In situ provision of drinking water to grazing dairy cows improves milk production.

AIMS: To determine the effect of providing water within the area grazed by dairy cows on milk yield and quality, compared to requiring cows to walk to a distant water trough, on a dairy farm in the Pampa region of Argentina during summer. METHODS: Holstein dairy cows were allocated to two herds with similar parity, days in milk and milk production. They were grazed in one paddock that was divided in two, with a fixed water trough at one end. Cows were moved twice daily to grazing plots within the paddock. Control cows (n=66) could only access water from the fixed trough, whereas supplemented cows (n=67) also received water from a mobile trough within the grazing plot. Milk production of each cow, and water consumption of the two herds were measured daily over 62 days. Milk composition for each herd was determined weekly from Days 18 to 60 of the study, and grazing behaviour was observed between 08:00 and 16:00 hours on Days 11-15, 19-22 and 39-43. RESULTS: Over the 62 days of the study, supplemented cows produced 1.39 (SE 0.11) L/cow/day more milk than Control cows (p=0.027). Estimated mean daily water intake was 50.4 (SE 2.1) L/cow/day for supplemented cows and 58.2 (SE 2.7) L/cow/day for Control cows (p=0.004). Percentage total solids in milk was higher for supplemented (12.5 (SE 0.06)%) than Control (12.4 (SE 0.04)%) cows (p=0.047). During the periods of behavioural observation, a higher percentage of cows in the water supplemented than the Control herd were observed in the grazing area (p=0.012). CONCLUSIONS AND CLINICAL RELEVANCE: This preliminary study demonstrated that provision of water to dairy cows within the grazing plot was beneficial for milk production and composition, and may be associated with longer periods spent within the grazing area, during hot weather in the Pampa region of Argentina.

Neuroendocrine Regulation of Metabolism.

Given the current environment in most developed countries, it is a challenge to maintain a good balance between calories consumed and calories burned, although maintenance of metabolic balance is key to good health. Therefore, understanding how metabolic regulation is achieved and how the dysregulation of metabolism affects health is an area of intense research. Most studies focus on the hypothalamus, which is a brain area that acts as a key regulator of metabolism. Among the nuclei that comprise the hypothalamus, the arcuate nucleus is one of the major mediators in the regulation of food intake. The regulation of energy balance is also a key factor ensuring the maintenance of any species as a result of the dependence of reproduction on energy stores. Adequate levels of energy reserves are necessary for the proper functioning of the hypothalamic-pituitary-gonadal axis. This review discusses valuable data presented in the 2015 edition of the International Workshop of Neuroendocrinology concerning the fundamental nature of the hormonal regulation of the hypothalamus and the impact on energy balance and reproduction.

Cellular proliferation rate and insulin-like growth factor binding protein (IGFBP)-2 and IGFBP-3 and estradiol receptor alpha expression in the mammary gland of dairy heifers naturally infected with gastrointestinal nematodes during development.

Mammary ductal morphogenesis during prepuberty occurs mainly in response to insulin-like growth factor-1 (IGF-1) and estradiol stimulation. Dairy heifers infected with gastrointestinal nematodes have reduced IGF-1 levels, accompanied by reduced growth rate, delayed puberty onset, and lower parenchyma-stroma relationship in their mammary glands. Immunohistochemical studies were undertaken to determine variations in cell division rate, IGF-1 system components, and estradiol receptors (ESR) during peripubertal development in the mammary glands of antiparasitic-treated and untreated Holstein heifers naturally infected with gastrointestinal nematodes. Mammary biopsies were taken at 20, 30, 40, and 70 wk of age. Proliferating cell nuclear antigen immunolabeling, evident in nuclei, tended to be higher in the parenchyma of the glands from treated heifers than in those from untreated. Insulin-like growth factor binding proteins (IGFBP) type 2 and type 3 immunolabeling was cytoplasmic and was evident in stroma and parenchyma. The IGFBP2-labeled area was lower in treated than in untreated heifers. In the treated group, a maximal expression of this protein was seen at 40 wk of age, whereas in the untreated group the labeling remained constant. No differences were observed for IGFBP3 between treatment groups or during development. Immunolabeling for α ESR (ESR1) was evident in parenchymal nuclei and was higher in treated than in untreated heifers. In the treated group, ESR1 peaked at 30 wk of age and then decreased. These results demonstrate that the parasite burden in young heifers negatively influence mammary gland development, affecting cell division rate and parameters related to estradiol and IGF-1 signaling in the gland.

Metritis in dairy cows: risk factors and reproductive performance.

