Detection of Mycobacterium avium subspecies paratuberculosis: comparing fecal culture versus serum enzyme-linked immunosorbent assay and direct fecal polymerase chain reaction.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in cattle. The disease causes diarrhea, reduced milk production, poor reproductivity, emaciation, and eventually death. Culture on Herrold's egg yolk agar is considered to be the definitive test for diagnosis of Johne's in cattle. This method has moderate sensitivity (30 to 50%) and is 100% specific; however, it can take up to 16 wk due to the slow growth of MAP. Currently, serum ELISA is used to screen herds for Johne's disease, but positive tests must be confirmed culturally or by PCR. The current research sought to evaluate an in-house direct fecal PCR procedure and directly compare it to ELISA using culture as the gold standard. Serum and fecal samples were collected from cows (n = 250) with unknown Johne's status. Fecal samples were processed for culture on Herrold's egg yolk agar and direct PCR. Serum samples were tested using the Parachek serum ELISA. Overall, 67/250 [26.8%, 95% confidence interval (CI) 21.4 to 32.8] animals were culturally confirmed to be shedding MAP. The PCR and ELISA detected 74/250 (29.6%, 95% CI 24 to 35.7) and 25/250 (10%, 95% CI 6.6 to 14.4), respectively. Culture and PCR were able to detect more positive animals than ELISA. Overall, direct fecal PCR was 70.2% sensitive and 85.3% specific when using culture as the gold standard. The ELISA method was 31.3% sensitive and 97.8% specific. When culture reported <10 cfu, the sensitivity and specificity of PCR and ELISA were 57.1 and 85.3%, and 4.8 and 97.8%, respectively. When culture reported 10 to <40 cfu, the sensitivity of PCR and ELISA were 75 and 50%, respectively. When culture reported > or =40 cfu, the sensitivity of PCR and ELISA were 100 and 88.2%, respectively. Specificity could not be calculated at these levels because there were no negative samples. The direct PCR outperformed the ELISA in detecting animals potentially infected with MAP and was not significantly different when compared with culture. The direct fecal PCR method described here provides faster results than traditional culture and is more sensitive than ELISA at detecting animals suspected of Johne's disease. These data support the use of PCR as an alternative method for screening herds for prevalence and diagnosis of Johne's disease.

Two days of pulsatile GnRH infusion beginning 4 days before weaning in sows initiates a wave of follicular growth that is not sustained after weaning.

Intervals to estrus and ovulation in weaned sows depend partially on the diameter of ovarian follicles at weaning. The objective was to determine if follicular diameter in sows could be increased by a 48h period of GnRH infusion before weaning and whether this pre-weaning growth would advance follicular development after weaning. The posterior vena cava was cannulated in eight sows at 10+/-1 day after farrowing. Sows were randomly assigned to receive intravenous treatment with either 2mL of GnRH (1microg/mL; n=4) or 2mL of saline (n=4) every 0.5h for 48h beginning 94h before weaning. The average follicular diameter and the number of follicles within diameter classes were determined daily by ultrasonography. Serum LH concentrations increased on the first infusion day but serum LH was equal to control on the last infusion day (P<0.077). The GnRH infusion increased the average diameter of ovarian follicles (P<0.001). Serum estradiol increased (P<0.001) and serum FSH decreased (P<0.016) coincident with GnRH-induced follicular development but these changes were reversed within 24h after the end of the infusion period. Follicles that grew in response to GnRH regressed and were replaced by a new population of follicles within 4 days after weaning. Within the experimental model for the present study, a GnRH infusion increased follicular growth in lactating sows but follicles could not be sustained beyond the end of GnRH infusion.

Timed artificial insemination of two consecutive services in dairy cows using prostaglandin F2alpha and gonadotropin-releasing hormone.

