Fasting-induced changes in ovulation rate, plasma leptin, gonadotropins, GH, IGF-I and insulin concentrations during oestrus in ewes

The aim of this experiment was to study the changes in the hormonal status and ovulation rate (OR) evoked by starvation during the follicular phase of the oestrous cycle in ewes. To achieve this goal, 12 female crossbreed sheep were synchronized and then half of them were fasted from the 12th to the 16th day of the oestrous cycle. On the 16th day, analysis of hormones and insulin-like growth factor-I (IGF-I) were performed in 10-min intervals. Then, on the 6th day of the following oestrous cycle, the OR in all ewes was determined by laparoscopy. Fasting reduced significantly (P < 0.05) the OR in ewes (1.25 +/- 0.50) in comparison with control (1.75 +/- 0.50). The drop in the OR was coincident with a significant (P < 0.001) decrease in the plasma concentration and pulse amplitude of leptin (0.29 +/- 0.08 ng/ml versus control 0.53 +/- 0.14 ng/ml), the plasma level of luteinizing hormone (LH) (0.19 +/- 0.06 IU/l versus 0.25 +/- 0.09 IU/l in control; P < 0.05) and the mean frequency of LH pulses (2.0/h versus 2.5/h in control). Fasting resulted also in a significant (P < 0.05) decrease in the plasma concentration and pulse amplitude of follicle stimulating hormone (FSH) in comparison with the control. Simultaneously, a significant (P < 0.001) drop in the IGF-I concentration in the fasted ewes (4.78 +/- 0.91 ng/ml) was found in comparison with control (7.63 +/- 1.85 ng/ml). Also the level of insulin were significantly (P < 0.001) lower in the fasted (178.99 +/- 39.08 pM/l respectively) than in the control sheep (302.66 +/- 49.01 pM/l respectively). Meanwhile, a double increase in the growth hormone (GH) pulses frequency and an augmentation in its plasma concentrations as a result of starvation was found. The obtained results shows that the acute fasting exerts an inhibitory effect on the ovulation rate in ewes coincident with suppression in leptin, FSH and LH secretion and changes in signalization mediated by GH.     

Effect of cerebroventricular infusion of insulin and (or) glucose on hypothalamic expression of leptin receptor and pituitary secretion of LH in diet-restricted ewes

The objective was to determine the effect of central infusion of insulin and (or) glucose on hypothalamic expression of leptin receptor and pituitary secretion of LH in the ewe. Twenty-two ovariectomized ewes (32 wk of age) were fitted with two lateral cerebroventricular (LCV) cannulae and fed 33% of NRC requirements for 8 wk. Ewes (n> or =5/group) were then infused, via LCV cannulae, with artificial cerebrospinal fluid (aCSF) or aCSF containing physiological concentrations of insulin (INS), glucose (GLU), or INS + GLU; the mass of each increasing linearly from Day 0 (mass = 0 units/h) to Day 8 (mass of INS = 80 mIU/hr and GLU = 10 mg/hr). Jugular serum was collected every 12 min for 4 hr on Days 0, 2, and 4. Ewes treated with INS or INS + GLU had greater (P<0.06) mean concentrations of LH than aCSF treated ewes on Day 2 (13.8+/-1.8 and 12.5+/-1.3 > 8.0+/-3.3 ng/ml). Furthermore, on Day 4, concentrations of LH in INS treated ewes exceeded that (P<0.07) of aCSF treated ewes (14.8+/-2.0 > 7.4+/-3.0 ng/ml). Expression of NPY mRNA did not differ between treatments (P = 0.87). Leptin receptor mRNA expression was dramatically reduced (P<0.0002) in INS+GLU versus aCSF treated ewes. These data provide evidence to suggest that insulin may be an important component of hypothalamic mechanisms regulating secretion of LH and expression of leptin receptors in undernourished ruminants.     

Gonadotropin concentrations, follicular development, and luteal function in pituitary stalk-transfected ewes treated with bovine follicular fluid.