The objectives of this study were to assess the risk factors for metritis, its effects on milk yield and on reproductive performance, and the efficacy of ceftiofur therapy in Holstein dairy cows. Cows (n=303) from a commercial dairy herd in Argentina were studied. Cows were scored for body condition, and blood samples were collected on d -14, 7, 21, 31, 41, and 50 relative to parturition. Cows having a watery, purulent, or brown, and fetid vaginal discharge (VD) and rectal temperature ≤ 39.2°C were diagnosed as having clinical metritis, and those having a similar VD and rectal temperature >39.2°C were diagnosed as having puerperal metritis. Both clinical and puerperal metritis cows were randomly assigned to control (no treatment) or ceftiofur group (2.2mg/kg×3 consecutive days). Cure was declared if clear VD was observed at 21 d in milk (DIM). Blood samples were analyzed for nonesterified fatty acids, ß-hydroxybutyrate, and blood urea nitrogen using commercial kits, and for insulin-like growth factor-1, insulin, and leptin by RIA. Data were analyzed with PROC MIXED, GENMOD, PHREG, and LIFETEST from SAS (SAS Institute Inc., Cary, NC). The risk for metritis increased with dystocia, retained fetal membranes, and dead calf [AOR (adjusted odds ratio)=2.58, 95% CI: 1.189-5.559], and as prepartum nonesterified fatty acids levels increased (AOR=1.001, 95% CI: 0.999-1.002). Conversely, risk decreased as prepartum insulin-like growth factor-1 increased (AOR=0.65, 95% CI: 0.349-1.219). Cows having either clinical or puerperal metritis produced less milk by 90 DIM than did healthy cows (2,236 ± 172 vs. 2,367 ± 77 vs. 2,647 ± 82 kg, respectively). Cows with puerperal metritis had lower risk for pregnancy by 100 DIM (AOR=0.189, 95% CI: 0.070-0.479) and a lower hazard rate for pregnancy by 150 DIM (hazard rate: 0.753, 95% CI: 0.621-0.911), and took longer to get pregnant (129 vs. 111 vs. 109 d, for puerperal metritis, clinical metritis, and healthy cows, respectively). Ceftiofur treatment was not associated with cure rate or milk yield but was related to increased risk for pregnancy at timed artificial insemination (AOR=2.688, 95% CI: 0.687-10.832), and for lower risk of reproductive cull (AOR=0.121, 95% CI: 0.014-1.066). In conclusion, abnormal calving and negative energy balance are associated with increased risk for metritis. Metritis, especially puerperal metritis, correlates with reduced milk production and poor reproductive performance. Finally, the likelihood for having a normal VD (indicative of cure) increased 2.6% for every day of increase in postpartum time and was 2 times higher for cows with clinical metritis than for those with puerperal metritis.

Mecanismos de tumorogénesis hipofisaria: Estudio de factores angiogénicos y de proliferación / Mechanisms of Pituitary Tumorigenesis: Study of Angiogenic and Proliferation factors

Introducción y Objetivos: La angiogénesis es un proceso fundamental en el desarrollo tumoral. Sin embargo, se han encontrado discrepancias en el patrón angiogénico de los tumores hipofisarios. Nos propusimos estudiar la expresión de VEGF y FGF2 y su importancia en la vascularización de los adenomas hipofisarios, cuantificar los vasos con los marcadores CD31 y CD34 y determinar el índice de proliferación con PCNA y Ki67. Materiales y Métodos: Se estudiaron 76 macroadenomas hipofisarios que fueron intervenidos quirúrgicamente. Los adenomas se clasificaron según su secreción hormonal. A partir de cortes histológicos se realizó inmunohistoquímica para los marcadores de endotelio CD31 y CD34; y Ki-67 para estudio de proliferación celular. Por western blot se midieron VEGF, CD31 y PCNA. Se efectuaron comparaciones con glándulas normales. Resultados: El nivel de expresión de VEGF, hallado en todas las muestras analizadas, resultó mayor en los prolactinomas resistentes respecto a los demás tipos de adenomas hipofisarios. Esta proteína localizó en las células endoteliales de los vasos como así también en citoplasmas y núcleos de células tumorales. El 56 % de las muestras resultaron positivas para FGF2, mostrando localización citoplasmática y en matriz extracelular. Obtuvimos una fuerte correlación positiva entre VEGF y CD31 en las muestras tumorales, sin encontrar correlación lineal entre PCNA y VEGF, ni Ki-67 y VEGF en las muestras estudiadas. El área vascular resultó mayor en los tejidos normales que en los tumores utilizando CD34 como marcador de vasos. Conclusión: La importancia del estudio de la angiogénesis en los adenomas hipofisarios radica en la necesidad de hallar marcadores moleculares que predigan el comportamiento tumoral. Pudimos demostrar la expresión de los factores angiogénicos VEGF y FGF2 en estos adenomas, y la existencia de correlación lineal entre VEGF y CD31. Nuestros resultados son indicativos de existencia de angiogénesis en los adenomas hipofisarios por lo que su bloqueo podría plantearse como una estrategia alternativa para los casos resistentes a las terapias convencionales.