Timed artificial insemination (TAI) protocols use PGF(2alpha) and GnRH injections to synchronize ovulation. The objective was to evaluate the PGPG protocol (d 0, PGF(2alpha); d 3, GnRH; d 11, PGF(2alpha); d 13, GnRH and TAI) for first TAI and also examine methods for second TAI in nonpregnant cows. A factorial test of the first PGF(2alpha) and first GnRH injections within the PGPG protocol was performed (the last PGF(2alpha) and GnRH injections were deemed essential to the TAI). Lactating dairy cows (n = 804) in a commercial herd were assigned to 1 of 5 first-TAI treatments, which were PGPG, GPG (d 0, no treatment; d 3, GnRH; d 11, PGF(2alpha); d 13, GnRH and TAI), PPG (d 0, PGF(2alpha); d 3, no treatment; d 11, PGF(2alpha); d 13, GnRH and TAI), and PG (d 0, no treatment; d 3, no treatment; d 11, PGF(2alpha); d 13, GnRH and TAI); the Ovsynch protocol (GnRH, 7 d, PGF(2alpha), 2 d, GnRH and TAI) was the positive control. For resynchronization, cows received either GnRH or the control (no injection) on d 22 after TAI. Nonpregnant cows on d 28 were then treated with PGF(2alpha) on d 29, GnRH on d 31, and TAI [i.e., resynchronization treatments of ReGPG (received GnRH on d 22) and RePG (did not receive GnRH on d 22)]. Pregnancy rates for PGPG, GPG, PPG, PG, and Ovsynch were similar at d 28 after first TAI. Analyses of multiple explanatory factors by logistic regression detected an effect of uterine or ovarian abnormality on the d-28 pregnancy rate (normal more likely to be pregnant). Day-42 pregnancy rates were affected by uterine or ovarian abnormality (normal more likely to be pregnant), postpartum disease occurrence (healthy cows more likely to be pregnant), milk production, and days in milk. Treatment was not significant for the d-42 pregnancy rate. Effects of postpartum disease, milk production, and days in milk on the d-42 pregnancy rate were apparently manifested through their effects on embryonic loss between d 28 and 42 of pregnancy. High-producing cows that received TAI early postpartum were most likely to experience embryonic loss. Day-42 pregnancy rates after the resynchronization treatment were affected by an interaction of the first synchronization treatment with the resynchronization treatment. We concluded that although PGPG can be used for TAI, a simpler TAI protocol that includes the last 2 injections (PGF(2alpha), 2 d; GnRH and TAI) would be equally effective.

Partial feed restriction decreases growth hormone receptor 1A mRNA expression in postpartum dairy cows.

Uncoupling of the growth hormone (GH) axis in early postpartum dairy cows is correlated with a decrease in liver GH receptor (GHR) 1A mRNA and a decrease in liver GH receptor protein. Postpartum recoupling of the GH axis is also correlated with GHR 1A mRNA and GHR protein. We hypothesized that dry matter intake (DMI) partially controls the increase in GHR 1A mRNA postpartum. Prepartum Holstein dairy cows (n = 11) were offered feed ad libitum. After calving, 6 cows were fed 70% of their expected DMI (feed restriction) for 14 d and 5 cows were fed ad libitum (control). Both groups were fed ad libitum after d 14. Liver was biopsied prepartum and on d 1, 7, 14, and 21 postpartum; blood was sampled throughout the experimental period. Rate of increase in postpartum milk production was less for feed-restricted cows. The GHR 1A mRNA decreased from prepartum to d 1 postpartum and subsequently increased. Rate of postpartum increase in GHR 1A mRNA was less in feed-restricted cows. Diminished GHR 1A persisted for at least 7 d after feed-restricted cows returned to ad libitum feeding. Liver insulin-like growth factor-I mRNA concentrations decreased from prepartum to d 1 as well, but were similar for feed restricted and control thereafter. We concluded that DMI partially controls GHR 1A mRNA expression in early postpartum dairy cows and that the decrease in GHR 1A in response to feed restriction persisted for at least 1 wk after ad libitum feeding was restored.

GH-releasing peptide-6 overcomes refractoriness of somatotropes to GHRH after feeding.

After a meal, somatotropes are temporarily refractory to growth hormone-releasing hormone (GHRH), the principal hormone that stimulates secretion of growth hormone (GH). Refractoriness is particularly evident when free access to feed is restricted to a 2-h period each day. GH-releasing peptide-6 (GHRP-6), a synthetic peptide, also stimulates secretion of GH from somatotropes. Because GHRH and GHRP-6 act via different receptors, we hypothesized that GHRP-6 would increase GHRH-induced secretion of GH after feeding. Initially, we determined that intravenous injection of GHRP-6 at 1, 3 and 10 microg/kg body weight (BW) stimulated secretion of GH in a dose-dependent manner. Next, we determined that GHRP-6- and GHRH-induced secretion of GH was lower 1 h after feeding (22.5 and 20 ng/ml respectively) than 1 h before feeding (53.5 and 64.5 ng/ml respectively; pooleds.e.m.=8.5). However, a combination of GHRP-6 at 3 microg/kg BW and GHRH at 0.2 microg/kg BW synergistically induced an equal and massive release of GH before and after feeding that was fivefold greater than GHRH-induced release of GH after feeding. Furthermore, the combination of GHRP-6 and GHRH synergistically increased release of GH from somatotropes cultured in vitro. However, it was not clear if GHRP-6 acted only on somatotropes or also acted at the hypothalamus. Therefore, we wanted to determine if GHRP-6 stimulated secretion of GHRH or inhibited secretion of somatostatin, or both. GHRP-6 stimulated secretion of GHRH from bovine hypothalamic slices, but did not alter secretion of somatostatin. We conclude that GHRP-6 acts at the hypothalamus to stimulate secretion of GHRH, and at somatotropes to restore and enhance the responsiveness of somatotropes to GHRH.