Two experiments, each arranged as a 2 x 2 factorial, were conducted in ewes to examine direct effects of bovine follicular fluid (bFF) on follicular development and luteal function and to further characterize follicular development and luteal function after pituitary stalk transection (SS). In Exp. 1, ewes were sham-operated or SS on d 6 of an estrous cycle and received 5 ml of saline or bFF three times daily on d 5 through 11 of the same cycle. In Exp. 2, all ewes were SS on d 6 of an estrous cycle and treated with saline or bFF three times daily on d 5 through 11 and with ovine FSH (60 micrograms; NIADDK-oFSH-16) or saline (1.2 ml) from d 7 to 11. In Exp. 2, ewes were ovariectomized on d 11 to assess effects of treatments on follicular development and luteal function. In both experiments, concentrations (ng/ml) of FSH on d 7 were suppressed (P less than or equal to .005) by bFF compared with saline (.50 +/- .17 vs 1.63 +/- .15) and remained suppressed (P less than or equal to .005) through d 11 (.46 +/- .12 vs 1.54 +/- .12). Replacement therapy (oFSH) restored concentrations of FSH. Concentrations of LH were not affected by bFF but were elevated (P less than or equal to .05) 1 d after SS (d 7; .88 +/- .09 vs .56 +/- .09) and remained elevated (P less than or equal to .05; 1.31 +/- .20 vs .65 +/- .11) from d 6 through 11. Concentrations of progesterone were unaffected by SS.(ABSTRACT TRUNCATED AT 250 WORDS)     

A chicken leptin-specific radioimmunoassay

Recombinant chicken leptin was used to produce an antiserum in order to develop a specific and sensitive radioimmunoassay (RIA) for chicken leptin in plasma and serum. We have used either murine or chicken leptin as tracer and competition curves were performed using recombinant chicken leptin. Variations in leptin plasma levels in different chicken strains and various nutritional states were correlated with the physiological status. Leptin plasma concentrations were regulated by the nutritional state with higher levels in the fed state as compared to the fasted state (3.36 +/- 0. 13 versus 2.78 +/- 0.11 ng/ml) and being dependent upon the age. Higher leptin levels were found in 22 week-old as compared to 15 week-old layer chickens (2.709 +/- 0.172 versus 1.478 +/- 0.102 ng/ml). We have also shown that the multispecies leptin RIA kit (LINCO Inc.) underestimated leptinemia compared to the chicken leptin- specific RIA reported here. In conclusion the RIA developed in the present study is specific to the chicken and thus may be considered as powerful tool for investigating the physiological significance of leptin in chickens.     

Plasma leptin concentration in adult cattle: effects of breed,adiposity, feeding level,and meal intake

An ovine-specific RIA, shown to be reliable for bovine leptin determination, was used to study the effects of breed, body fatness, feeding level, and meal intake on plasma leptin level in adult cattle. Eighteen fat Charolais, fat Holstein, and lean Holstein adult cows were either well-fed (130% of maintenance energy requirements [MER]) or underfed (60% of MER) for 3 wk. The breed tended to have a small effect on plasma leptin level, which was decreased by 70% (P < 0.05) in lean compared to fat Holstein cows. A strong curvilinear relationship was found between mean adipocyte volume and plasma leptin concentrations in well-fed (r = +0.95) and underfed (r = +0.91) cows. Underfeeding caused a significant decrease in plasma leptin levels from 8.0+/-3.1 to 6.1+/-2.3 ng/mL (P < 0.01). Nine adult Holstein cows initially fed at 130% of MER (control) were underfed to 21% of MER for 7 d, and five of them were refed to 237% of MER for 21 d. Plasma leptin measured 1 h before meal distribution was decreased from 5.9+/-0.4 to 3.8+/-0.2 ng/mL (P < 0.01) by underfeeding and increased to reach 8.8+/-1.0 ng/mL (P < 0.01) after refeeding. It was positively related to plasma glucose (r = +0.52, P < 0.01) and negatively related to plasma NEFA (r = -0.67, P < 0.001). Plasma leptin measured 4 h after meal distribution was positively related to feeding level and to plasma 3-OH-butyrate (r = +0.61, P < 0.005) and negatively related to plasma NEFA (r = -0.56, P < 0.01). Differences between pre- and postprandial leptin concentrations showed a decrease after meal intake in control and well-fed cows (-7 and -19%, P < 0.01, respectively) and an increase in underfed cows (+12%, P < 0.01). Leptin response to meal intake was positively related to glucose response (r = +0.66, P < 0.001) and negatively related to 3-OH-butyrate response (r = -0.78, P < 0.001). By using the "multispecies" commercial RIA, leptin concentrations were lower and we observed similar physiological responses, although less related to other hormones or metabolites. These data provide evidence, first, that a specific RIA for ruminant leptin determination is necessary to better understand leptin regulation, and second, that plasma leptin is strongly related to adipose cell size and positively related to feeding level in adult cattle, and that an effect of meal intake could be mediated by glucose and(or) ketone bodies.     

