition and M-PPN had greater concentrations of insulin during the breeding season (BCS x PPN; P < .02). Cows with a BCS of 6 at parturition had the lowest concentrations of NEFA; however, cows on H-PPN had greater concentrations of NEFA (BCS x PPN; P < .03). Location, BCS, PPN, and day affected luteal activity (P < .002). Location differences in luteal activity were associated with the interval from calving to the start of breeding. In general, a greater percentage of cows with BCS of 5 or 6 at calving had luteal activity by the end of the breeding season. Concentrations of metabolites in blood during breeding were not predictive of luteal activity. We conclude that BCS at parturition and postpartum nutrition influence concentrations of glucose, insulin, and NEFA in blood and the onset of luteal activity in primiparous beef cows.

Effect of gonadotropin-releasing hormone (GnRH) pulse frequency on serum and pituitary concentrations of luteinizing hormone and follicle-stimulating hormone, GnRH receptors, and messenger ribonucleic acid for gonadotropin subunits in cows.

Thirty-two nutritionally anestrous cows were used to determine the effect of the frequency of exogenous GnRH pulses on ovarian follicular growth, serum concentrations of LH and FSH, and concentrations of LH, FSH, GnRH receptors (GnRH-R), messenger RNA (mRNA) for GnRH-R, and mRNA for gonadotropin subunits in the pituitary. Cows were randomly assigned to one of four treatments: 2 micrograms GnRH infused (i.v.) continuously during 1 h, 2 micrograms GnRH infused during 5 min once every hour, 2 micrograms GnRH infused during 5 min once every fourth hour, or saline (control) for 13 days. Infusion of GnRH every hour increased LH concentrations in serum (P < 0.05), but FSH concentrations were not affected by GnRH infusion. Luteal activity (LA) was assessed by the presence of corpora lutea and/or serum progesterone greater than 1 ng/ml. Six of eight cows infused with GnRH every hour had LA by day 13, whereas only 25% of cows infused either continuously or with a pulse every fourth hour had LA by day 13. None of the control cows had LA during the experiment (P < 0.01). Concentrations of LH and FSH in the pituitary were significantly reduced when GnRH was infused hourly or continuously. Concentrations of common alpha and FSH beta mRNA were not influenced by treatment. However, continuous infusion of GnRH decreased (P < 0.05) LH beta mRNA subunit. Concentrations of GnRH-R (P < 0.1) and GnRH-R mRNA (P < 0.05) were reduced when GnRH was infused continuously. We concluded that pulsatile secretion of LH is necessary for follicular growth and LA in beef cattle, and GnRH treatment differentially regulates LH and FSH gene transcription and serum concentrations of LH and FSH in cattle.