Insulin restores GH responsiveness during lactation-induced negative energy balance in dairy cattle: effects on expression of IGF-I and GH receptor 1A.

Early lactation in dairy cattle is a period of severe negative energy balance (NEB) characterized by reduced blood glucose and insulin concentrations and elevated blood GH concentrations. The liver is refractory to GH during NEB and this uncoupling of the GH-IGF axis results in diminished plasma concentrations of IGF-I. Our objectives were to examine the effects of insulin administration during the immediate postpartum period on plasma IGF-I and GH concentrations and to examine the hepatic expression of total GH receptors (all GH receptor transcripts), GH receptor 1A (GHR 1A) and IGF-I. In addition, we examined adipose tissue for total GH receptor and IGF-I mRNA levels to establish the effects of chronic hyperinsulinemia on an insulin-responsive peripheral tissue. Holstein cows (n=14) were subjected to either a hyperinsulinemic-euglycemic clamp (insulin; INS) or saline infusion (control; CTL) for 96 h starting on day 10 postpartum. Insulin was infused i.v. (1 micro g/kg body weight per h), blood samples were collected hourly, and euglycemia was maintained by infusion of glucose. Insulin concentrations during the infusions were increased 8-fold in INS compared with CTL cows (2.33+/-0.14 vs 0.27+/-0.14 ng/ml (S.E.M.); P<0.001) while blood glucose concentrations were not different between treatments (45.3+/-2.2 vs 42.5+/-2.2 mg/dl; P>0.1). Plasma IGF-I increased continuously during the insulin infusion, and reached the highest concentrations at the end of the clamp, being almost 4-fold higher in INS compared with CTL cows (117+/-4 vs 30+/-4 ng/ml; P<0.001). Hepatic expression of GHR 1A and IGF-I mRNA was low in CTL cows, but was increased 3.6-fold (P<0.05) and 6.3-fold (P<0.001) respectively in INS cows. By contrast, in adipose tissue the changes in gene expression in response to insulin were reversed with decreases in both total GHR and IGF-I mRNA. The expressions of GHR 1A and IGF-I mRNA in liver tissue were correlated in INS (r=0.86; P<0.05), but not CTL cows (r=0.43; P>0.1). Insulin appears to be a key metabolic signal in coupling the GH-IGF axis, thus orchestrating a marked elevation in circulating IGF-I concentrations.

Thyrotropin-releasing hormone mediates serotonin-induced secretion of GH in cattle.

Serotonin stimulates secretion of growth hormone (GH) in cattle, but the mechanism is unknown. In rats, thyrotropin-releasing hormone (TRH) mediates serotonin-induced secretion of GH. We hypothesized that the same is true in cattle. Cattle were fed for 2h daily to synchronize secretion of GH, such that concentrations of GH were high before and low after feeding. Our first objective was to determine whether or not feeding suppresses serotonin receptor agonist (quipazine) induced secretion of GH. Holstein steers were injected with quipazine (0.2 mg/kg BW) either 1 h before or 1 h after feeding. Quipazine-induced secretion of GH which did not differ in magnitude before and after feeding. If TRH mediates serotonin-induced secretion of GH, then magnitude of TRH-induced secretion of GH should not be different before and after feeding (our second objective). Sixteen meal-fed Holstein steers were injected with 0.3 microg TRH/kg BW either 1 h before or 1 h after feeding. Indeed, magnitude of TRH-induced secretion of GH before and after feeding was not different. Our third objective was to inhibit endogenous TRH with 3,5,3'-triiodothyronine (T(3)) and examine basal, GH-releasing hormone (GHRH)-, TRH- and quipazine-induced secretion of GH. Sixteen Holstein steers were injected daily with either T(3) (3 or 6 microg/kg BW) or vehicle for 20 days and then challenged sequentially with vehicle or GHRH, TRH, or quipazine. T(3) did not affect basal, GHRH- or TRH-induced secretion of GH, but reduced basal secretion of thyroxine. T(3) reduced but did not completely block quipazine-induced secretion of GH. In conclusion, TRH mediates, in part, serotonin-induced secretion of GH in cattle.

Feeding-induced increases in insulin do not suppress secretion of growth hormone.