Introduction and objectives: Angiogenesis is an essential process in tumor development. Nevertheless, discrepancies in the angiogenic pattern of pituitary tumors, in terms of hormonal phenotype, size or invasiveness have been found. Our aim was to study the expression of VEGF and FGF2 growth factors, and their importance in the vascularization of pituitary adenomas. We also quantified blood vessels with the endothelial cell markers CD31 and CD34 determining the vascular area, and the proliferation rate through PCNA and Ki67 index. Materials and Methods: We studied 76 pituitary macroadenomas that were surgically resected in the period between 2006 and 2010 from a total of 276 patients with this pathology. Adenomas were classified into prolactinomas (PRL), somatotropinomas (GH), corticotropinomas (ACTH), non-functioning (NF) and plurihormonal (Ph) according to their hormonal secretion. Samples were collected in formalin, embedded in paraffin, and immunohistochemistry was performed from histological sections for endothelial markers CD31 and CD34; and for Ki-67 to study cell proliferation. VEGF, CD31 and PCNA were measured by Western blot. We compared results with normal glands (N=6). Results: VEGF expression levels, found in all of the samples analyzed, were higher in resistant prolactinomas than in other pituitary adenomas. This protein was detected in endothelial cells of blood vessels and in tumor cells cytoplasms and nuclei. Fifty-six percent of samples were positive for FGF2, the other potent angiogenic factor studied, showing cytoplasmatic and extracellular matrix localization. We obtained a strong positive correlation between VEGF and CD31 in tumor samples, but we did not find lineal correlation between PCNA and VEGF, or between Ki-67 and VEGF in the samples studied. The vascular area was higher in normal tissues than in tumors when CD34 was used as endothelial cell marker. Conclussion: The importance of studying angiogenesis in pituitary adenomas lies in the need to find molecular markers that can predict tumor behavior. We could demonstrate the expression of VEGF and FGF2, two potent angiogenic factors, and the existence of linear correlation between VEGF and CD31. Our results are indicative of the existence of angiogenesis in pituitary adenomas; therefore the blockage of angiogenesis might be proposed as an alternative strategy for cases of resistance to standard therapy.

Mecanismos de tumorogénesis hipofisaria: Estudio de factores angiogénicos y de proliferación / Mechanisms of Pituitary Tumorigenesis: Study of Angiogenic and Proliferation factors

Introducción y Objetivos: La angiogénesis es un proceso fundamental en el desarrollo tumoral. Sin embargo, se han encontrado discrepancias en el patrón angiogénico de los tumores hipofisarios. Nos propusimos estudiar la expresión de VEGF y FGF2 y su importancia en la vascularización de los adenomas hipofisarios, cuantificar los vasos con los marcadores CD31 y CD34 y determinar el índice de proliferación con PCNA y Ki67. Materiales y Métodos: Se estudiaron 76 macroadenomas hipofisarios que fueron intervenidos quirúrgicamente. Los adenomas se clasificaron según su secreción hormonal. A partir de cortes histológicos se realizó inmunohistoquímica para los marcadores de endotelio CD31 y CD34; y Ki-67 para estudio de proliferación celular. Por western blot se midieron VEGF, CD31 y PCNA. Se efectuaron comparaciones con glándulas normales. Resultados: El nivel de expresión de VEGF, hallado en todas las muestras analizadas, resultó mayor en los prolactinomas resistentes respecto a los demás tipos de adenomas hipofisarios. Esta proteína localizó en las células endoteliales de los vasos como así también en citoplasmas y núcleos de células tumorales. El 56 % de las muestras resultaron positivas para FGF2, mostrando localización citoplasmática y en matriz extracelular. Obtuvimos una fuerte correlación positiva entre VEGF y CD31 en las muestras tumorales, sin encontrar correlación lineal entre PCNA y VEGF, ni Ki-67 y VEGF en las muestras estudiadas. El área vascular resultó mayor en los tejidos normales que en los tumores utilizando CD34 como marcador de vasos. Conclusión: La importancia del estudio de la angiogénesis en los adenomas hipofisarios radica en la necesidad de hallar marcadores moleculares que predigan el comportamiento tumoral. Pudimos demostrar la expresión de los factores angiogénicos VEGF y FGF2 en estos adenomas, y la existencia de correlación lineal entre VEGF y CD31. Nuestros resultados son indicativos de existencia de angiogénesis en los adenomas hipofisarios por lo que su bloqueo podría plantearse como una estrategia alternativa para los casos resistentes a las terapias convencionales.(AU)

Introduction and objectives: Angiogenesis is an essential process in tumor development. Nevertheless, discrepancies in the angiogenic pattern of pituitary tumors, in terms of hormonal phenotype, size or invasiveness have been found. Our aim was to study the expression of VEGF and FGF2 growth factors, and their importance in the vascularization of pituitary adenomas. We also quantified blood vessels with the endothelial cell markers CD31 and CD34 determining the vascular area, and the proliferation rate through PCNA and Ki67 index. Materials and Methods: We studied 76 pituitary macroadenomas that were surgically resected in the period between 2006 and 2010 from a total of 276 patients with this pathology. Adenomas were classified into prolactinomas (PRL), somatotropinomas (GH), corticotropinomas (ACTH), non-functioning (NF) and plurihormonal (Ph) according to their hormonal secretion. Samples were collected in formalin, embedded in paraffin, and immunohistochemistry was performed from histological sections for endothelial markers CD31 and CD34; and for Ki-67 to study cell proliferation. VEGF, CD31 and PCNA were measured by Western blot. We compared results with normal glands (N=6). Results: VEGF expression levels, found in all of the samples analyzed, were higher in resistant prolactinomas than in other pituitary adenomas. This protein was detected in endothelial cells of blood vessels and in tumor cells cytoplasms and nuclei. Fifty-six percent of samples were positive for FGF2, the other potent angiogenic factor studied, showing cytoplasmatic and extracellular matrix localization. We obtained a strong positive correlation between VEGF and CD31 in tumor samples, but we did not find lineal correlation between PCNA and VEGF, or between Ki-67 and VEGF in the samples studied. The vascular area was higher in normal tissues than in tumors when CD34 was used as endothelial cell marker. Conclussion: The importance of studying angiogenesis in pituitary adenomas lies in the need to find molecular markers that can predict tumor behavior. We could demonstrate the expression of VEGF and FGF2, two potent angiogenic factors, and the existence of linear correlation between VEGF and CD31. Our results are indicative of the existence of angiogenesis in pituitary adenomas; therefore the blockage of angiogenesis might be proposed as an alternative strategy for cases of resistance to standard therapy.(AU)