Effect of body fat mass and nutritional status on 24-hour leptin profiles in ewes

This study was designed to determine the effect of feeding or fasting of fat or thin ewes on 24-h leptin profiles. Ewes were assigned, based on ultrasonic assessments of last-rib subcutaneous fat measurements, into fat (fat thickness > 1 cm; mean = 1.52 +/- 0.03 cm; range 1.14 to 2.18 cm) or thin (fat thickness < 1 cm; mean = 0.25 +/- 0.03 cm; range 0.03 to 0.84 cm) groups. Fat and thin ewes were then assigned to either fed or fasted (deprived of feed) groups consisting of five ewes per group. Thus, four groups existed and were designated as fat-fed, fat-fasted, thin-fed, and thin-fasted. Fed ewes had ad libitum access to feed throughout the study. Fasted ewes were prohibited access to feed beginning 48 h preceding the experiment. Plasma samples were collected for leptin analysis from ewes every 15 min for 24 h beginning 48 h after the initiation of feed restriction or the congruent interval in fed ewes. Data were subjected to CLUSTER pulse analysis procedures. Profiles of plasma concentrations of leptin were episodic in nature and did not differ in a diurnal manner. Fed ewes had greater mean concentrations of leptin, area under the curve, number of peaks, peak height, peak nadir, and a shorter interval between peaks than fasted ewes (P < or = 0.05). Fat ewes had greater mean concentrations of leptin, area under the curve, number of peaks, peak height, peak nadir, and a shorter interval between peaks than thin ewes (P < 0.02). There also was a tendency for a body condition x treatment interaction for number of peaks (P = 0.073) and interval between peaks (P = 0.056). These results provide evidence that plasma concentrations of leptin are episodic in nature and are influenced by nutritive state and fat thickness over the ribs, but display no circadian variation.     

Plasma leptin concentrations in cats: reference range, effect of weight gain and relationship with adiposity as measured by dual energy X-ray absorptiometry

The aims of our study were to determine a reference range for plasma leptin in healthy, normal-weight cats and to measure the effect of weight gain on plasma leptin levels. To increase our understanding of the association between leptin and feline obesity, we investigated the relationship between plasma leptin and measures of adiposity in cats. Twenty-six normal-weight cats were used to determine the reference range for feline leptin using a multispecies radioimmunoassay. In the second part of the study, plasma leptin concentrations were determined in 16 cats before and after approximately 10 months of spontaneous weight gain. Dual energy X-ray absorptiometry scans (DEXA) were performed after weight gain. The tolerance interval for plasma leptin concentrations was 0.92-11.9 ng/ml Human Equivalent (HE) with a mean concentration of 6.41+/-2.19 ng/ml HE. In part two of the study, 16 cats gained on average 44.2% bodyweight over 10 months. The percentage of body fat in obese cats ranged from 34.2 to 48.7%. Mean plasma leptin concentrations increased from 7.88+/-4.02 ng/ml HE before weight gain to 24.5+/-12.1 ng/ml HE after weight gain, (P<0.001). Total body fat and body fat per cent were the strongest predictors of plasma leptin in obese cats (r=0.8 and r=0.78, P<0.001, respectively). In conclusion, plasma leptin concentrations increased three-fold in cats as a result of weight gain and were strongly correlated with the amount of adipose tissue present. Despite elevated leptin levels, cats continued to eat and gain weight, suggesting decreased sensitivity to leptin. This investigation into the biology of leptin in cats may aid the overall understanding of the role of leptin and the development of future treatments to help prevent and manage feline obesity.     

Serum leptin concentrations, luteinizing hormone and growth hormone secretion during feed and metabolic fuel restriction in the prepuberal gilt