Secretion of growth hormone (GH) is reduced for several hours after feeding when access to feed is restricted to a 2-hr period each day. We hypothesized that increased secretion of insulin after feeding inhibits release of GH from the anterior pituitary gland. Our objectives were to determine whether: 1) alloxan prevents concentrations of insulin from increasing after feeding steers; 2) concentrations of GH remain high after feeding alloxan-treated steers; and 3) GH-releasing hormone (GHRH) stimulates greater release of GH in alloxan-treated, than in control, steers after feeding. Steers were injected iv with either saline (control) or with alloxan (110 mg/kg) (n = 4 per group). Concentrations of insulin were not different (P = 0.61) between control and alloxan-treated steers before feeding (87.5 +/- 33.6 pmol/l). However, alloxan prevented insulin from increasing (P < 0.001) after feeding (131.8 pmol/1) compared with control steers (442.0 pmol/l) (pooled SEM = 47.5). Overall, GH was higher (P < 0.05) in alloxan-treated (6.4 ng/ml) than in control steers (3.7 ng/ml) (pooled SEM = 0.7), but GH decreased (P < 0.001) after feeding in both groups. Iv injection of GHRH stimulated release of GH 1 hr before, but not when injected 1 hr after feeding (P < 0.001). In addition, net areas under the GH curve were not significantly different between control and alloxan-treated groups. We conclude that increased concentrations of insulin after feeding do not mediate feeding-induced suppression of GH secretion in steers.

Neuroregulation of growth hormone secretion in domestic animals.

Growth hormone (GH) is essential for postnatal somatic growth, maintenance of lean tissue at maturity in domestic animals and milk production in cows. This review focuses on neuroregulation of GH secretion in domestic animals. Two hormones principally regulate the secretion of GH: growth hormone-releasing hormone (GHRH) stimulates, while somatostatin (SS) inhibits the secretion of GH. A long-standing hypothesis proposes that alternate secretion of GHRH and SS regulate episodic secretion of GH. However, measurement of GHRH and SS in hypophysial-portal blood of unanesthetized sheep and swine shows that episodic secretion of GHRH and SS do not account for all episodes of GH secreted. Furthermore, the activity of GHRH and SS neurons decreases after steers have eaten a meal offered for a 2-h period each day (meal-feeding) and this corresponds with reduced secretion of GH. Together, these data suggest that other factors also regulate the secretion of GH. Several neurotransmitters have been implicated in this regard. Thyrotropin-releasing hormone, serotonin and gamma-aminobutyric acid stimulate the secretion of GH at somatotropes. Growth hormone releasing peptide-6 overcomes feeding-induced refractoriness of somatotropes to GHRH and stimulates the secretion of GHRH. Norepinephrine reduces the activity of SS neurons and stimulates the secretion of GHRH via alpha(2)-adrenergic receptors. N-methyl-D,L-aspartate and leptin stimulate the secretion of GHRH, while neuropeptide Y stimulates the secretion of GHRH and SS. Activation of muscarinic receptors decreases the secretion of SS. Dopamine stimulates the secretion of SS via D1 receptors and inhibits the secretion of GH from somatotropes via D2 receptors. Thus, many neuroendocrine factors regulate the secretion of GH in livestock via altering secretion of GHRH and/or SS, communicating between GHRH and SS neurons, or acting independently at somatotropes to coordinate the secretion of GH.

Pituitary adenylate cyclase-activating polypeptide induces secretion of growth hormone in cattle.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a hypothalamic neuropeptide that stimulates release of growth hormone (GH) from cultured bovine anterior pituitary gland cells, but the role of PACAP on the regulation of in vivo secretion of GH in cattle is not known. To test the hypothesis that PACAP induces secretion of GH in cattle, meal-fed Holstein steers were injected with incremental doses of PACAP (0, 0.1, 0.3, 1, 3, and 10 microg/kg BW) before feeding and concentrations of GH in serum were quantified. Compared with saline, injection of 3 and 10 microg PACAP/kg BW increased peak concentrations of GH in serum from 11.2 ng/ml to 23.7 and 21.8 ng/ml, respectively (P < 0.01). Peak concentrations of GH in serum were similar in steers injected with 3 or 10 microg PACAP/kg BW. Meal-fed Holstein steers were then injected with 3 microg/PACAP/kg BW either 1 hr before feeding or 1 hr after feeding to determine if PACAP-induced secretion of GH was suppressed after feeding. Feeding suppressed basal concentrations of GH in serum. Injection of PACAP before feeding induced greater peak concentrations of GH in serum (19.2 +/- 2.6 vs. 11.7 +/- 2.6 ng/ml) and area under the response curve (391 +/- 47 vs. 255 +/- 52 ng. ml(-1) min) than injection of PACAP after feeding, suggesting somatotropes become refractory to PACAP after feeding similar to that observed by us and others with growth hormone-releasing hormone (GHRH). We concluded that PACAP induces secretion of GH and could play a role in regulating endogenous secretion of GH in cattle, perhaps in concert with GHRH.

