Ovariectomy in young prepubertal dairy heifers causes complete suppression of mammary progesterone receptors.

Mammary growth and development depends on ovarian steroids and particularly interaction of estrogen and progesterone with their intracellular receptors. The objectives of this study were to determine the effect of ovariectomy on the expression of protein and messenger RNA for estrogen receptor-alpha (ESR1) and progesterone receptor (PGR) and their relation to mammary ductal development and cell proliferation. Prepubertal Holstein heifers 2, 3, or 4 mo of age were randomly assigned to one of 2 treatments, ovariectomized (OVX; n = 8) or sham operated (INT; n = 12). Mammary parenchymal (PAR) tissue samples were harvested 30 d after surgery. Localization and quantitation of ESR1 and PGR in PAR were determined by immunohistochemistry and quantitative multispectral imaging. Relative messenger RNA expression of ESR1 and PGR in PAR was measured by quantitative real time polymerase chain reaction. We observed the complete absence of PGR-positive epithelial cell nuclei and reduced PGR transcript abundance in mammary parenchyma of OVX heifers. The percent of epithelial cells expressing ESR1 did not differ by treatment but was decreased with age. However, average intensity of ESR1 expression per cell was reduced in OVX heifers. The abundance of Ki67 labeled epithelial cells and stromal cells was reduced after ovariectomy. These data suggest that reduced mammary development after ovariectomy may be mediated by loss of PGR expression and reduced ESR1 expression in positive cells. A presumptive relationship with ovarian-derived circulating estradiol remains unresolved, but data suggest other ovarian-derived agents may play a role. Use of specific antagonists to manipulate expression or action of PGR and ESR1 receptors should provide direct evidence for roles of these receptors in prepubertal bovine mammary development.

Effect of staged ovariectomy on measures of mammary growth and development in prepubertal dairy heifers.

Previous studies in prepubertal heifers suggest that the magnitude of reduction in mammary parenchymal growth in response to ovariectomy varies with the age at which surgery is performed. We hypothesized that ovarian secretions are essential for initiating mammary development but not required to maintain allometric mammary growth in prepubertal dairy heifers. The objectives of this study were to determine the effect of staged ovariectomy during the prepubertal period on mammary growth and tissue composition and the expression of selected genes. Prepubertal Holstein heifers at 2, 3 or 4 months of age were randomly assigned to one of two treatments, ovariectomized (OVX; n = 12) or sham operated (INT; n = 12). Mammary parenchyma (PAR) and fat pad (MFP) were harvested 30 days after surgery. Proximate composition of PAR and MFP (DNA, protein and lipid) as well as expression of the selected estrogen-responsive genes stanniocalcin1 (STC1), tissue factor pathway inhibitor precursor (TFPI) and proliferating cell nuclear antigen (PCNA) were determined in PAR and MFP by quantitative real-time PCR. The relative amount of epithelium and proportion of epithelia cell nuclei expressing the proliferation marker Ki67 were determined by histological and immunohistochemical analyses, respectively. MFP mass was not impacted by treatment but was decreased with age as was lipid content and concentration (P ⩽ 0.01). The mass of mammary PAR was reduced in OVX and increased with age (P ⩽ 0.01). Parenchymal tissue tended to have less total DNA, protein and lipid in OVX heifers. Parenchymal tissue concentrations of protein and DNA were increased with age and there was an age × treatment interaction. Treatment had no effect on either the Ki67 labeling index or percent epithelial area. The relative abundances of STC1, TFPI and PCNA mRNA in PAR were reduced in OVX. We did not find a significant impact of ovariectomy on mRNA expression when surgery was performed at 2 months compared with surgery at 3 or 4 months of age. However, having nearly undetectable PAR in two heifers ovariectomized at the earliest period (2 months of age) suggests that early ovariectomy is especially detrimental to subsequent parenchymal development.

Effect of exogenous somatotropin in Holstein calves on mammary gland composition and proliferation.