Two experiments were conducted to determine 1) the effect of acute feed deprivation on leptin secretion and 2) if the effect of metabolic fuel restriction on LH and GH secretion is associated with changes in serum leptin concentrations. Experiment (EXP) I, seven crossbred prepuberal gilts, 66 +/- 1 kg body weight (BW) and 130 d of age were used. All pigs were fed ad libitum. On the day of the EXP, feed was removed from four of the pigs at 0800 (time = 0) and pigs remained without feed for 28 hr. Blood samples were collected every 10 min from zero to 4 hr = Period (P) 1, 12 to 16 hr = P 2, and 24 to 28 hr = P 3 after feed removal. At hr 28 fasted animals were presented with feed and blood samples collected for an additional 2 hr = P 4. EXP II, gilts, averaging 140 d of age (n = 15) and which had been ovariectomized, were individually penned in an environmentally controlled building and exposed to a constant ambient temperature of 22 C and 12:12 hr light: dark photoperiod. Pigs were fed daily at 0700 hr. Gilts were randomly assigned to the following treatments: saline (S, n = 7), 100 (n = 4), or 300 (n = 4) mg/kg BW of 2-deoxy-D-glucose (2DG), a competitive inhibitor of glycolysis, in saline iv. Blood samples were collected every 15 min for 2 hr before and 5 hr after treatment. Blood samples from EXP I and II were assayed for LH, GH and leptin by RIA. Selected samples were quantified for glucose, insulin and free fatty acids (FFA). In EXP I, fasting reduced (P < 0.04) leptin pulse frequency by P 3. Plasma glucose concentrations were reduced (P < 0.02) throughout the fast compared to fed animals, where as serum insulin concentrations did not decrease (P < 0.02) until P 3. Serum FFA concentrations increased (P < 0.02) by P 2 and remained elevated. Subcutaneous back fat thickness was similar among pigs. Serum IGF-I concentration decreased (P < 0.01) by P 2 in fasted animals compared to fed animals and remained lower through periods 3 and 4. Serum LH and GH concentrations were not effected by fast. Realimentation resulted in a marked increase in serum glucose (P < 0.02), insulin (P < 0.02), serum GH (P < 0.01) concentrations and leptin pulse frequency (P < 0.01). EXP II treatment did not alter serum insulin levels but increased (P < 0.01) plasma glucose concentrations in the 300 mg 2DG group. Serum leptin concentrations were 4.0 +/- 0.1, 2.8 +/- 0.2, and 4.9 +/- 0.2 ng/ml for S, 100 and 300 mg 2DG pigs respectively, prior to treatment and remained unchanged following treatment. Serum IGF-I concentrations were not effected by treatment. The 300 mg dose of 2DG increased (P < 0.0001) mean GH concentrations (2.0 +/- 0.2 ng/ml) compared to S (0.8 +/- 0.2 ng/ml) and 100 mg 2DG (0.7 +/- 0.2 ng/ml). Frequency and amplitude of GH pulses were unaffected. However, number of LH pulses/5 hr were decreased (P < 0.01) by the 300 mg dose of 2DG (1.8 +/- 0.5) compared to S (4.0 +/- 0.4) and the 100 mg dose of 2DG (4.5 +/- 0.5). Mean serum LH concentrations and amplitude of LH pulses were unaffected. These results suggest that acute effects of energy deprivation on LH and GH secretion are independent of changes in serum leptin concentrations.     

N-methyl-D, L-aspartate induces a transient increase in LH secretion in the seasonally anestrous ewe

The primary objective of this study was to determine the LH response to an excitatory amino acid agonist, N-methyl-D, L-aspartate (NMA) in the seasonally anestrous ewe. In experiment 1, 3 i.v. injections of NMA were given; doses of 0.5, 1.5 and 4.5 mg/kg BW were tested. LH response to NMA depended on the dose. There was little response to the lowest dose. All animals responded to the first injection of the intermediate and the highest doses (mean pulse amplitude: 9.2 +/- 0.4 and 6.8 +/- 1.2 ng ml, respectively). The responses to the second or third injections of both doses were variable and were either absent or reduced compared to that of the first. In experiments 2 and 3, ewes were given 3 injections of normal saline (NS) followed by 3 injections of NMA (1.25 and 4.5 mg/kg BW, respectively) at 2 hr intervals. The last injection of NMA was followed 2 hr later by an injection of GnRH (3.0 ng/kg BW). In experiment 2, the first NMA injection induced an immediate LH pulse (mean pulse amplitude: 8.0 +/- 1.6 ng/ml) in all ewes, however, the second and third injections induced LH pulses in only 25% and 75% (mean pulse amplitude: 2.2 and 2.4 +/- 0.6 ng/ml) of the ewes, respectively. In experiment 3, NMA increased mean LH release (P less than 0.05) after all injections, but responsiveness to the third injection was reduced in some ewes. GnRH injections induced LH release in all ewes in experiments 2 and 3 (mean pulse amplitude: 6.9 +/- 1.8 and 6.4 +/- 2.2 ng/ml, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sandwich enzyme-linked immunosorbent assay of canine leptin

Leptin is the ob gene product secreted from adipocytes in mammals, and thereby its plasma level reflects body fat content. To establish an assay method for leptin in the dog, rabbit anti-canine leptin antibody was obtained using canine recombinant leptin as an antigen. This antibody reacted to canine leptin much stronger than mouse, rat and human leptins. Sandwich enzyme-linked immunosorbent assay (ELISA) using this antibody was developed. The serum leptin levels of 13 healthy dogs were in a range from 1.4 to 5.6 ng/ml with the mean +/- SEM of 3.0 +/- 0.3 ng/ml.     