Synchronisation of oestrus in dairy cows using prostaglandin F2alpha, gonadotrophin-releasing hormone, and oestradiol cypionate.

An oestrous synchronisation protocol was developed for use in lactating dairy cows using PGF(2alpha), GnRH, and oestradiol cypionate (ECP). In experiment 1, lactating dairy cows received two injections of PGF(2alpha) (on days 0 and 11) (PP; n=10) or two injections of PGF(2alpha) (days 0 and 11) and 100 microg of GnRH on day 3 (PGP; n=10). In experiment 2, cows were treated with PGP (n=7), or PGP and 1 mg of ECP at the same time (PGPE(0); n=7) or 1 day after the second PGF(2alpha) injection (PGPE(1); n=7). In experiment 3, 101 lactating dairy cows in a commercial herd were assigned to one of three treatments; PP, PGP, or PGPE(1). Follicular growth was measured by ultrasound in experiments 1 and 2. Every cow (experiments 1, 2, and 3) was blood sampled at selected intervals for progesterone and oestradiol assays and inseminated at oestrus. In experiment 1, a higher percentage of GnRH-treated cows ovulated after the first PGF(2alpha) injection (90% versus 50%; P<0.05). The GnRH-treated cows tended to have a larger dominant follicle present at the time of the second PGF(2alpha) injection (16.5+/-0.5 mm versus 15.0+/-0.7 mm; P<0.10). The percentage of cows that ovulated after the second PGF(2alpha) injection was similar (60%). In experiment 2, cows treated with ECP had higher peak preovulatory concentrations of oestradiol in plasma (6.99+/-0.63 versus 3.63+/-0.63; P<0.01) following the second PGF(2alpha) injection and a higher percentage ovulated (86% versus 43%; P<0.05). A higher percentage of PGPE(1)-treated cows in experiment 3 were observed in standing oestrus and ovulated after the second PGF(2alpha) injection (standing oestrus, 26.4, 34.3, and 62.6%, P<0.01; ovulated, 56, 63, and 78%, P<0.05; PP, PGP, and PGPE(1), respectively). In conclusion, the PGP protocol increased the number of cows that ovulated after the first PGF(2alpha) injection and produced a more mature dominant follicle at the time of the second PGF(2alpha) injection. Adding ECP to PGP (PGPE(1)) enhanced the expression of oestrus and increased ovulation percentage. The combination of PGP and ECP is potentially a new method to routinely synchronise oestrus and ovulation in dairy cows.

Effect of photoperiod on hepatic growth hormone receptor 1A expression in steer calves.

Photoperiod manipulation, specifically a long-day photoperiod (LDPP), increases milk production in lactating cattle. We have previously reported that the galactopoietic effect of LDPP is associated with an increase in circulating IGF-I, which seems to occur independently of changes in concentrations of GH, IGFBP-2, and IGFBP-3. This study tested the hypothesis that LDPP increases the expression of GH receptor (GHR) 1A messenger RNA (mRNA) in the liver. Two groups of Holstein steer calves (98 +/- 4 d old) were maintained indoors and exposed to LDPP (16-h light: 8-h dark; n = 6) or short-day photoperiod (SDPP; 8-h light: 16-h dark; n = 6) for 60 d. Calves were individually fed a grain- and alfalfa-based diet. Jugular blood samples were collected weekly and via cannula at 15-min intervals for a 4-h period on d 1, 26, and 55 of the study to monitor pulsatile hormone secretion. Serum was harvested and assayed for IGF-I, prolactin (PRL), and GH using RIA. Liver biopsies were obtained at 3-wk intervals to quantify changes in hepatic IGF-I and GHR 1A mRNA using real-time PCR. Steer BW increased during the study but did not differ between treatments. No differences in ADG or total DMI were observed. Relative to SDPP, calves on LDPP had higher (P < 0.05) serum IGF-I concentrations. Concentrations of PRL increased (P < 0.01) in calves exposed to LDPP compared with calves exposed to SDPP. Differences (P < 0.05) in pulsatile GH secretion were also detected. Hepatic IGF-I and GHR 1A mRNA were positively correlated with circulating IGF-I concentrations, and although both increased with time, they were not affected by photoperiod treatment. These results confirm that LDPP increases circulating concentrations of IGF-I, but this occurs independently of changes in IGF-I synthesis and GHR 1A mRNA expression in the liver. Therefore, our hypothesis that LDPP increases the expression of GHR 1A mRNA in the bovine liver is rejected.