Clinical endometritis in an Argentinean herd of dairy cows: risk factors and reproductive efficiency.

The objectives of this study were to assess the clinical and metabolic risk factors for clinical endometritis, the likelihood for having a normal vaginal discharge during postpartum, and the effects of endometritis on milk yield, reproductive efficiency, and metabolic status in Holstein cows. The study was conducted in a commercial dairy herd (Cordoba, Argentina) where 303 Holstein cows were enrolled. Cows were body condition scored (1 to 5) and tail bled on -14, 7, 21, 31, 41, and 50 d relative to parturition. Cows having a vaginal discharge with presence of pus between 21 and 41 d postpartum (dpp) were diagnosed as having clinical endometritis. Plasma blood samples were analyzed for nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHBA), and blood urea nitrogen using commercial kits and insulin-like growth factor 1, insulin, and leptin by RIA. Data were analyzed with PROC MIXED, PROC GENMOD, and PROC PHREG of SAS (SAS Institute Inc., Cary, NC). Abnormal calving and puerperal metritis increased the risk for endometritis [adjusted odds ratio (AOR)=2.21 for both]. High prepartum NEFA and high postpartum BHBA increased the risk for endometritis (AOR=1.003 and 1.001, respectively), whereas high prepartum blood urea nitrogen reduced it (AOR=0.853). Cut-offs of 456.6 µM NEFA and 402.5 µM BHBA had sensitivities of 0.69 and 0.58, and specificities of 0.88 and 0.86, respectively. The likelihood for having normal vaginal discharge increased with time (∼1% × dpp) and with normal calving. Cows with endometritis had higher milk yield than normal herdmates (27.8±0.9 vs. 25.7±0.4 kg/d), lower risk for pregnancy by 100 dpp (AOR=0.10), higher nonpregnancy risk by 200 dpp (AOR=2.87), and higher risk for culling than normal cows (AOR=2.28). Cows with endometritis had a lower hazard rate (0.44) for pregnancy and had approximately 70 d longer calving-to-conception intervals. Finally, endometritis had no effect on metabolic hormones. In conclusion, the risk for clinical endometritis increases with abnormal calving and puerperal metritis, as prepartum NEFA and postpartum BHBA concentrations increase. Prepartum NEFA and postpartum BHBA could be useful for the prediction of endometritis. Last, clinical endometritis has detrimental effects on reproductive efficiency, as affected cows take longer to get pregnant and are at higher risk for culling.

Comparison of three methods for gastrointestinal nematode diagnosis determination in grazing dairy cattle in relation to milk production.

Development of resistance to anthelmintic drugs has motivated the search for diagnostic methods to identify animals for targeted selective treatments. We compared three methods for the diagnosis of nematode infection in relation to milk production in a fully grazing dairy herd of 150 cows in the humid Pampa (Argentina). Animals had feces, blood and milk sampled during the first postpartum month for EPG, pepsinogen and anti-Ostertagia antibody determination, respectively. With the results obtained two groups of cows, divided in high and low parasite burden, were conformed for each method, and milk production was then compared between groups. When cows were separated by the EPG method (EPG=0 (N=106) vs. EPG>0 (N=44)) a difference of nearly 800 l of milk per cow per lactation was found (P<0.05). On the other hand, milk production between groups separated by Pepsinogen (mUtyr ≤ 1000 vs. mUtyr > 1000) or by anti-Ostertagia (ODR ≤ 0.5 vs. ODR > 0.5) results did not differ. Interestingly, proportion of cows in each group differed between methods (P<0.0001), and the anti-Ostertagia method yielded significantly more cows in the high index group compared to results using the EPG or Pepsinogen method. No correlations were found between parasite indexes determined by the different methods. High parasite burden estimation found may be ascribed to the production system, fully grazing all year round, and to the sampling time, at the beginning of lactation with cows in negative energy balance and depressed immunity. The fact that the cows were born and reared outside, on pasture with continuous nematode larvae exposure, may also account for the results obtained. In conclusion, EPG counting during the first postpartum month may be a useful tool for the diagnosis of production impairment induced by high nematode burden in adult grazing dairy cows. The anthelmintic treatment of only the EPG-positive recently calved cows would improve milk production, while reducing selective pressure on nematode population for the development of resistance.

