Mouse bioassay to assess oestrogenic and anti-oestrogenic compounds: hydroxytamoxifen, diethylstilbestrol and genistein

Young intact and adult gonadectomized C3H male and female mice were utilized as bioassay models to detect endocrine disruption chemicals. Animals treated with oestradiol (E) or progesterone (Prog) or E plus Prog were used to assess steroid hormone agonist and antagonist activities of 4-OH-tamoxifen (TAM), diethylstilbestrol (DES) and genistein (GEN) by bioassay. The stimulation or inhibition of mammary growth by TAM depended on sex, the state of animal (intact or gonadectomized), hormonal treatment (Prog, E, E plus Prog) and dose of TAM. TAM stimulated mammary growth in untreated ovariectomized (OV-X) females and in Prog-treated intact males and OV-X females. In intact males, mammary growth was increased by TAM at dose 0.1-1 microg day(-1) and decreased at dose 10 microg day(-1). Mammary growth was inhibited by TAM in Prog-treated intact females and in E or E plus Prog-treated intact and gonadectomized males and females. Uterine weights were increased by TAM in both untreated and treated (E, Prog, E plus Prog) intact and OV-X females; however, seminal vesicle weights were decreased by TAM in both untreated and treated (E, Prog, E plus Prog) intact males. DES alone affected mammary growth (an inverted-U-shaped dose-response curve) both in male and female mice. DES increased uterine weights; however, seminal vesicle weights were decreased. GEN increased mammary and uterine growth in OV-X females, GEN plus Prog stimulated mammary growth in intact males. The results obtained in these studies show clearly that only a bioassay consisting of several endpoints reflective to the mechanism of oestrogen and anti-oestrogen action has the ability to evaluate activities of a molecule.     

Mouse bioassay for in vivo screening of oestrogen and progesterone antagonists

This study tested and compared the anti-proliferative and proliferative activities of two anti-oestrogens and three anti-progestins on four separate mouse model systems: young intact and adult ovariectomized (OV-X) females, and young intact and adult castrated males. Pure steroidal anti-oestrogen ICI 182,780 (ICI) decreased mammary and uterine growth stimulated by endogenous hormones in young intact females and by exogenous hormones [progesterone (Prog), 17beta-oestradiol (E) or E plus Prog] in both young intact and adult ovariectomized (OV-X) females. Non-steroidal anti-oestrogen EM-800 (EM), on the other hand, had no effect on mammary and uterine growth stimulated by endogenous hormones in young intact females and in adult OV-X females. Uterine growth was even stimulated by EM alone, and a combination of EM plus Prog not only stimulated uterine growth but also mammary growth (an oestrogenic agonistic activity). However, EM showed anti-oestrogenic activities in both mammary and uterine tissues in females treated with E or E plus Prog. In males, ICI and EM decreased mammary growth stimulated by exogenous hormones (E or E plus Prog) in both young intact and adult castrated animals. In young intact, but not in adult castrated males, ICI increased seminal vesicle growth affected by both endogenous and exogenous (Prog, E or E plus Prog) hormones. EM, on the other hand, decreased seminal vesicle weights in E or E plus Prog and increased its weights in Prog-treated young intact males. Thus, under certain conditions EM possess mixed agonist and antagonist activity in the mammary gland, uterus and seminal vesicles. Norethindrone acetate (NA)-stimulated mammary growth was decreased by anti-progestins onapristone (ON), RU 46556 (RU), and RU 38486 (MI) by 34-59% in females and by 35-93% in males. Uterine weights of NA-treated females were decreased by ON and RU by 29-55% but not by MI. In NA-treated young intact males, seminal vesicle weights were stimulated by RU (by 63%) and not affected by ON and MI. In NA-treated adult castrated males, seminal vesicle weights were decreased by ON, increased by RU and not affected by MI. The results obtained in these and our earlier studies show clearly that mouse four-model systems could serve as in vivo tool for the detection of steroid hormone agonist and antagonist activities of natural and man-made chemicals.     

