J.V. Nørgaard

Cellular Mechanisms in Regulating Mammary Cell Turnover During Lactation and Dry Period in Dairy Cows

The mechanisms involved in regulating mammary cell turnover during the pregnancy-lactation cycle in dairy cows are unclear. The objective of present experiment was to describe expression of genes encoding proteins known to be involved in pathways regulating mammary cell proliferation, apoptosis, differentiation, cell survival, and tissue remodeling. Mammary gland biopsies were taken 7 times during the pregnancy-lactation cycle of 10 dairy cows, and samples were analyzed by immunohistochemistry and real-time PCR. Cell proliferation was greatest during the dry period and apoptosis was high in early dry period and early lactation. Based on Fas (tumor necrosis factor receptor superfamily member 6), Fas ligand, and caspase-3, caspase-8, and caspase-9 gene expression, no indication was found of a stage-dependent shift between the extrinsic and intrinsic pathways leading to apoptosis. Gene expression of microsomal glutathione S-transferase (mGST) did not vary significantly, whereas B-cell leukemia/lymphoma 2 (Bcl-2) and BCL2-associated X protein (Bax) gene expression was greatest during the dry period and early lactation and coincided with high cell turnover. Gene expression of early response genes c-Fos, c-Jun, and c-Myc correlated to neither rate of cell proliferation nor plasma concentration of insulin-like growth factor (IGF)-I and insulin. Gene expression of nuclear factor of kappa light chain gene enhancer in B-cells (NFkappaB) and NFkappaB inhibitor alpha was greatest in the periparturient period, and NFkappaB gene expression coincided with an anticipated need for cell survival factors. Expression of transforming growth factor beta (TGF-beta) receptor 1 and 2 mRNA was greatest in early lactation, whereas TGF-beta1 did not vary significant during the pregnancy-lactation cycle. Even though our results on the TGF-beta system did not comply with other studies, the gene expression pattern of the TGF-beta receptors indicates a role in regulating apoptosis in early lactation. Signal transducer and activator of transcription 5 (STAT5) gene expression was high in the periparturient period, which suggests a role for STAT5 in regulation of mammary cell proliferation and differentiation in dairy cows. Expression of tissue-plasminogen activator, plasminogen activator inhibitor-1, and IGF binding protein 5 genes was greatest in early lactation, suggesting a role for IGF binding protein 5 in coordinating regulation of apoptosis and tissue remodeling.

Emissions of Sulfur-Containing Odorants, Ammonia, and Methane from Pig Slurry: Effects of Dietary Methionine and Benzoic Acid

Supplementation of benzoic acid to pig diets reduces the pH of urine and may thereby affect emissions of ammonia and other gases from slurry, including sulfur-containing compounds that are expected to play a role in odor emission. Over a period of 112 d, we investigated hydrogen sulfide (H(2)S), methanethiol (MT), dimethyl sulfide (DMS), dimethyl disulfide (DMDS), and dimethyl trisulfide (DMTS), as well as ammonia and methane emissions from stored pig slurry. The slurry was derived from a feeding experiment with four pig diets in a factorial design with 2% (w/w) benzoic acid and 1% (w/w) methionine supplementation as treatments. Benzoic acid reduced slurry pH by 1 to 1.5 units and ammonia emissions by 60 to 70% for up to 2 mo of storage, and a considerable, but transitory reduction of methane emissions was also observed after 4 to 5 wk. All five volatile sulfur (S) compounds were identified in gas emitted from the slurry of the control treatment, which came from pigs fed according to Danish recommendations for amino acids and minerals. The emission patterns of volatile S compounds suggested an intense cycling between pools of organic S in the slurries, with urinary sulfate as the main source. Diet supplementation with methionine significantly increased all S emissions. Diet supplementation with benzoic acid reduced emissions of H(2)S and DMTS compared with the control slurry and moderately increased the concentrations of MT. Sulfur gas emissions were influenced by a strong interaction between methionine and benzoic acid treatments, which caused a significant increase in emissions of especially MT, but also of DMDS. In conclusion, addition of 2% benzoic acid to pig diets effectively reduced ammonia volatilization, but interactions with dietary S may increase odor problems.

Absorption and metabolism of benzoic acid in growing pigs.

Dietary benzoic acid (BA) supplementation causes a pronounced reduction in urinary pH but only small changes in blood pH. The present study aimed to investigate the portal absorption profile, hepatic metabolism of BA, and renal excretion of hippuric acid (HA) underlying the relatively small impact of BA on systemic acid-base status. Eight growing pigs (BW = 63 +/- 1 kg at sampling) fitted with permanent indwelling catheters in the abdominal aorta, hepatic portal vein, hepatic vein, and mesenteric vein were allocated to 4 sampling blocks and randomly assigned to control (CON; nonsupplemented diet) or BA supplementation (B; control diet + 1% BA top-dressed). Feed intake was restricted to 3.6% of BW and the ration divided into 3 equally sized meals offered at 8-h intervals. Blood pH (7.465 and 7.486 +/- 0.004) and urinary pH (4.99 and 7.01 +/- 0.09) were less (P = 0.03 and P < 0.01) in B compared with CON. The arterial concentration, net portal flux, and net hepatic uptake of BA increased (P < 0.01) in B compared with CON. The net portal flux of BA increased (P < 0.01) after feeding with B, but remained positive (P < 0.01) at all sampling times (n = 8). Recovery of dietary BA as increased net portal flux and hepatic uptake of BA was 87 +/- 5% and 89 +/- 15%, respectively. The recovery of dietary BA as urinary excretion of BA and HA was 0.08 +/- 0.02% and 85 +/- 7%, respectively. It is concluded that the small impact of BA supplementation on systemic acid-base status was caused by a protracted BA absorption and efficient hepatic extraction and glycine conjugation in combination with efficient renal clearance of HA. Together, these physiological mechanisms prevented major BA and HA accumulation in body fluids.