Experimental and clinical studies on plasma leptin in obese dogs

Leptin is a protein synthesized and secreted primarily by adipocytes, and the circulating leptin concentration is elevated in obese humans and rodents. Recently, we have established a sandwich enzyme-linked immunosorbent assay for canine leptin. In the present study, plasma leptin concentrations were measured in experimentally developed obese beagles and in clinically obese dogs. When 5 male beagles were given a high-energy diet for 3 months, all of them became obese and the plasma leptin concentration significantly increased from 2.4+/-1.2 to 4.9+/-0.9 ng/ml, positively correlating with body fat content estimated by the deuterium oxide dilution method (r=0.87). The leptin concentrations of plasma samples collected from 59 dogs in veterinary practices were compared with their body condition scores (BCS). The plasma leptin concentrations of obese dogs were 9.7+/-0.7 and 12.3+/-1.5 ng/ml at BCS=4 and BCS=5, respectively, which were significantly higher than those of optimal (BCS=3) dogs (2.7+/-0.3 ng/ml). There was no significant effect of sex and breed. A weak positive correlation (r=0.37) was found between the plasma leptin concentration and age, probably due to the lesser content of visceral fat in puppies younger than 1 year old. These results indicate that plasma leptin is a good index of adiposity in dogs regardless of breed, age and sex, and may be useful for quantitative assessment of obesity in small animal practice.     

Effects of estradiol-17 beta, naloxone and gonadotropin releasing hormone on postpartum secretion of luteinizing hormone in fall-lambing ewes

The interaction among exogenous estradiol-17 beta, naloxone and gonadotropin releasing hormone (GnRH) in the control of luteinizing hormone (LH) secretion was studied in intact postpartum ewes nursing their offspring. One-half of 30 fall-lambing ewes were implanted subcutaneously with an estradiol-17 beta containing Silastic capsule between postpartum d 1 and 12 which doubled their serum concentrations of estradiol (16.0 +/- .1 vs 8.4 +/- .1 pg/ml). Blood samples were collected from implanted and non-implanted ewes at 15-min intervals for 5 h on d 3, 8, 13, 20 and 28 postpartum. Pre-injection samples were collected for 1 h, and ewes were injected with saline, naloxone (NAL;1 mg/kg) or GnRH (100 micrograms/ewe). When averaged across all days and implant groups, serum LH in the three post-NAL samples was higher (P less than .05) than in the three pre-NAL samples (3.6 +/- 1.2 vs .6 +/- .2 ng/ml). Post-GnRH concentrations of serum LH were lower (P less than .05) in estradiol-implanted ewes than in non-implanted ewes on d 8 and 13, but there were no differences in any LH characteristics on d 20 and 28 after implant removal on d 12. In non-implanted ewes, serum LH responses to GnRH increased (P less than .05) eightfold from d 3 (3.8 +/- 1.4 ng/ml) to d 8 (31.6 +/- 1.4 ng/ml), remained elevated through d 20, but declined by d 28 (10.8 +/- 1.4 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Early pregnancy alters the metabolic responses to restricted nutrition in sheep

This study investigated whether a 27-day period of nutrition at half-maintenance during early pregnancy (up to Day 14) could alter maternal endocrine responses. Forty-six ewes were fed all or half of their maintenance requirements and slaughtered on Day 14 of the oestrous cycle or pregnancy. We used real time RT-PCR to study gene expression of growth hormone receptor (GHR) and leptin in adipose tissue and GHR, GHR1A and of the insulin-like growth factor I (IGF-I) in the liver. Blood profiles of metabolites and metabolic hormones were also determined. Throughout the experiment, underfed animals presented lower body weight and body condition, greater plasma concentrations of non-esterified fatty acids (NEFA), and lower plasma concentrations of leptin, compared to adequately fed animals. Undernutrition affected the patterns of gene expression in adipose and hepatic tissues, and the responses differed between pregnant and non-pregnant ewes. In adequately fed ewes, pregnancy up-regulated leptin mRNA expression in adipose tissue, a response that was impaired in underfed ewes. The hepatic expression of IGF-I mRNA was increased by pregnancy in underfed animals while no effect was observed in adequately fed ewes. It remains to be determined whether the changes in the endocrine milieu are paralleled by modifications in uterine gene expression that could alter the environment of the embryo during early pregnancy.     