Effects of estrogen and progesterone on the development of the mammary gland and the associated blood vessels in ovariectomized mice.

Effects of estrogen and progesterone on the first abdomino-inguinal mammary gland and the associated blood vessels in ovariectomized mice were investigated morphometrically, and light and electron microscopically. Although there were no significant differences in the area of the fat pad of the mammary gland among the experimental groups, the area of the mammary parenchyma and diameters of the blood vessels supplying the mammary gland, i.e., A. et V. circumflexa ilium profunda and A. et V. epigastrica caudalis superficialis, reached the maximal value in ovariectomized mice treated with estradiol (E) + progesterone (P). Similarly, the blood capillaries around buds and ducts of the mammary gland were most densely distributed in E + P treated mice. In the adipose tissues of the mammary stroma in intact mice, fat cells were of multilocular type in the peripheral regions around the main vessel, and of unilocular type in the other part. In E and E + P treated mice, however, fat cells were mostly of unilocular type. These findings suggest that the formation of mammary fat tissues may occur in advance of that of the mammary parenchyma. By TEM, epithelial cells of the mammary parenchyma contained a large number of mitochondria and ribosomes, well-developed Golgi apparatus and rER, and large lipid droplets. Endothelial cells of blood capillaries displayed numerous pinocytotic vesicles, longer marginal folds and microvillous processes. Each organelle in these two cell types increased in number or developed to a greater degree in E + P treated mice than the other experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of spaying and timing of spaying on survival of dogs with mammary carcinoma

The risk of developing mammary gland tumors in dogs is significantly decreased by ovariohysterectomy at an early age. However, previous studies have not found a benefit to ovariohysterectomy concurrent with tumor removal in dogs with established mammary gland tumors, suggesting that the progression of these tumors is independent of continued estrogen stimulation. The purpose of this study was to evaluate the effect of spaying and of the timing of spaying on survival in dogs with mammary gland carcinoma. Signalment, spay status and spay age, tumor characteristics, treatment. survival, and cause of death of 137 dogs with mammary gland carcinoma were analyzed. The dogs were classified into 3 groups according to spay status and spay time: intact dogs, dogs spayed less than 2 years before tumor surgery (SPAY 1), and dogs spayed more than 2 years before their tumor surgery (SPAY 2). Dogs in the SPAY 1 group lived significantly longer than dogs in SPAY 2 and intact dogs (median survival of 755 days, versus 301 and 286 days, respectively, P = .02 and .03). After adjusting for differences between the spay groups with regard to age, histologic differentiation, and vascular invasion, SPAY 1 dogs survived 45% longer compared to dogs that were either intact or in the SPAY 2 group (RR = .55; 95% CI .32-.93; P = .03). This study reveals ovariohysterectomy to be an effective adjunct to tumor removal in dogs with mammary gland carcinoma and that the timing of ovariohysterectomy is important in influencing survival.     

Identification of the bovine alpha1-acid glycoprotein in colostrum and milk

Alpha1-acid glycoprotein (AGP) is an immunomodulatory protein expressed by hepatocytes in response to the systemic reaction that follows tissue damage caused by inflammation, infection or trauma. This paper presents the detection of bovine AGP (boAGP) in mammary secretions (colostrum and milk) and mammary gland tissue. Bovine AGP was detected by Western blotting in all the samples analysed, and could be quantified in colostrum at 162 (+/- 63.7) microg/mL and 114.5 (+/- 67.8) microg/mL during the first 12 h and 24 h respectively. In mature milk, the boAGP concentration clearly decreased and was no longer detectable using the Radial Immunodiffusion (RID) technique. The concentration of mature milk boAGP was therefore semi-quantified using an anion-exchange chromatographic procedure that allowed the concentration of the protein to be determined. The presence of AGP in bovine milk was confirmed by the internal sequence analysis performed following purification to homogeneity of the protein from milk. The concentration of AGP in bovine milk with low SCC (< 250,000) was very similar to that from bovine milk with high SCC (> 250,000). In order to investigate the origin of AGP in bovine milk, a search for mRNA was carried out in somatic cells and mammary gland tissue: mRNA expression of the boAGP gene was detected in mammary gland tissue, but not in somatic cells. Finally, the cDNA sequence of the boAGP was determined, and is hereby presented.     

