Sandra J. Bertics

Distribution and ovarian control of progestin-metabolizing enzymes in various rat hypothalamic regions☆

The three principal hypothalamic progesterone metabolizing enzyme activities, namely the progesterone 5 alpha-reductase and 5 alpha-dihydroprogesterone NADH- and NADPH-linked 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) activities, were examined in discrete rat hypothalamic subsections throughout the estrous cycle and from ovariectomized rats treated with estradiol benzoate or vehicle. The regions studied included the median eminence, the medial preoptic area and the ventromedial and arcuate nuclei. The enzyme assays were performed using radiolabeled steroid substrates and reverse isotopic dilution analysis. While all four hypothalamic regions obtained from intact cycling animals possessed substantial amounts of these three enzyme activities, the median eminence generally had the highest activity levels (2- to 4-fold greater) except during estrus. The other three regions usually had comparable levels. No significant fluctuations were observed in any enzyme activity over the estrous cycle. After ovariectomy, there was a significant decrease (approximately 35%) in the level of the NADPH-linked 3 alpha-HSOR activity in the median eminence compared to the level observed in intact cycling animals, suggesting ovarian control. Estrogen treatment for 3 days did not restore this enzyme level to that observed in intact animals. The NADPH-linked 3 alpha-HSOR activity from the other three hypothalamic regions, as well as the NADH-linked 3 alpha-HSOR and the 5 alpha-reductase activities from all four brain regions, did not change significantly after ovariectomy. These results indicate that the median eminence possesses an increased capacity for progesterone metabolism relative to the other hypothalamic regions tested, and that the NADPH-linked 3 alpha-HSOR activity in this region may be under ovarian control.

Relationships between body condition score change, prior mid-lactation phenotypic residual feed intake, and hyperketonemia onset in transition dairy cows

Extensive efforts have been made to identify more feed-efficient dairy cows, yet it is unclear how selection for feed efficiency will influence metabolic health. The objectives of this research were to determine the relationships between residual feed intake (RFI), a measure of feed efficiency, body condition score (BCS) change, and hyperketonemia (HYK) incidence. Blood and milk samples were collected twice weekly from cows 5 to 18 d postcalving for a total of 4 samples. Hyperketonemia was diagnosed at a blood β-hydroxybutyrate (BHB) ≥1.2 mmol/L and cows were treated upon diagnosis. Dry period, calving, and final blood sampling BCS was recorded. Prior mid-lactation production, body weight, body weight change, and dry matter intake (DMI) data were used to determine RFI phenotype, calculated as the difference between observed DMI and predicted DMI. The maximum BHB concentration (BHBmax) for each cow was used to group cows into HYK or not hyperketonemic. Lactation number, BCS, and RFI data were analyzed with linear and quadratic orthogonal contrasts. Of the 570 cows sampled, 19.7% were diagnosed with HYK. The first positive HYK test occurred at 9 ± 0.9 d postpartum and the average BHB concentration at the first positive HYK test was 1.53 ± 0.14 mmol/L. In the first 30 d postpartum, HYK-positive cows had increased milk yield and fat concentration, decreased milk protein concentration, and decreased somatic cell count. Cows with a dry BCS ≥4.0, or that lost 1 or more BCS unit across the transition to lactation period, had greater BHBmax than cows with lower BCS. Prior-lactation RFI did not alter BHBmax. Avoiding over conditioning of dry cows and subsequent excessive fat mobilization during the transition period may decrease HYK incidence; however, RFI during a prior lactation does not appear to be associated with HYK onset.

The effect of increasing concentrations of dl-methionine and 2-hydroxy-4-(methylthio) butanoic acid on hepatic genes controlling methionine regeneration and gluconeogenesis

Metabolizable methionine (Met) concentrations can be increased by feeding rumen-protected dl-Met or the isopropyl ester of 2-hydroxy-4-(methylthio) butanoic acid (HMBi). Hepatic responses to increasing concentrations of metabolizable Met as a result of supplementation of different Met sources have not been comparatively examined. The objective of this experiment was to examine the regulation of key genes for Met metabolism, gluconeogenesis, and fatty acid oxidation in response to increasing concentrations of dl-Met or 2-hydroxy-4-(methylthio) butanoic acid (HMB) in bovine primary hepatocytes. Hepatocytes isolated from 4 Holstein calves less than 7d old were maintained as monolayer cultures for 24h before addition of treatments. Cells were then exposed to treatments of dl-Met or HMB (0, 10, 20, 40, or 60 µM) in Met-free medium for 24h and collected for RNA isolation and quantification of gene expression by quantitative PCR. Expression of betaine-homocysteine methyltransferase (BHMT), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), and 5,10 methylenetetrahydrofolate reductase (MTHFR) genes, which catalyze regeneration of Met from betaine and homocysteine, decreased linearly with increasing dl-Met concentration. We observed similar effects with increasing HMB concentration, except expression of MTHFR, which was not altered. Expression of Met adenosyltransferase 1A (MAT1A), which catalyzes the first step of Met metabolism to generate S-adenosylmethionine (SAM), a primary methyl donor, was decreased with increasing dl-Met or HMB concentration. Expression of S-adenosylhomocysteine hydrolase (SAHH) was decreased linearly with increasing HMB concentration, but not altered by dl-Met. Increasing concentrations of dl-Met and HMB decreased cytosolic phosphoenolpyruvate carboxykinase (PCK1) expression, but did not alter the expression of mitochondrial phosphoenolpyruvate carboxykinase (PCK2) or pyruvate carboxylase (PC). Expression of glucose-6-phosphatase(G6PC) decreased linearly with increasing HMB concentration, but not altered by dl-Met. Neither dl-Met nor HMB altered the expression of carnitine palmitoyltransferase 1A(CPT1a). These findings demonstrate reduced necessity for Met regeneration with increased Met concentrations in the medium, regardless of the Met source. The lack of upregulation of gluconeogenesis indicates that increased dl-Met or HMB is not prioritized for glucose synthesis in primary bovine hepatocytes.