Gastrointestinal parasites presence during the peripartum decreases total milk production in grazing dairy Holstein cows.

Parasitism in cattle is known to impair growth and development. Recent findings suggest that productivity of adult animals is also affected, but little is known about the physiological mechanisms involved. Furthermore, development of nematode resistance to drugs makes imperative the search of management practices that avoid whole herd treatment. We undertook an epidemiological and endocrine study in a grass based dairy farm in Argentina to study the effect of parasites on milk production and the underlying mechanisms involved, and identify individual animals that would benefit from antiparasitic treatment. All the cows in the dairy were followed monthly for egg parasite output in feces. Samples were cultured for genera determination. Milk production and reproductive results were recorded and periodical bleedings for hormone determination were performed. Nematode egg output (EPG) was maximal in late Summer and Autumn and minimal in Spring in coincidence with the Ostertagia inhibition-disinhibition cycle as this genus had the highest prevalence in all the study. The highest proportion of positive samples was found in the high producing herd and maximal counts were found in the peripartal period. Milk production did not correlate with EPG mean values but, when cows were grouped by EPG positivity around parturition, a significant difference in total milk production between EPG null and positive cows was observed. Positive cows produced 7%, 12% or 15% less milk than null EPG cows, depending on the sampling month/s chosen for classification. The highest difference was seen when both prepartum and postpartum samples were taken into account. No difference in lactation length and a marginal effect on partum to first service interval were encountered. Endocrine studies revealed a decrease in serum growth hormone (GH), type I insulin-like growth factor (IGF-I) and prolactin during lactation in cows with positive EPG in the first postpartum sample with respect to null EPG cows at that time. GH levels decreased and prolactin and IGF-I levels increased in both groups of cows from month 0 to 6 in milk. Serum insulin levels remained stable throughout lactation and were similar in both groups of cows. In conclusion, EPG around parturition may be a useful tool for identifying cows that will have a decrease in productivity due to parasite effects and would possibly benefit from an antiparasitic treatment. Besides, our results suggest that detrimental effect of parasites on milk production may be mediated by GH, IGF-I and prolactin serum levels.

Effect of anthelmintics on reproductive performance and first-lactation culling rate in Holstein heifers.

Female Holstein calves were treated with ivermectin from birth to first oestrus to study the effect of parasitic burden and anthelmintic treatment on reproductive and productive performance. First oestrus, age at first service and age at calving were advanced by 30, 70 and 110 days, respectively (P<0.05), in ivermectin-treated animals compared with controls. No significant differences were observed in the conception rate, the number of services and the characteristics of the newborn calves and any problems at calving between the two groups. Daily milk yield, fat content in milk during first lactation, and the concentrations of growth hormone, insulin-like growth factor type 1, insulin and prolactin in serum were similar in both groups of cows. Culling during the first lactation was more common in untreated (47 per cent) than in treated (11 per cent) cows (P<0.05).

Hypothalamic orexin, OX1, alphaMSH, NPY and MCRs expression in dopaminergic D2R knockout mice.

In 5-month-old male and female dopamine receptor 2 (D2R) knockout mice food intake per animal was unaltered while food per g BW was increased. We wished to evaluate the effect of D2R disruption on different components of energy balance and food intake regulation. We determined hypothalamic orexin precursor (PPO) expression, its receptor OX1, serum leptin levels, hypothalamic leptin receptor (OBR), circulating and pituitary alpha MSH levels, as well as central MC3 and MC4 receptors and NPY mRNA in wildtype and D2R knockout mice (KO). Loss of D2R caused a marked increase in serum prolactin levels, to higher levels in females compared to male KO mice. On the other hand, it produced a female-specific increase in circulating alphaMSH, and hypothalamic alphaMSH content, while neurointermediate alphaMSH content was decreased in both sexes. No differences were found in hypothalamic NPY, MC3R or MC4R concentration. Hypothalamic PPO mRNA expression was significantly decreased only in female KOs, while OX1 mRNA was not different between genotypes. Serum leptin levels were also similar in both genotypes. Our results show that in female and not in male mice disruption of the D2R produces two potentially anorexigenic events: an increase in serum and hypothalamic alphaMSH, and a decrease in hypothalamic orexin expression. Very high prolactin levels, which are orexigenic, probably counterbalance these effects, so that food intake is slightly altered. In males, on the other hand, hypothalamic PPO, and serum or hypothalamic alphaMSH are not modified, and increased prolactin levels may account for increased food intake per g BW. These results suggest a sexually dimorphic participation of the D2R in food intake regulation.

Restorative effect of insulin-like growth factor-I gene therapy in the hypothalamus of senile rats with dopaminergic dysfunction.