Comparison of estrogen responsive genes in the mouse uterus, vagina and mammary gland

Female reproductive organs are mainly regulated by estrogen and progesterone. Specifically, the uterus, vagina and mammary gland show organ-specific mitosis and morphological changes during proliferative events, such as estrous cycle, gestation and lactation. The mechanism underlying these organ-specific estrogen-dependent events is still unknown. We examined, therefore, global gene expression in the mature uterus, vagina and mammary gland of ovariectomized adult mice 6 hr after an injection of 5 microg/kg 17beta-estradiol (E2) using a microarray method in order to identify primary E2-responsive genes. Half of the E2 up-regulated genes in the uterus were similar to those in the vagina. E2 up-regulated the expression of Insulin-like growth factor 1 (Igf-1) genes in the uterus and vagina. In the vagina, E2 up-regulated the expression of IGF binding proteins (Igfbp2 and Igfbp5). In the mammary gland, unlike the uterus and vagina, no gene showed altered expression 6 hr after the E2 exposure. These results suggest that expression of Igf-1 and morphogenesis genes is regulated by E2 in an organ-specific manner, and it is supported by the results of BrdU labeling showing E2-induced mitosis in the uterus and vagina except the mammary gland. The differences in organ specificity in response to E2 may be attributed by differences in gene expression regulated by E2 in female reproductive organs. The candidate estrogen-responsive genes in the uterus and vagina identified by profiling provide an important foundation understanding functional mechanisms of estrogen regulating morphogenesis and maintenance of each reproductive organ.     

Quantification of mammary gland tissue size and composition changes after weaning in sows

The objectives of this study were to characterize the tissue compositional changes in porcine mammary glands after weaning and to determine whether administration of estradiol alters the profile of these tissue changes. Forty-five primiparous sows were assigned randomly to one of two treatment groups after weaning, control or estrogen treated. Estrogen-treated sows received twice-daily injections of estradiol-17beta (0.125 mg/kg of BW); control sows received vehicle injections. Sows were weaned at d 21 of lactation and killed on either d 0 (d of weaning; n = 5) or on d 2, 3, 4, 5, or 7 after weaning (n = 4 per treatment on each day). Teat order relative to suckling behavior was observed on the day before weaning to determine which mammary glands the piglets suckled. Suckled and non-suckled glands were identified from the teat order observation, and individual mammary glands were collected at slaughter. Mammary glands were trimmed of skin and extraneous fat pad, individually weighed, and bisected to measure cross-sectional area. The remaining half of each gland was ground and stored at -20 degrees C for chemical analyses. Frozen tissue was used for measuring tissue DNA, DM, protein, fat, and ash contents. Suckled mammary glands of sows undergo significant and dramatic changes during the initial 7 d after weaning, with significant changes occurring even by d 2 after weaning. Mean cross-sectional area of parenchymal tissue in suckled mammary glands decreased from 59.7 +/- 2.1 cm2 on the day of weaning to 26.8 +/- 2.3 cm2 by d 7 after weaning (P < 0.0001). Mammary gland wet weight decreased from 485.9 +/- 22.0 g on the day of weaning to 151.5 +/- 24.8 g by d 7 after weaning (P < 0.0001), whereas DNA decreased from 838.8 +/- 46.2 g on the day of weaning to 278.4 +/- 52.5 g by d 7 after weaning (P < 0.0001). The changes in gland wet weight and DNA during the period of mammary gland involution in the sow represent loses of over two-thirds of the parenchymal mass and nearly two-thirds of the cells that were present on the day of weaning. Estrogen treatment did not affect overall mammary involution during the first 7 d after weaning. Mammary glands that were not suckled during lactation had no further loss of parenchymal tissue during the first 7 d after weaning. Mammary gland involution in the sow is a rapid process and is probably irreversible within 2 or 3 d after weaning.     