Decreased follicular size during late lactation caused by treatment with charcoal-treated follicular fluid delays onset of estrus and ovulation after weaning in sows.

The weaning to estrus and weaning to ovulation intervals in sows are controlled by ovarian follicular growth after weaning. Longer intervals could be caused by smaller diameter follicles at weaning that take more time to reach a preovulatory size. We addressed this hypothesis by decreasing the diameter of follicular populations before weaning and then measuring follicular development and interval to estrus and ovulation after weaning. The posterior vena cava, cranial to the entry of the ovarian vein, was cathetered for blood sampling and infusion in 20 sows at 12 +/- 1 d after farrowing. Sows were assigned randomly to receive either 30 mL of charcoal-treated follicular fluid (FF, n = 9; a treatment known to decrease serum FSH and follicular diameter) or 30 mL of saline (n = 11) by venous infusion thrice daily (0700, 1500, and 2300 h) for 96 h beginning at 14 +/- 1 d after farrowing. Sows were weaned 48 h after the last infusion. Blood samples were collected for FSH analysis thrice daily beginning on the day of catheterization and continuing until ovulation. Follicular diameter was determined once daily by transrectal ultrasonography. A treatment x time interaction was detected for serum FSH (P < 0.001) and follicular diameter (P < 0.001) because serum FSH and the diameter of follicular populations decreased in FF sows during the infusion period. After the infusion period, serum FSH rebounded in FF sows, and follicles resumed growth but grew at the same rate as those of saline-treated sows, thus failing to achieve equivalent diameters relative to saline-treated sows on a given day after weaning. As a result, sows treated with FF had longer (P < 0.05) weaning to estrus (6.1 +/- 0.4 d) and weaning to ovulation (8.6 +/- 0.5 d) intervals compared with saline-treated sows (4.7 +/- 0.4 d and 7.2 +/- 0.4 d, respectively). We conclude that the diameter of the follicular population at weaning is one factor that controls interval to estrus and ovulation in sows. Small follicles at weaning cannot undergo compensatory growth and require additional time to reach a preovulatory size.

Detection of Mycobacterium avium subspecies paratuberculosis in free-ranging bison (Bison bison) by PCR.

Bacterial culture is the 'gold standard' for detecting Mycobacterium avium subspecies paratuberculosis (MAP) infection, but is time consuming, laborious, and recovery of organism varies with species of animal tested. PCR has been used for detection of MAP DNA in feces and tissues. We used PCR to detect MAP DNA isolated from tissues from 25 free-ranging North American bison (Bison bison), each with clinical signs compatible with Johne's disease. We report the performance of PCR to detect MAP DNA in both frozen and paraffin-embedded ileum, jejunum, and ileocecal lymph node samples collected at the time of slaughter. Specific oligonucleotide primers for PCR amplification were derived from 16S rRNA sequence M. avium subspecies (MAs) and insertion elements IS1245 (MAs avium), IS901 (MAs avium), IS900 (MAP), and hspX (MAP). Genomic DNA samples were prepared three different ways; crude DNA from frozen tissues, crude DNA from paraffin-embedded tissues, and purified DNA from paraffin-embedded tissues. An animal was considered infected if MAP DNA was detected in at least two separate tissues using the IS900 primer set. Using these criteria, 25 of 25 bison tested were positive for MAP. The data indicate that these free-ranging bison have been infected by MAP.

Plasma hormones and expression of growth hormone receptor and insulin-like growth factor-I mRNA in hepatic tissue of periparturient dairy cows.

Growth hormone plays a central role in the change in nutrient metabolism that occurs during the initiation of lactation. The actions of growth hormone are mediated by the growth hormone receptor (GHR) whose mRNA is present in three alternatively spliced forms (GHR 1A, 1B, and 1C). Liver-specific GHR 1A mRNA is transiently decreased around parturition, but the exact timing of the decline is not known. Our objective was to generate a daily profile for total GHR (GHRtot; all GHR transcripts), GHR 1A, and IGF-I mRNA expression in liver of periparturient Holstein cows and evaluate these daily mRNA profiles relative to daily profiles for periparturient hormones and metabolites. Liver biopsies and blood samples (n = 139) were collected from 65 Holstein cows at the University of Missouri Dairy Farm. At least two cows were sampled on each day from 14 d before to 14 d after parturition. Total cellular RNA was isolated and reverse transcribed to cDNA. Target cDNA were measured by quantitative real-time polymerase chain reaction. Plasma was assayed for progesterone, estradiol, insulin, growth hormone, IGF-I, glucose, and nonesterified fatty acids. The GHR 1A mRNA declined 2 d before parturition, was lowest 3 to 4 d after parturition, and then increased. The IGF-I mRNA declined 1 d after parturition, was lowest 2 to 5 d after parturition and then increased. Total GHR mRNA was not affected by day. The decrease in GHR 1A mRNA was associated with a decrease in progesterone and an increase in estradiol shortly before parturition. A detailed profile of GHR 1A, IGF-I, and GHRtot mRNA expression during the periparturient period was provided. The decreases in GHR 1A and IGF-I during the transition period occurred immediately before (GHR 1A) or shortly after (IGF-I) parturition. Rapid changes in placental and ovarian steroids before parturition were coincident with changes in GHR 1A mRNA.