Insulin-like growth factor-I (IGF-I) is emerging as a powerful neuroprotective molecule that is strongly induced in the central nervous system after different insults. We constructed a recombinant adenoviral vector (RAd-IGFI) harboring the gene for rat IGF-I and used it to implement IGF-I gene therapy in the hypothalamus of senile female rats, which display hypothalamic dopaminergic (DA) neurodegeneration and as a consequence, chronic hyperprolactinemia. Restorative IGF-I gene therapy was implemented in young (5 months) and senile (28 months) female rats, which received a single intrahypothalamic injection of 3 x 10(9) plaque-forming units of RAd-betagal (a control adenoviral vector expressing beta-galactosidase) or RAd-IGFI and were killed 17 days post-injection. In the young animals, neither vector modified serum prolactin levels, but in the RAd-IGFI-injected senile rats a nearly full reversion of their hyperprolactinemic status was recorded. Morphometric analysis revealed a significant increase in the total number of tyrosine hydroxylase-positive cells in the hypothalamus of experimental as compared with control senile animals (5874+/-486 and 3390+/-498, respectively). Our results indicate that IGF-I gene therapy in senile female rats is highly effective for restoring their hypothalamic DA dysfunction and thus reversing their chronic hyperprolactinemia.

Increased pituitary vascular endothelial growth factor-a in dopaminergic D2 receptor knockout female mice.

Vascular endothelial growth factor (VEGF)-A is an important angiogenic cytokine in cancer and pathological angiogenesis and has been related to the antiangiogenic activity of dopamine in endothelial cells. We investigated VEGF expression, localization, and function in pituitary hyperplasia of dopamine D2 receptor (D2R)-knockout female mice. Pituitaries from knockout mice showed increased protein and mRNA VEGF-A expression when compared with wild-type mice. In wild-type mice, prolonged treatment with the D2R antagonist, haloperidol, enhanced pituitary VEGF expression and prolactin release, suggesting that dopamine inhibits pituitary VEGF expression. VEGF expression was also increased in pituitary cells from knockout mice, even though these cells proliferated less in vitro when compared with wild-type cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium proliferation assay, proliferating cell nuclear antigen expression, and [(3)H]thymidine incorporation. In contrast to other animal models, estrogen did not increase pituitary VEGF protein and mRNA expression and lowered serum prolactin secretion in vivo and in vitro in both genotypes. VEGF (10 and 30 ng/ml) did not modify pituitary cell proliferation in either genotype and increased prolactin secretion in vitro in estrogen-pretreated cells of both genotypes. But conditioned media from D2R(-/-) cells enhanced human umbilical vein cell proliferation, and this effect could be partially inhibited by an anti-VEGF antiserum. Finally, using dual-labeling immunofluorescence and confocal laser microscopy, we found that in the hyperplastic pituitaries, VEGF-A was mostly present in follicle-stellate cells. In conclusion, pituitary VEGF expression is under dopaminergic control, and even though VEGF does not promote pituitary cellular proliferation in vitro, it may be critical for pituitary angiogenesis through paracrine actions in the D2R knockout female mice.

Dehydroepiandrosterone (DHEA) modulates GHRH, somatostatin and angiotensin II action at the pituitary level.

In view of the present controversy related to the potential beneficial effects of clinical dehydroepiandrosterone (DHEA) treatments, and considering our own previous results that reveal an influence of this steroid in pituitary hyperplasia development in vivo in rats, we decided to evaluate the role of DHEA in prolactin and GH secretion, as well as in second messengers involved, in cultured rat anterior pituitary cells. DHEA (1 x 10(-5) to 1 x 10(-7) M) did not modify basal GH or prolactin release, and a prolactin inhibitory effect was observed only for androstenediol, a metabolite of DHEA. DHEA partially prevented dopamine (1 x 10(-6) M)-induced prolactin inhibition and facilitated the prolactin-releasing effect of 10(-8) M Ang II, without modifying the resulting Ca2+(i) mobilization. Furthermore, DHEA potentiated the GH release and cAMP production induced by 1 x 10(-8) M GHRH. Finally, DHEA partially reversed the inhibitory effect of 1 x 10(-8) M somatostatin on GH, but not prolactin, release. We conclude that DHEA in vitro, directly or indirectly through conversion into metabolites, is able to modulate the hormonal response of the pituitary to hypothalamic regulators. It can enhance pituitary prolactin release and induce GH secretion. These effects could help explain some of the side effects observed in prolonged DHEA treatments in vivo and should be taken into account when considering its use in human clinical trials.

Partial replacement of corn grain by hydrogenated oil in grazing dairy cows in early lactation.