The expression of carbonic anhydrase in canine mammary gland and mammary gland tumor

Carbonic anhydrase (CA), a metallo-enzyme containing zinc, broadly distributes in mammalian tissues and participates in physiological regulation such as respiration, acid-base balance, ion transport, bone resorption, as well as the development of tumor by the reversible hydration of carbon dioxide. However the expression of CA in the tissue of mammary gland tumor was not documented. In this study we examine the histolocalization and gene expression of CA in both normal canine mammary gland tissue and mammary gland tumor by histochemical examination, and RT-PCR. Four mRNA expression of CA isoenzymes, such as CA II, IV, VI and IX were found under RT-PCR analysis and different band patterns were found between normal canine mammary tissue and canine mammary gland tumor tissue. CA II, IV, VI and IX gene mRNA expression were found in the normal mammary gland tissue, indicating CA II, IV, VI and IX are likely to be the essential enzymes to maintain the normal physiological condition of canine mammary gland tissue cells. However the expression of CA IV was not found in the tissue of malignant mammary gland tumor that may become the marker for the prognostic recognition of canine mammary gland tumor.     

Bulk and trace elements in the accessory glands of buffalo bulls.

Little is known of the importance of mineral levels in buffalo semen or the relationship of the level of various elements to spermatozoan activity. To establish a basis upon which further studies could be conducted, the accessory glands (seminal vesicles, prostate and bulbo-urethrals) of 43 buffalo bulls (2-4 years old) with intact testes and eight steers (3-4 years old) were analysed for their content of bulk and trace elements. The ash obtained from known weights of various accessory glands was used to determine the concentrations of Na and K using emission flame photometry. P was determined chemically, whereas Ca, Mg, Cu, Fe and Zn were assayed by atomic absorption spectrophotometry. The accessory glands of intact bulls varied significantly in the concentration of all elements except Fe. Ca and Cu were relatively more concentrated in the seminal vesicles. The prostate had the highest concentration of Zn, while the highest concentrations of Na, K, Mg and P were found in the bulbo-urethral glands. Age of the bulls had no marked effect on any of the elements studied except Cu which appeared to decrease significantly in older bulls. Seasonal variation was significant for the levels of K, Ca and Zn. Castration appeared to result in a highly significant reduction of Zn concentration in all accessory glands.     

Decreased sperm number and motile activity on the F1 offspring maternally exposed to butyl p-hydroxybenzoic acid (butyl paraben)

Butyl p-hydroxybenzoic acid (butyl paraben, BP) is widely used as a preservative in food and cosmetic products. Routledge et al showed that BP is weakly estrogenic in both in vitro and in vivo (rat uterotrophic) analyses. We investigated whether maternal exposures to BP during gestation and lactation periods affected the development of the reproductive organs of the F1 offspring. Pregnant Sprague-Dawley rats were injected subcutaneously with 100 or 200 mg/kg of BP from gestation day (GD) 6 to postnatal day (PND) 20. In the group exposed to 200 mg/kg of BP, the proportion of pups born alive and the proportion of pups surviving to weaning were decreased. The body weights of female offspring were significantly decreased at PND 49. The weights of testes, seminal vesicles and prostate glands were significantly decreased in rats exposed to 100 mg/kg of BP on PND 49. In contrast, the weights of female reproductive organs were not affected by BP. The sperm count and the sperm motile activity in the epididymis were significantly decreased at doses of 100 and 200 mg/kg of BP. In accordance with the sperm count in the epididymis, the number of round spermatids and elongated spermatids in the seminiferous tubule (stage VII) were significantly decreased by BP. Testicular expression of estrogen receptor (ER)-alpha and ER-beta mRNA was significantly increased in 200 mg/kg of BP treated group at PND 90. Taken together, these results indicated that maternal exposure of BP might have adverse effects on the F1 male offspring.     