Pubertal mammary gland growth and development are hormonally regulated, but the details are poorly understood in calves. Our purpose was to evaluate the effects of exogenous growth hormone (GH) on the biochemical composition of the prepubertal mammary gland, mRNA expression of selected genes, and histological characteristics of the developing parenchyma (PAR). In this experiment, 19 calves (7 ± 4 d of age) were randomly assigned to 1 of 2 treatments: bovine somatotropin (bST, 500 mg; n = 10) or placebo (Sal; 0.9% saline; n = 9). Animals were treated every 3 wk beginning on d 23. Calves were assigned to an early (65 d; tissue harvested after 2 treatment injections) or late collection time (107 d; tissue harvested after 4 treatment injections). Calves were fed milk replacer and calf starter for 8 wk and starter and hay thereafter. Parenchyma and mammary fat pad (MFP) from one udder half were harvested for analysis of protein, lipid, and DNA. Additional tissues were preserved for histological analysis or snap-frozen for quantitative real-time PCR. Somatotropin treatment did not significantly alter the mass of PAR or MFP or the general pattern of development of epithelial structures. Significant increases were observed in protein/100 kg of body weight (BW), total protein, DNA concentration, DNA/100 kg of BW, and total DNA in 107-d calves, and a significant treatment by day interaction was observed for DNA and lipid concentrations in PAR. In MFP, a significant decrease was observed in protein/100 kg of BW in bST-treated calves and in total MFP protein in 65-d calves. A treatment by day interaction was found for total protein, DNA, and protein/100 kg of BW. In PAR, relative expression of ATPase-binding cassette 3 and growth hormone receptor were reduced by bST and both were lower in 107-d-harvest calves. Epithelial cell retention of bromodeoxyuridine (BrdU; possible indicator of stem-like cells) was greatest in 65-d bST-treated calves, and a significant time of sampling response and treatment × time interaction were observed. Expression of the proliferation marker protein Ki67 was numerically higher in bST-treated calves but the difference was nonsignificant. Retention of the BrdU label was reduced in 107-d calves. Exogenous growth hormone given to calves may affect mammary tissue composition and epithelial cell gene expression in subtle ways but exogenous supplementation with bST alone is not likely to alter overall development patterns or affect the mass of mammary parenchymal tissue. Whether such subtle changes have an effect on subsequent development or function is unknown.

Developmental histology, segmental expression, and nutritional regulation of somatotropic axis genes in small intestine of preweaned dairy heifers.

Components of the somatotropic axis and nutrition regulate intestinal development and maturation of enterocytes. We measured gene expression in the mucosal layer of small intestine of preweaned dairy heifers to test the hypothesis that feeding increased amounts of protein and fat alters expression of somatotropic axis genes. Twenty-four newborn Holstein heifers were randomly assigned to 1 of 4 milk replacer (MR) diets: (1) 20% CP, 20% fat MR (DM basis) fed at 450 g/d (CON); (2) 28% CP, 20% fat MR fed at 970 g/d (HPLF); (3) 28% CP, 28% fat MR fed at 970 g/d (HPHF); and (4) 28% CP, 28% fat MR fed at 1,460 g/d (HPHF+). Dry calf starter (20% CP, 1.43% fat) was offered free choice. At 64 +/- 3 d of age heifers were killed and intestinal tissues were harvested for RNA isolation and histological examination. We measured the mRNA expression of growth hormone receptor (GHR), insulin-like growth factor-I (IGF-I), IGF-I receptor (IGF-IR), and IGF binding proteins (IGFBP)-1 to -6 in duodenum, jejunum, and ileum by quantitative real-time reverse transcription PCR. Expression of IGFBP-3 mRNA was lowest in the duodenum of HPHF+ and greatest in the ileum of the CON group, whereas expression of IGFBP-4 mRNA was greatest in the jejunum of the HPHF+ group. Expression of IGFBP-5 mRNA was greatest in the CON and lowest in the HPHF+. However, overall diet did not affect expression of GHR, IGF-I, IGF-IR, or IGFBP-1, -2, and -6. Expression of somatotropic axis genes differed among small intestinal locations. The GHR, IGF-IR, IGFBP-1, and IGFBP-5 mRNA were greatest in the ileum. Duodenum produced less IGF-IR, IGF-I, and IGFBP-5 mRNA. Villi were shortest in the ileum, but there was no difference in villus height between the duodenum and jejunum. There was no difference in crypt depth or villus circumference between locations. In conclusion, some components of the somatotropic axis in preweaned dairy heifers are differentially expressed in regions of the small intestine, and the gene expression tended to be affected by dietary protein and fat.