Effect of feeding flax or linseed meal on progesterone clearance rate in ovariectomized ewes

Ovariectomized ewes (n=22; 68.76+/-2.34 kg initial body weight; 2.9+/-0.1 initial body condition score) were individually fed one of three diets: (1) control (phytoestrogen-free; n=7), (2) flax containing diet (n=8), or (3) linseed meal (LSM) containing diet (n=7) to investigate the rate of progesterone (P4) clearance. On day 20 of feeding (day 0=initiation of treatment), a P4 releasing device (CIDR) was placed in the vagina and jugular blood samples were obtained prior to CIDR insertion and 15, 30, 60, and 120 min following CIDR insertion. Further, blood samples were obtained daily between days 21 and 24. On day 25, blood samples were retrieved prior to CIDR removal and 2, 5, 10, 15, 30, 60, 120, and 360 min following CIDR removal. There was no difference in initial or final body weight or body condition score and there were no time by diet interactions on P4 clearance. The fractional rate of P4 uptake measured prior to CIDR insertion through day 4 following insertion tended to be greater (P=0.07) in LSM fed ewes (508.75+/-71.37%/min) compared to flax (295.39+/-66.76%/min) and control fed (287.54+/-71.37%/min) ewes. Diet tended (P=0.10) to influence P4 clearance rate when measured from prior to CIDR removal through 120 min following CIDR removal with LSM fed ewes having a greater (1.26+/-0.2) fractional rate constant than flax (0.929+/-0.09) and control fed (0.922+/-0.09) ewes. Flax fed ewes also had more (P<0.01) omega-3 fatty acids and total fatty acids in plasma. Reports of increased pregnancy rates in dairy cows fed flax may relate to P4 metabolism.     

Growth hormone release after N-methyl-D,L-aspartate in sheep: dose response and effect of an opioid antagonist

The objectives of our experiments were 1) to determine the effect of N-methyl-D,L-aspartate (NMA), an agonist of the neuroexcitatory amino acids aspartate and glutamate, on growth hormone (GH) release in ovariectomized ewes, and 2) to determine the effect of naloxone, an opioid antagonist, on the GH response to NMA. Jugular blood was collected via venipuncture at 12-min intervals for 2 h before and 2 h after i.v. injection of NMA. In Exp. 1, ewes received either 0, 6, 12 or 24 mg NMA/kg BW dissolved in .9% saline solution (n = 4 per treatment). Growth hormone concentrations were similar (P greater than .1) between groups prior to injection (9.8 +/- .7 ng/ml; mean +/- SEM) and were unaffected (P greater than .1) by saline treatment. In contrast, 6, 12 or 24 mg NMA/kg BW increased mean GH concentration by 210% (P less than .04), 273% (P less than .02) and 234% (P less than .02), respectively. In Exp. 2, ewes received NMA (6 mg/kg BW) 5 min after either saline (n = 4) or naloxone (1 mg/kg BW; n = 4) pretreatment. Serum GH concentrations averaged 7.0 +/- 1.1 ng/ml before pretreatment and increased similarly (238%; P greater than .1) in both groups following NMA. In summary, NMA increased GH concentrations in ovariectomized ewes by some mechanism that does not involve opioid receptors that are antagonized by naloxone.     

Effects of estradiol on gonadotrophin release, estrus and ovulation in CIDR-treated beef cattle