Pathologie changes in endocrine glands and certain other tissues of lambs implanted with the synthetic growth promotant zeranol.

As doses of zeranol implants (0 (control), 12, 24, 48, and 96 mg) were increased, there were increased reaction and activity in target organs (such as urogenital tract and mammary, adrenal, hypophyseal, and thyroid glands) of castrated male sheep (wethers). Hyperplasia and transitional and squamous transformation in the prostate were mild (1+) in the wethers given 12- and 24-mg doses, moderate to marked (2.5+) in the wethers given 48-mg doses, and severe (4+) in the wethers given 96-mg doses. Papillary proliferation and fibrosis increased correspondingly in the seminal vesicle. Changes in the distal penile urethra increased from papillary hyperplasia in the wethers given a 24-mg dose to 100% squamous transformation in the wethers given a 96-mg dose. Mammary gland development was noticeable in the wethers given a 24-mg dose and increased thereafter to progressive alveolar growth and secretory activity in the wethers given 48- and 96-mg doses. Along with a progressive increase of adrenal gland weight and adrenal gland/thyroid gland ratios over the controls, the principals had hypertrophy and hyperplasia of the adrenal cortex. Mean adrenal cortex widths for control wethers and wethers given 24-, 48-, and 96-mg doses were 2,089, 2,140 (adjusted value, 2,194), 2,416, and 2,425 mum, respectively. Mean adrenal gland weights for control wethers and wethers given 12-, 24-, 48-, and 96-mg doses were 2.50, 2.61, 2.53, 2.70, and 2.78 g. respectively. Hyperplasia (nodule formation) plus exhaustive and pyknotic changes of the adrenal cortex increased similarly with increasing zeranol dose. After the thyroid gland weights decreased (2.19, 2.04, 2.00, and 1.72 g, respectively, for control wethers and wethers given 12-, 48-, and 96-mg doses), secretory activity of thyroid epithelial cells decreased. In the glandular portion of the hypophysis, secretory activity and proliferation of eosinophilic cells increased with the larger zeranol doses (48 and 96 mg). There was a corresponding decrease in the number of basophils. These changes are consistent with increased somatotropin and adrenocorticotropin secretion and decreased thyrotropin secretion. Muscle and ligament structures appeared looser and widened in the wethers given the 96-mg dose, and fat cell formation was increased in the muscles along ligament muscle junctions.     

Mammary gland growth as influenced by litter size in lactating sows: impact on lysine requirement

Twenty-eight primiparous sows were used to determine the effect of litter size on the growth of mammary glands and nursing pigs during lactation. Litter size was set to 6, 7, 8, 9, 10, 11, or 12 pigs by cross-fostering immediately after birth. Four sows were allotted to each litter-size group. Sows were allowed to consume a daily maximum of 13.6 Mcal ME and 46.3 g of lysine during lactation. Sows were slaughtered on d 21 (20.6+/-1.1) of lactation. Mammary glands were collected at slaughter and trimmed of skin and the extraneous fat pad. Each gland was separated, weighed, and ground for chemical analysis. Dry matter, dry fat-free tissue (DFFT), crude protein, ash, and DNA contents were measured. Only glands known to have been nursed were included in the data set. Wet and dry weights and the amounts of DFFT, protein, DNA, ash, and fat in individual nursed mammary glands linearly decreased (P<.05) as litter size increased. Percentages of DFFT, protein, and DNA were quadratically affected (P<.05) by litter size on d 21 of lactation. Total mammary wet and dry weights and total DFFT, protein, DNA, fat, and ash amount of all nursed mammary glands of each sow were increased as litter size increased (P<.05). Changing litter size from 6 to 12 pigs resulted in 2,098, 432, 253, 227, 4.4, 178, and 20 g increases in the amounts of total mammary wet weight, dry weight, DFFT, protein, DNA, fat, and ash, respectively, on d 21 of lactation. Litter weight gain was 18.1 kg greater in sows with 12 pigs than in sows with 6 pigs. Sows with a larger litter size had a greater increase in total mass of mammary gland tissue and litter weight but had lower growth of individual nursed mammary glands and individual pigs than sows with the smaller litter size. The need for nutrients to support additional mammary gland and litter growth as litter size increases should be considered when estimating nutrient requirements for lactating sows. Sows need an additional .96 g lysine per day to account for mammary gland growth for each pig added to a litter.     