Growth rate and changes of the somatotropic axis in beef cattle administered exogenous bovine somatotropin beginning at two hundred, two hundred fifty, and three hundred days of age.

To determine the effects of bovine somatotropin (bST) treatment beginning at 3 ages on the growth rate and components of the somatotropic axis, 40 beef cattle (200 +/- 21 d of age) were randomly assigned to 1 of 4 treatments (10 animals/treatment). Three of the treatment groups received bST (33 mug/kg of BW) daily beginning at 200, 250, or 300 d of age until all animals reached 400 d of age; the fourth group served as controls (0 bST). Animals were housed in pens (5 animals per pen; 2 pens per treatment) and fed a diet formulated for an ADG of 1.2 kg/d. Feed intake (per pen) was measured daily, and BW was determined weekly. Blood samples (10 mL) and ultrasound measurements were collected at 200, 250, 300, 350, and 400 d of age. Serum concentrations of ST and IGF-I were determined by RIA and IGFBP-2 and -3 by ligand blot procedures. Overall, cattle gained 284.0 +/- 14.7 kg of BW with a treatment x week interaction (P < 0.01), such that during the treatment period ADG was 11.6, 8.7, and 15.8% greater (P < 0.05) in cattle treated with bST beginning at 200, 250, and 300 d, respectively, relative to controls during the same time frame. Average DMI was 13.6% less (P < 0.05) in bST-treated cattle than in controls. Increases in ADG coupled with a reduction in DMI resulted in 11.7, 14.0, and 26.4% increases (P < 0.01) in the efficiency of gain (G:F) in bST-treated cattle beginning at 200, 250, and 300 d of age, respectively, compared with contemporary controls. Backfat thickness increased (P < 0.05) over time, but the magnitude of the increase was less in the bST-treated cattle (treatment x week interaction; P < 0.05). Area of the LM increased (P < 0.05) over time but was similar across treatment groups. Serum concentrations of ST, IGF-I, and IGFBP-3 increased (P < 0.05), whereas IGFBP-2 decreased (P < 0.05) over time. The changes in the components of somatotropic axis were more pronounced in bST-treated cattle compared with controls, with the greatest magnitude of response in animals that began bST treatment at 300 d of age. In conclusion, the exogenous bST-induced growth response was greater in animals that began to receive bST administration at 300 d of age and received it for a shorter period (100 d) compared with animals that received bST beginning at 200 or 250 d of age.

Evidence of avian pneumovirus spread beyond Minnesota among wild and domestic birds in central North America.

To detect avian pneumovirus (APV) in central North America, nasal turbinates or choanal deft tissues from domestic turkeys and wild birds were examined for the presence of APV RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas serum samples from domestic turkeys were analyzed for APV antibodies by enzyme-linked immunosorbent assay (ELISA). In 2002, the seroprevalence of disease in domestic turkeys in Minnesota remained high (42.3% of the flocks). In addition, there is evidence the disease has spread to turkey flocks in North Dakota (8.2%), South Dakota (7%), Iowa (10%), and Wisconsin (8.6%) as detected by RT-PCR and/or ELISA. House sparrows and ring-billed gulls sampled in Minnesota and snow geese from Saskatchewan, Canada, were found to harbor APV RNA. Sequence analysis of wild bird APV strains showed high amino acid sequence identity among wild bird isolates (<97%) and between wild bird and turkey viral isolates (93.2%-99.3%). This study demonstrated that APV infections were present in domestic turkey flocks and wild birds outside the state of Minnesota; however, the role of wild birds in spreading APV to domestic turkeys remains unclear.