The effects of estradiol-17beta (E-17beta) or estradiol benzoate (EB) on gonadotrophin release, estrus and ovulation in beef cattle were evaluated in two experiments. In experiment 1, 16 ovariectomized cows received a previously used CIDR insert from days 0 to 7 and 1mg of EB on day 8; they also received 5mg of E-17beta on days 0 or 1, or 5mg of E-17beta+100mg of progesterone on day 0. There was only an effect of time (P<0.0001) on plasma concentrations of progesterone, estradiol, FSH, and LH. Following treatment with E-17beta, plasma FSH concentrations were suppressed for approximately 36 h, whereas plasma LH concentrations were reduced (P<0.05) for 6 h, but surged within 24 h. Injecting 1mg of EB 24 h after CIDR removal decreased (P<0.02) plasma LH concentrations for 6h, followed by an LH surge at 18 h. In experiment 2, ovary-intact heifers (n=40) received a used CIDR and 5mg of E-17beta+100mg of progesterone on day 0. On day 7, CIDR were removed, PGF given, and heifers received nothing (control) or 1mg of EB 12, 24, or 36 h later. In these groups, plasma LH peaked (mean+/-SEM) 78.0+/-23.0, 37.8+/-8.5, 44.4+/-10.3, and 51.0+/-5.1 h after CIDR removal (means, P<0.001; variances, P<0.001) and intervals from CIDR removal to ovulation were 102.0+/-6.7, 63.6+/-3.6, 81.6+/-3.5, and 78.0+/-4.1h (P<0.05). The interval from CIDR removal to ovulation was shorter and less variable in EB-treated groups; the interval from EB to ovulation was shortest (P<0.05) in the 12-h group. In summary, E-17beta or EB decreased both FSH and LH, but LH increased after 6h (despite elevated progesterone concentrations). Following CIDR removal, 1mg of EB effectively synchronized LH release, and ovulation (in intact cattle), but the interval from CIDR removal to EB treatment affected the time of ovulation.     

Development of an endocrine challenge test to investigate subfertility in ewes

Simultaneous or sequential injection of 250 ng gonadotrophin releasing hormone (GnRH) and 25 micrograms oestradiol benzoate, with luteinizing hormone (LH) measurements at 0, +20 min (after GnRH) and +16 h (after oestradiol), enabled investigation of the positive feedback effects on the hypothalamus and pituitary. Control ewes had pretreatment LH values of 3.1 +/- 1.2 ng/ml with an increment of 3.2 +/- 2.3 ng/ml 20 min after GnRH. Subfertile ewes, in spite of elevated pretreatment LH concentrations (15.8 +/- 9.5 ng/ml) in eight out of 10 ewes, had increments of 1.4-84 ng/ml after GnRH. Control ewes had LH increments of 3-75 ng/ml 16 h after oestradiol. Subfertile ewes with pretreatment LH concentrations less than 15 ng/ml also responded to oestradiol whereas those with initial LH concentrations 16-40 ng/ml had no further LH increment. Subsequent administration of 1000 iu pregnant mares' serum gonadotrophin (PMSG), with measurement of LH and oestradiol at 0, +24, +30, +48, +54, and +72 h, allowed assessment of ovarian response and hypothalamus-pituitary function. Five control ewes were sampled up to 30 h post-PMSG and only 1 had oestradiol concentrations greater than 10 pg/ml. Sampling up to 72 h in another five control ewes resulted in oestradiol concentrations greater than 10 pg/ml. Increments in LH concentration greater than 3 ng/ml were recorded in control and subfertile ewes with oestradiol concentrations greater than 10 pg/ml. The use of these endocrine challenge tests enabled positive diagnosis of abnormality on 8 out of 10 occasions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of long-term consumption of sewage solids on blood, milk and tissue elemental composition of breeding ewes

Fine-wool ewes received for 2 yr a complete pelleted basal diet (11% protein) or the basal diet fortified with 3.5% cottonseed meal (CSM; 12% protein) or gamma-irradiated (1 megarad) dried solids (SS; 12% protein) from primary (undigested) sewage (Las Cruces, New Mexico, municipal sewage). Five ewes fed each diet were sampled to determine Ag, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb and Zn in blood, milk and tissues. Tissues and blood were sampled at slaughter 40 d after weaning of lambs. Mean whole blood mineral concentrations were similar (P less than .05) among treatments 3 d postpartum; however, at 42 d after lambing (mean +/- SE) both basal- (54 +/- 2 micrograms/ml) and sewage-fed (54 +/- 2 micrograms/ml) ewes had elevated (P less than .05) blood Ca compared with ewes fed CSM (46 +/- 2 micrograms/ml). No biologically important differences were detected in the concentrations of elements in milk. Ewes fed SS had lower (P less than .05) blood Fe than animals in the other groups. Sewage-fed ewes also had higher (P less than .05) liver Fe (1,092 +/- 100 micrograms/g) than basal-fed ewes (626 +/- 100 micrograms/g); whereas Fe in CSM-fed ewes (873 +/- 100 micrograms/g) was similar to both. Basal-fed animals had 1.1- to 1.3 times more (P less than .05) liver Mg and two- to threefold higher liver Na than CSM or SS. Livers from SS-fed ewes had higher concentrations (P less than .05) of Cd (1.5- to 1.6-times) and Pb (1.4- to 1.9-times) than livers from CSM- or basal-fed ewes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of exogenous estradiol and progesterone on plasma concentrations of leptin in ewes in non-breeding season