Measurement of Carbonic Anhydrase Isozyme VI (CA-VI) in Bovine Sera,Saliva, Milk and Tissues

Concentrations of bovine carbonic anhydrase isozyme VI (CA-IV) in bovine serum, saliva, normal milk, colostrum, submandibular gland, liver, and mammary gland were determined. CA-VI was purified from bovine saliva and an antibody to CA-VI was generated. The concentrations of CA-VI in the saliva (7.8 ± 7.9 μg/ml), serum (2.1± 5.7 ng/ml), milk (7.9 ± 12.1 ng/ml), submandibular gland (284.7 μg/g protein), liver (921.0 ± 180.7 ng/g protein) and mammary gland (399.6 ± 191.2 ng/g protein) were determined by ELISA. No seasonal change in CA-VI levels was observed in normal milk. The concentration of CA-VI in colostrum (day 1 post partum) was 119 ng/ml and decreased rapidly by 1 month following birth. Mammary gland contained much smaller amounts than the submandibular gland. CA-VI mRNA was detected in the liver and mammary gland of cow by RT-PCR. The ELISA used in this study proved to be a precise and sensitive method for determining CA-VI concentrations in saliva, serum, milk and tissue specimens from cows. The ELISA may enable the study of changes in CA-VI associated with hereditary or metabolic disorders of the salivary gland, mammary gland and liver using small samples of saliva, serum or milk.     

Perioperative desmopressin prolongs survival in surgically treated bitches with mammary gland tumours: a pilot study

Desmopressin (1-deamino-8-d-arginine vasopressin, also known as DDAVP) is a safe haemostatic compound capable of inhibiting lymph node and lung metastasis in a mouse model of mammary tumour manipulation and surgical excision. The aim of this study was to test the efficacy and safety of perioperative DDAVP (1microg/kg) in surgically treated bitches with mammary gland tumours (MGT). Twenty-one, otherwise healthy, intact bitches, with malignant MGT stage III or IV were randomly allocated to DDAVP (n=11) or placebo (n=10) groups. En bloc mastectomy of the affected gland/s was performed. DDAVP had a significant beneficial effect on disease-free period (P<0.01) and overall survival time (P<0.05). No side effects were seen in any of the cases. Whatever the mechanism of action, it seems that DDAVP may have a novel use in cancer surgery to minimise spread or survival of residual malignant cells. Additional, large scale controlled trials are required to fully evaluate this adjuvant pharmacological protocol.     

Protective effect of CpG-DNA against mastitis induced by Escherichia coli infection in a rat model

A mastitis model in rats, induced by Escherichia coli infection, was established and the protective effect of Cytosine-phosphate-Guanosine (CpG)-DNA was determined. An E. coli suspension containing either 2 x 10(3) colony forming units (CFU)mL(-1)(EL group), 2 x 10(5)CFU mL(-1) (EH group), or (as controls) 100 microL phosphate buffer saline (CON group), was inoculated into the mammary glands 72 h after parturition. The rats were euthanased 24 h post-infection. The histopathological changes in mammary tissue in the EL group were mild, whereas the structural changes in the EH group were severe and polymorphonuclear leukocytes (PMNs) had accumulated in the mammary alveoli. Interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha and N-acetyl-beta-d-glucosaminidase (NAGase) were significantly increased in the mammary tissue from the EH group but not significantly changed in the EL group. On the basis of these findings, the potential protective effect of CpG-DNA on mammary glands was tested using a 2 x 10(5)CFU mL(-1) suspension. An intramuscular injection of either CpG-DNA (200 microg) or PBS (100 microL) was given immediately after parturition. At 72 h post-partum, 2 x 10(5)CFU mL(-1)E. coli (100 microL) were inoculated into the mammary glands of all rats. At pre-infection (0 h), and 8, 16, 24, 48 and 72 h after inoculation six rats were euthanased. CpG-DNA induced more rapid migration of PMNs from the blood to mammary tissue at the initial stage of infection, stimulated the secretion of IL-6 and TNF-alpha at different time points, reduced viable E. coli in mammary tissues and decreased the activity of NAGase. CpG-DNA also promoted the expression of its specific receptor TLR-9 mRNA in mammary tissue. The study showed that CpG-DNA protected against E. coli mastitis in this rat model.     