Thirty-six grazing dairy cows were used to determine milk production and composition, and dry matter and energy intake when corn grain was partially replaced by hydrogenated oil in the concentrate. Four additional cows, each fitted with a ruminal cannula, were used in a crossover design to evaluate effects of supplemental fat on rumen environment and pasture digestion. All cows grazed mixed pastures with an herbage allowance of 30 kg dry matter/cow per day. The control group was fed a concentrate containing corn grain (4.49 kg dry matter/cow per day) and fishmeal (0.37 kg dry matter/cow per day), whereas the other group (fat) received a concentrate containing corn grain (2.87 kg dry matter/cow per day), fishmeal (0.37 kg dry matter/cow per day) and fat (0.7 kg dry matter/cow per day). The fat was obtained by hydrogenation of vegetable oils (melting point 58 to 60 degrees C, 30.3% C16:0, 34.9% C18:0, 21.8% C18:1, 3.3% C18:2). Supplemental fat increased milk production (control = 23.7 vs. fat = 25.0 kg/cow per day), fat-corrected milk (control = 22.5 vs. fat = 24.5 kg/cow per day), milk fat content (control = 3.64% vs. fat = 3.86%) and yields of milk fat (control = 0.86 vs. fat = 0.97 kg/cow per day) and protein (control = 0.74 vs. fat = 0.78 kg/cow per day). Milk percentages of protein, lactose, casein, cholesterol, and urea nitrogen were not affected. Pasture DMI and total DMI of pasture and concentrate and estimated energy intake were unchanged. No differences in loss of body weight or body condition score were detected. Plasma concentrations of nonesterified fatty acids, somatotropin, insulin, and insulin-like growth factor were not affected by supplemental fat. Concentrations of plasma triglyceride and total cholesterol were increased by supplemented fat, and no changes in plasma glucose and urea nitrogen were observed. The acetate-to-propionate ratio was higher in rumen fluid of cows that consumed fat (fat = 3.39 vs. control = 3.27). In situ pasture NDF degradation was not affected. The partial replacement of corn grain with fat improved the productive performance of early-lactation cows grazing spring pastures. No negative effects of supplemental fat on ruminal fiber digestion were detected.

Dehydroepiandrosterone treatment attenuates oestrogen-induced pituitary hyperplasia.

The physiological importance of and therapeutic interest in dehydroepiandrosterone (DHEA) has been predominantly in relation to its action as an inhibitor of the promotion and progression of several kinds of tumours, including those of breast, prostate, lung, colon, liver and skin tissues. The aim of the present study was to determine the role of DHEA in diethylstilboestrol (DES)-induced pituitary hyperplasia. Female Sprague-Dawley rats were divided into four treatment groups: DES (implanted s.c. with a 20 mg DES pellet), DHEA (two 50 mg DHEA pellets), DHEA/DES (both DHEA and DES pellets), and controls (not implanted). Every week, all rats were weighed and cycled, and jugular blood samples were obtained. After 7 weeks, rats were killed. Hypophyses were removed and weighed, and serum prolactin, GH, IGF-I and leptin levels were assayed by RIA. DHEA cotreatment reduced pituitary enlargement by 39% in DES-treated rats. It also reduced the hyperprolactinaemia (280.4+/-43.6 ng/ml for DHEA/DES vs 823.5+/- 127.1 ng/ml for DES) and partially reversed the loss of body weight induced by DES. DHEA treatment did not modify the effects of DES on serum GH, IGF-I and leptin levels. But DHEA per se also increased pituitary weight and induced hyperprolactinaemia, although to a lesser degree than DES. We conclude that DHEA administration has beneficial effects on oestrogen-induced pituitary hyperplasia and hyperprolactinaemia, but the fact that DHEA per se also induces diverse hormonal effects and a slight pituitary enlargement limits its use as a possible therapeutic drug.

Supplementation with partially hydrogenated oil in grazing dairy cows in early lactation.

Effects of partially hydrogenated oil on performance, loss of body weight and body condition score, and blood metabolite and hormone concentrations were evaluated in 37 multiparous Holstein cows in grazing conditions during the first 100 d of lactation. Six additional Holstein cows, each fitted with a ruminal cannula, were allocated to a replicated 3 x 3 Latin square to evaluate effects of supplemental fat on rumen environment and pasture digestion. All cows grazed mixed pastures based on alfalfa (Medicago sativa) and orchardgrass (Dactylis glomerata L.) and received 5.4 kg/d of a basal concentrate to which 0, 0.5, or 1 kg/cow per day of partially hydrogenated oil (melting point 58 to 60 degrees C) containing 30.3, 34.9, 21.8, and 3.3% of C16:0, C18:0, C18:1, and C182, respectively, was added. Feeding 1 kg/d of supplemental fat increased fat-corrected milk from 23.4 to 26.3 kg/d, milk fat content from 3.44 to 3.78%, and milk fat yield from 0.87 to 1.03 kg/d compared to control. Milk protein percentage and yield were not affected. Cows fed 1 kg/d of fat increased the content and yield of C16:0 and C18:0 in milk compared with cows fed no added oil. Dry matter intake (DMI) from pasture decreased from 17.8 kg/d for control cows to 13.6 kg/d for cows fed 1 kg of oil, whereas DMI from concentrate was higher for cows fed 1 kg/d of fat (6.0 kg/d) than for controls (5.2 kg/d). Supplemental fat did not affect total dry matter or estimated energy intake and did not change losses of body weight or body condition scores. Plasma concentrations of nonesterified fatty acids, insulin, somatotrophin, and insulin-like growth factor-I did not differ among treatments. Concentration of plasma triglycerides was lowered from 318.5 to 271.2 mg/dl, whereas plasma cholesterol was elevated from 185.0 to 235.8 mg/dl in cows receiving 1 kg/d of supplemental fat compared with controls. Responses to lipolytic or insulin challenges were not affected by feeding oil. Supplemental fat did not affect the digestion of pasture fiber. The addition of energy in the form of partially hydrogenated fat to early lactation dairy cows fed primarily on pasture increased the yield of fat-corrected milk and milk fat content when it represented about 11% of the total metabolizable energy requirement of cows, without affecting milk protein content. The partial hydrogenation of a byproduct of the oil industry apparently prevented detrimental effects of fat supplementation on ruminal digestion.