Growth hormone (GH) binding and expression of GH receptor 1A mRNA in hepatic tissue of periparturient dairy cows.

Growth hormone (GH) plays a role in metabolic adaptations that occur during lactogenesis. Liver GH receptor transcript (GHR 1A) is transiently decreased near parturition and may reduce GH-dependent signaling leading to low blood insulin-like growth factor I (IGF-I) concentrations in periparturient dairy cattle. We hypothesized that the decrease in GHR 1A mRNA at parturition was associated with decreased GH binding (i.e., GHR protein concentration) in liver. Blood and liver biopsy samples were collected from 12 Holstein cows on d -12 +/- 1, 3, and 17 relative to parturition. Total cellular RNA was isolated from a sub-sample of liver. Quantitative real-time polymerase chain reactions were used to measure GHR 1A, total GHR, IGF-I, and cyclophilin mRNA. Microsomal membranes were isolated from the remaining liver tissue and assayed for 125I-bGH binding. Plasma was assayed for GH and IGF-I concentrations. Liver GHR 1A mRNA, specific 125I-bGH binding to liver membranes, liver IGF-I mRNA, and plasma IGF-I concentrations were lower on d 3 relative to d -12. The GHR 1A mRNA, 125I-bGH binding, and plasma GH concentrations increased on d 17 but liver IGF-I mRNA and plasma IGF-I concentrations did not change between d 3 and 17. Total GHR mRNA and cyclophilin mRNA amounts were similar on d -12, 3, and 17. Across all days, 125I-bGH specific binding in liver was highly correlated with liver GHR 1A mRNA (R2 = 0.68) but not with total GHR mRNA. Saturation binding analysis showed that GHR concentration (Bmax) in liver on d 3 had decreased to only 5% of the amount on d -12. We conclude that decreased GHR 1A mRNA leads to decreased GHR protein concentration in liver. Reduced GHR in liver likely contributes to a decrease in liver IGF-I production and reduced concentrations of IGF-I in blood of periparturient cows.

Effect of dietary energy and somatotropin on components of the somatotropic axis in Holstein heifers.

The somatotropic axis, consisting of growth hormone (GH), GH receptor (GHR), insulin-like growth factor (IGF)-I, IGF binding proteins (IGFBP), and IGF receptors, controls growth and mammary development in heifers. Manipulation of the axis with recombinant bovine somatotropin (rbST) improves heifer growth and reduces age at first calving. The effects of rbST are influenced by dietary energy through partially understood mechanisms. The objective was to characterize the somatotropic axis in Holstein heifers fed a diet for either low or high rate of gain and treated with or without rbST. Heifers (120 d of age) were assigned to one of 2 diets to gain either 0.8 kg/d (low, n = 18) or 1.2 kg/d (high, n = 20). Within each diet, half of the heifers (n = 9 for low and n = 10 for high) received daily rbST injections (25 microg/kg of body weight). Treatments and diets continued until slaughter (2 mo after puberty). Blood was collected 2x per week, and a frequent sampling window was performed 1 d before slaughter. Liver was collected at slaughter. Feeding a high diet or treating with rbST increased serum IGF-I and decreased serum IGFBP-2. The observed changes in serum IGF-I and IGFBP-2 were reflected in their respective liver mRNA amounts. Feeding a high diet decreased serum GH concentrations after rbST injection, but the stimulatory effect of rbST on serum IGF-I was nonetheless greater in high-diet heifers. The differential IGF-I response may be explained by greater GHR 1A in the liver of high-diet heifers. We conclude that a high-gain diet modifies the somatotropic axis in rbST-treated heifers by decreasing serum GH but increasing serum IGF-I after rbST treatment. Greater IGF-I (indicative of an increased GH response) may be a consequence of greater GHR 1A expression in the liver.

Feeding reduces activity of growth hormone-releasing hormone and somatostatin neurons.