The elimination of serum-resistant Escherichia coli from experimentally infected single mammary glands of healthy cows.

Neutrophils isolated from mammary glands stimulated with a staphylococcal culture filtrate efficiently killed serum-resistant strains of Escherichia coli. This study was extended and it was shown that an infusion of wide ranging numbers (5 X 10(1) to 5 X 10(6)) of the same strains of E coli into a single mammary gland resulted in bacterial growth, which was eliminated following neutrophil infiltration. This elimination occurred before the appearance of any clinical signs. Once bacterial kill had started in the gland, it continued in the milk after withdrawal from the gland. These results offer an explanation of why causative microbial agents cannot be isolated from some cases of clinical mastitis.     

Effect of recombinant human thyroid stimulating hormone on serum thyroxin and thyroid scintigraphy in euthyroid cats

This study investigated the thyroidal response to administration of recombinant human thyroid stimulating hormone (rhTSH) by means of serum total thyroxine (TT(4)) concentration and pertechnetate uptake by the thyroid gland in six healthy euthyroid spayed female cats. A pertechnetate scan was performed on day 1 to calculate thyroid/salivary gland (T/S) uptake ratio. On day 3, 25 microg rhTSH was injected intravenously. Six hours later the thyroid scan was repeated as on day 1. Blood was drawn for serum TT(4) measurement prior to injection of rhTSH and performance of the pertechnetate scan. Statistically significant differences in mean serum TT(4) concentration, T/S uptake ratio before and 6h after rhTSH administration and T/S uptake ratio between left and right lobes were noted. We can conclude that 25 microg rhTSH increases pertechnetate uptake in the thyroid glands of cats, this should be taken into account when thyroid scintigraphy after rhTSH administration is interpreted.     

Hormone interactions confer specific proliferative and histomorphogenic responses in the porcine mammary gland

Mammary gland growth and morphogenesis are regulated by interactions between hormones as much as by their individual actions. The effect of these interactions on the mammary gland phenotype in species other than rodents is relatively undefined. We investigated the individual and combined effects of estrogen (E), progestin (P), and prolactin (PRL) on mammary gland development in gilts. Pigs were shown to have a ductal-lobular parenchyma that underwent hormone-stimulated progression of terminal ductal lobular unit (TDLU) morphogenesis similar to that in the human breast. Ovariectomy plus hypoprolactinemia abolished mammary gland growth. Estrogen alone stimulated mammary epithelial cell proliferation, terminal bud formation, and the progression of TDLU1 structures to a TDLU2 morphotype. Maximal epithelial cell proliferation, DNA content, parenchymal area, and morphological development of the porcine mammary gland were realized following treatment with E + PRL or E + P + PRL. In contrast, P alone did not promote epithelial cell proliferation, TDLU type progression, mammary gland growth, or morphogenesis. These data indicate that interactions between E and PRL are the main determinants of growth and morphogenesis in the porcine mammary gland.     