Disruption of the D2 dopamine receptor alters GH and IGF-I secretion and causes dwarfism in male mice.

We determined the consequences of the loss of D2 receptors (D2R) on the GH-IGF-I axis using mice deficient in functional dopamine D2 receptors by targeted mutagenesis (D2R(-/-)). Body weights were similar at birth, but somatic growth was less in male D2R(-/-) mice from 1-8 months of age and in D2R(-/-) females during the first 2 months. The rate of skeletal maturation, as indexed by femur length, and the weight of the liver and white adipose tissue were decreased in knockout male mice even though food intake was not altered. The serum GH concentration was significantly decreased during the first 2 months in knockout female and male mice, and IGF-I and IGF-binding protein-3 levels were lower in knockout mice. PRL was significantly higher in knockout mice, and females attained higher levels than males. Pituitaries from adult knockout mice had impaired basal GH release and a lower response to GHRH in vitro. We propose that the D2R participates in GHRH/GH release in the first month of life. In accordance, the D2R antagonist sulpiride lowered GH levels in 1-month-old wild-type mice. Our results indicate that lack of D2R alters the GHRH-GH-IGF-I axis, and impairs body growth and the somatotrope population.

Metabolic cues for puberty onset in free grazing Holstein heifers naturally infected with nematodes.

Leptin is a new plausible candidate for the molecular link between nutritional status and the reproductive axis. In previous studies we described that continuous natural nematode infections in heifers retarded growth and delayed the onset of puberty, and that the insulin-like growth factor I (IGF-I) was involved. In the present study we monitored the leptin levels during development in heifers naturally parasitized versus those chronically treated with ivermectin and we investigated whether growth hormone (GH) accounted for the differences in IGF-I previously noted. Insulin levels were also measured. Prolactin hormone was recorded as an indicator of immune system activation. We found a direct correlation between leptin and body weight during development and a prepubertal surge of the hormone 2 weeks before the first progesterone peak that indicates the onset of puberty. This suggests that leptin may act as a signal for this event. Insulin did not vary during growth and prepuberty. On the other hand, GH as not responsible for diminished IGF-I levels in parasitized animals as levels were similar in both groups. The GH levels were high at birth and then diminished rapidly and remained constant during development and puberty. The last hormone studied, prolactin, followed seasonal changes of sunlight duration and presented sporadic bursts in infected animals. These were related to high nematode infection and are probably involved in the immune response of the host.

GABA(B) receptors in anterior pituitary cells. Mechanism of action coupled to endocrine effects.

The activation of pituitary GABA(B) receptors by the specific agonist baclofen inhibits pituitary hormone secretion in vitro. Here we studied the mechanism of action of GABA(B) receptors in rat adenohypophysis. Anterior pituitary cells were obtained by trypsinization and were either plated for hormonal studies and cAMP determination or incubated in FURA 2AM for calcium measurements. Baclofen (BACL: 1 x 10(-5) M) significantly inhibited basal and thyrotropic releasing hormone (TRH)-stimulated (1 x 10(-7) M) PRL secretion in anterior pituitary cells from proestrous rats. In the presence of pertussis toxin (PTX: 150 ng/ml, 20 h), which leads to the uncoupling of the G(i/o)-protein from the receptor, both effects of BACL were abolished while the effect of dopamine (DA: 1 x 10(-8) M), used as an inhibitory control, was reduced from 70 to 25%. PTX also reversed BACL-induced inhibition of gonadotropin-releasing hormone (GnRH)-elicited luteinizing hormone (LH) secretion in anterior pituitary cells from 15-day-old female rats. In addition, though working in a pituitary mixed cell population, in which only some cell types possess GABA(B) receptors, BACL (1 x 10(-5) M) attenuated the forskolin-induced (0.5 microM) increase in cAMP. This effect was prevented by co-incubation with the antagonist 2 hydroxysaclofen and by preincubation with PTX. BACL (5 x 10(-5) M) and DA (5 x 10(-7) M) inhibited basal intracellular calcium concentrations ([Ca(2+)](i)) in pituitary cells and the effect of the latter was significantly stronger. The effect of BACL on [Ca(2+)](i) was abolished after preincubation with PTX. In the presence of the potassium channel blocking agents barium (200 microM and 1 mM) and tetraethylammonium (10 mM), BACL was still able to inhibit [Ca(2+)](i). Blockade of voltage-sensitive calcium channels (VSCC) with either verapamil (5 x 10(-6) M) or nifedipine (1 x 10(-6) M) completely abolished the effect of BACL on [Ca(2+)](i). In the presence of 12.5 mM potassium concentration baclofen significantly inhibited [Ca(2+)](i). In conclusion, our results describe the negative coupling of adenohypophyseal GABA(B) receptors to VSCC through PTX-sensitive G-proteins. These characteristics suggest a resemblance of these receptors to the typical presynaptic GABA(B) sites described in the central nervous system.