Secretion of growth hormone (GH) is synchronized among castrate male cattle (steers) around feeding when access to feed is restricted to a 2-hr period each day. Typically, concentrations of GH increase before and decrease after feeding. Our objectives were to determine whether i) concentrations of GH decrease in blood after start of feeding; ii) activity of immunoreactive growth hormone-releasing hormone (GHRH-ir) neurons decreases in the arcuate nucleus (ARC) after feeding; iii) activity of immunoreactive somatostatin (SS-ir) neurons in the periventricular nucleus (PeVN) and ARC increase after feeding; and iv) GHRH stimulates release of GH to a similar magnitude at 0900 and at 1300 hr, in steers fed between 1000 and 1200 hr. Blood samples were collected at 20-min intervals from 0700 to 1300 hr. Groups of steers were euthanized at 0700, 0900, 1100, and 1300 hr (n = 5 per group). Dual-label immunohistochemistry was performed on free-floating sections of hypothalami using antibodies directed against Fos and Fos-related antigens (Fos/FRA) as a marker of neuronal activity in immunoreactive GHRH and SS neurons. Concentrations of GH were high before and decreased after feeding. The percentage of SS-ir neurons containing Fos/FRA-ir in the PeVN was 50% lower (P<0.01) at 1100 hr and 36% lower (P<0.05) at 1300 hr than at 0900 hr. There was no change in percentage of SS-ir neurons containing Fos/FRA-ir in the ARC. The percentage of GHRH-ir neurons containing Fos/FRA-ir in the ARC was 66% lower (P<0.05) at 1100 hr and 65% lower (P<0.05) at 1300 hr than at 0700 hr. In contrast, the number of GHRH-ir neurons increased from 0700 to 1300 hr. GHRH-induced release of GH was suppressed at 1300 hr compared with 0900 hr. In conclusion, reduced basal and GHRH-induced secretion of GH after feeding was associated with decreased activity of GHRH neurons in the ARC and decreased activity of SS neurons in the PeVN.

Short communication: relationship between body growth and mammary development in dairy heifers.

Our objective was to determine if prepubertal rate of body weight (BW) gain, independent of diet, was related to mammary development of dairy heifers. Data from two studies recently conducted at Michigan State University were used to identify factors, within a dietary treatment group, that would account for variation in first lactation milk production or amount of mammary parenchymal DNA at the time of puberty. Factors analyzed for variation in milk production during first lactation were: postpartum BW, prepubertal BW gain, gestational BW gain, postpartum BW gain, body condition score (BCS) at breeding, and BCS at calving. Factors analyzed for variation in mammary parenchymal DNA at puberty were: BW at slaughter, age at puberty, prepubertal BW gain and body protein and body fat content at slaughter. For both analyses, prepubertal BW gain did not account for any of the variation in mammary development. The only significant covariate for the milk production model (r2 = 0.44) was BCS at breeding. Similarly, the only significant covariate in the parenchymal DNA model (r2 = 0.22) was body fat content at slaughter. These results suggest that, within a dietary treatment, heifers that grow faster do not have impaired mammary development, and increased body fatness may be a better predictor of impaired mammary development than BW gain.

Somatostatin inhibits alpha-2-adrenergic-induced secretion of growth hormone-releasing hormone.

The purpose of this experiment was to determine the role of growth hormone-releasing hormone (GHRH) and somatostatin (SRIH) neurons in mediating alpha(2)-adrenergic receptor-induced stimulation of growth hormone (GH) secretion in cattle. Our first objective was to determine if stimulation of alpha(2)-adrenergic receptors increases activity of GHRH neurons in the arcuate nucleus (ARC) and/or decreases activity of SRIH neurons in periventricular (PeVN) and ARC nuclei. Clonidine (an alpha(2)-adrenergic agonist) or vehicle (saline) were injected i.v. into steers and dual-label immunohistochemistry was performed to quantify the number of GHRH and SRIH neurons expressing Fos and Fos-related antigens (Fos/FRA) as markers of neuronal activity. Clonidine increased concentrations of GH in serum and decreased activity of SRIH neurons in the PeVN, but not in the ARC. Clonidine did not alter activity of GHRH neurons in the ARC. Our second objective was to determine if clonidine decreases secretion of SRIH from perifused slices of hypothalami, which contain perikarya and terminals of GHRH and SRIH neurons, and from explants of hypophysial stalk alone, which contain only terminals of GHRH and SRIH neurons. Clonidine failed to alter release of GHRH or SRIH from hypothalamic slices, but stimulated release of GHRH from explants of hypophysial stalk. Blockade of SRIH receptors enabled clonidine to stimulate release of GHRH from slices of hypothalami, but also stimulated release of SRIH. These results suggest that alpha(2)-adrenergic-induced secretion of GH occurs via a dual mechanism involving inhibition of SRIH neurons in the PeVN and direct stimulation of GHRH release from axon terminals in the median eminence.