Bactericidal activity of macrophages against Streptococcus uberis is different in mammary gland secretions of lactating and drying off cows

The aim of this study was to compare the ability of milk macrophages and macrophages from the mammary gland secretions during the mid-dry period for their interaction with the mastitis-causing Streptococcus uberis. We also aimed to determine if S. uberis induced the release of the cytokine tumour necrosis alpha (TNF-alpha) and the bactericidal moiety nitric oxide (NO) from milk macrophages of lactating cows and macrophages from the mammary gland secretions at the mid-dry period. Macrophages were isolated from the mammary gland secretions of cows during the mid-lactation or mid-dry period, and compared with blood monocytes for their interaction with the important mastitis-causing pathogen S. uberis. When infected in vitro with S. uberis, milk macrophages from lactating cows with S. uberis released modest amounts of the cytokine tumour necrosis factor alpha (TNF-alpha) (139 pg/ml) and the bactericidal moiety nitric oxide (NO) (3-4 microM of nitrite). Blood monocytes from lactating cows released significantly higher amounts of TNF-alpha (345 +/- 143 pg/ml) and NO (7 +/- 2 microM of nitrite) after interaction with S. uberis, compared to milk macrophages (P < 0.01 for both TNF-alpha and NO). Stimulation of blood monocytes with the cytokine interferon-gamma (IFN-gamma) enhanced significantly the release of NO and TNF-alpha, but IFN-gamma did not significantly enhance the production of NO and TNF-alpha by milk macrophages from lactating cows. Milk macrophages from all lactating cows failed to kill S. uberis efficiently, and this lack of killing was unaffected by prior treatment with gamma interferon (IFN-gamma) (P > 0.05). Rather, S. uberis multiplied significantly inside infected milk macrophages from lactating cows, with a two-fold increase in bacterial numbers at 2 h post-infection. Milk macrophages from lactating cows were able however, to kill a significant proportion (50-60%, P < 0.01) of phagocytosed Staphylococcus aureus. Blood monocytes from all cows were found to exert significant bactericidal activity against S. uberis. There were no significant differences in the bactericidal activity of milk macrophages obtained from lactating cows with low somatic cell counts (SCC; < 10(5) ml(-1)) compared with those with a mildly elevated SCC (> 10(5) ml(-1)) (P > 0.05). In contrast, mammary gland secretion macrophages isolated from the same cows in the mid-dry period killed a significant proportion of phagocytosed S. uberis (50-65% of ingested S. uberis killed, P < 0.01) although cytokine production in response to in vitro bacterial infection was low. We conclude that the bactericidal activity of mammary gland secretion macrophages against a virulent strain of S. uberis is low during the lactation period. In addition, our data indicate that S. uberis is not a strong inducer of NO and TNF-alpha in macrophages from the milk or mammary gland secretions of cows during the drying off period. Finally, IFN-gamma does not activate milk macrophages or macrophages from cows during the lactating period or mammary gland secretions during the drying off period.     

Neurophysiologic maps of the cutaneous innervation of the external genitalia of the ewe

The area of skin supplied by the afferent fibers in a peripheral nerve is called the cutaneous area (CA) of that nerve. The CA responsive to movement of wool or hair in the genital regions were mapped in 17 ewes, with the identifications of the peripheral nerves and of the spinal nerves contributing to the pudendal plexus being checked at necropsy. Differences were found in the origins and extent of CA of the cutaneous branches from the sacral plexus. The CA of the caudal rectal nerves and of a nerve that passed caudally between the caudal vertebrae and the ventral sacrococcygeus muscle lay lateral to the anus and in the adjacent skin of the tail. The CA of the proximal cutaneous branch and of the distal cutaneous branch from the pudendal nerve (or plexus) overlapped craniocaudally (by approx one-half) the CA of the distal cutaneous branch extending ventrally and ending just caudal to the ipsilateral mammary gland. The deep perineal nerve innervated the skin immediately lateral to the anus and vulva. The dorsal nerve of the clitoris innervated hairs on the ipsilateral half of the vulva. Other fibers in the pudendal nerve were presumed to pass into the mammary branch of the nerve. They innervated the skin ventral to the vulva, the ipsilateral mammary gland, and (in some ewes) areas of the skin cranial to the mammary gland. The CA of the genitofemoral nerve included the ipsilateral teat and the inguinal fossa.(ABSTRACT TRUNCATED AT 250 WORDS)