Estradiol and progesterone may play a role in controlling leptin secretion by utilizing their receptors in adipocytes and the genomic mechanisms of the leptin gene. This study was conducted to evaluate the effects of exogenous sex steroids on the blood leptin concentrations in ewes in the non-breeding season. Multiparous ewes were fed to maintenance level for their live weights. Blood samples were collected at 12-h intervals from Days -3 to -1 to determine the basal leptin levels (pre-injection period). From Day 0 to Day 5 (injection period), blood sampling continued at 12-h intervals, and the ewes were injected intramuscularly at 24-h intervals with oil, 50 mg progesterone in oil, 1 mg of estradiol in oil, or both steroids in oil. Leptin was measured using a sensitive and specific radioimmunoassay based on recombinant bovine leptin. Overall, plasma concentrations of leptin were not affected by any of the steroid treatments, and there were no differences in the value of leptin between the pre-injection and injection periods among the 4 groups. Therefore, the exogenous estrogen and progesterone used in this study do not have a strong effect on the blood leptin concentrations of ewes in the non-breeding season.     

Influence of dietary endophyte (Neotyphodium coenophialum)-infected tall fescue (Festuca arundinacea) seed on fecal shedding of antibiotic resistance-selected Escherichia coli O157:H7 in ewes

The objectives were to determine the effects of short-term feeding of a toxic endophyte (Neotyphodium coenophialum)-infected tall fescue seed (Festuca arundinacea, cultivar 'Kentucky 31') on fecal shedding and intestinal concentrations of Escherichia coli O157:H7 and the concentrations of prolactin, cortisol, and NEFA in experimentally inoculated ewes. Twelve ewes (mean BW = 46 +/- 2 kg) were fed a diet containing either high endophyte-infected (HI-E) or low endophyte-infected (LO-E) tall fescue seed for 7 d. Each diet consisted of 50% (as-fed basis) tall fescue seed. Ewes were experimentally inoculated with antibiotic resistance-selected E. coli O157:H7 on d 1 of the feeding treatment, and fecal shedding of inoculated pathogens was monitored daily on d 2 to 6. On d 7, ewes were weighed and euthanized, and tissues and contents were sampled from the ileum, cecum, and rectum for quantitative enumeration of E. coli O157:H7. Urine was collected at euthanization to determine total ergot alkaloid concentrations. Ewes fed HI-E had lower (P < 0.001) DMI than did ewes fed LO-E (0.8 and 1.6 +/- 0.1 kg/d of DMI for HI-E and LO-E ewes, respectively); consequently, there was a tendency (P = 0.06) for HI-E ewes to lose 0.3 +/- 0.4 kg of BW/d and LO-E ewes to gain 0.2 +/- 0.4 kg of BW/d during the 7 d. Urinary ergot alkaloids were increased (P < 0.001) in ewes fed HI-E (47.8 +/- 9.4 ng/mg of creatinine) compared with those fed LO-E (6.2 +/- 9.4 ng/mg of creatinine). Prolactin tended (P = 0.06) to be decreased in ewes fed HI-E (7.2 +/- 7.0 ng/mL) compared with those fed LO-E (27.7 +/- 7.0 ng/mL). Fecal shedding of E. coli O157:H7 tended (P = 0.06) to be increased in HI-E ewes [5.4 cfu (log10)/g of feces] compared with LO-E ewes [4.5 cfu (log10)/g of feces]. The population of E. coli O157:H7 in luminal contents from the ileum, cecum, and rectum did not differ (P > 0.36) between treatments. Treatment did not influence (P = 0.30) the occurrence of E. coli O157:H7 in cecal or rectal tissues; however, ileal tissues from HI-E ewes tended (P = 0.12) to have an increased incidence of E. coli O157:H7. Concentrations of NEFA tended (P = 0.12) to be greater in HI-E ewes than in LO-E ewes, whereas cortisol was similar (P = 0.49) for HI-E and LO-E ewes. We conclude that short-term feeding of HI-E tall fescue seed may alter the concentrations of prolactin and NEFA, and may increase fecal shedding of E. coli O157:H7 in experimentally inoculated ewes.