Estrogenic activity of bovine milk high or low in equol using immature mouse uterotrophic responses and an estrogen receptor transactivation assay.

BACKGROUND Cows milk contain phytoestrogens especially equol depending on the composition of the feed ration. However, it is unknown whether milk differing in equol exhibits different estrogenicity in model systems and thereby potentially in humans as milk consumers. METHODS The estrogenicity of high and low equol milk (HEM and LEM, respectively) and purified equol was investigated in immature female mice including mRNA expression of six estrogen-sensitive genes in uterine tissue. Extracts of HEM and LEM were also tested for estrogenicity in vitro in an estrogen receptor (ER) reporter gene assay with MVLN cells. RESULTS The total content of phytoestrogens was approximately 10 times higher in HEM compared with LEM, but levels of endogenous milk estrone and 17beta-estradiol were similar in the two milk types (503-566 and 60-64.6pg/ml, respectively). There was no difference in uterine weight between mice receiving LEM and HEM, and no difference from controls. Equol (50 times the concentration in HEM) was not uterotrophic. The ERbeta mRNA expression was down-regulated in the uteri of HEM mice compared with LEM and controls, but there was no difference between milk types for any of the other genes. Extracts of HEM showed a higher estrogenicity than extracts of LEM in MVLN cells, and there was a dose-dependent increase in estrogenicity by equol. CONCLUSION The higher in vitro estrogenicity of HEM was not reflected as a higher uterine weight in vivo although the down-regulation of ERbeta in uterine tissue of HEM mice could suggest some estrogenic activity of HEM at the gene expression level.

Effect of pregnancy and feeding level on cell turnover and expression of related genes in the mammary tissue of lactating dairy cows.

Milk yield is reduced by pregnancy, and the present experiment was conducted to study the biological basis for the negative effect of pregnancy on milk yield. A total of 16 dairy cows were fed at either a normal or a low feeding level (eight cows per treatment), and half of them were inseminated after approximately 3 months of lactation and the other half were not inseminated. Mammary biopsies were taken at approximately 9 months of lactation. The milk yield of pregnant cows was reduced by 2.6 kg/day, and lactation persistency was reduced already from the time of insemination. Low feeding level reduced the milk yield by 9.8 kg/day from week 8 to week 39 of lactation, whereas no interaction between pregnancy and feeding level was found. Cell proliferation (Ki-67) and apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL) were unaffected by feeding level, and pregnancy tended to reduce cell proliferation but had no effect on apoptosis. Reduced cell proliferation may explain the reduced lactation persistency in pregnant cows. Transcription of oestrogen receptor α, progesterone receptor A and B, and long and short isoforms of the prolactin receptor were higher in pregnant cows compared with non-pregnant cows. Feeding level did not mediate changes in transcription of genes. Transcription of other cell-turnover-related genes (IGF-I, IGF binding protein-5, caspase-3) as well as genes related to the secretory activity of the cells (α-lactalbumin and acetyl CoA carboxylase α) was not affected by pregnancy or by feeding level.

Continuous lactation effects on mammary remodeling during late gestation and lactation in dairy goats.

The present study aimed to 1) elucidate whether continuous milking during late gestation in dairy goats negatively affects mammary remodeling and hence milk production in the subsequent lactation, and 2) identify the regulatory factors responsible for changes in cell turnover and angiogenesis in the continuously lactating mammary gland. Nine multiparous dairy goats were used. One udder half was dried off approximately 9 wk prepartum (normal lactation; NL), and the other udder half of the same goat was milked continuously (continuous lactation; CL) until parturition or until the half-udder milk yields had dropped to below 50 g/d. Mammary biopsies were obtained from each udder half just before the NL gland was dried off (before dry period), within the first 2 wk after drying-off (early dry period, samples available only for NL glands), in the mid dry period, within the last 2 wk before parturition (late dry period), and at d 1 (the day of parturition), 3, 10, 60, and 180 of lactation. Mammary morphology was characterized in biopsies by quantitative histology, and cell turnover was determined by immunohistochemistry (terminal deoxynucleotidyl transferase dUTP nick end labeling and Ki-67). Transcription of genes encoding factors involved in mammary epithelial cell (MEC) turnover and vascular function was quantified by quantitative reverse transcription PCR. Results demonstrated that omitting the dry period was possible in goats but was not as easy as claimed before. Renewal of MEC was suppressed in CL glands, which resulted in a smaller MEC population in the subsequent lactation. At the time of parturition (and throughout lactation), the mammary glands subjected to CL had smaller alveoli, more fully differentiated MEC, and a substantially larger capillary fraction compared with NL glands. The continuously lactating gland thus resembled a normally lactating gland in an advanced stage of lactation. None of the studied genomic factors could account for these treatment differences. The described characteristics in CL glands compared with NL glands indicated a gland maintained in lactation for a longer period.

Requirement of standardized ileal digestible valine to lysine ratio for 8- to 14-kg pigs.

The objective was to define the Val requirement for weaned piglets in the context of reducing the dietary protein content. A dose-response experiment was conducted to estimate the standardized ileal digestible (SID) Val to Lys ratio required to support the optimum growth of post-weaned piglets. In this study, 96 pigs weighing 8 kg were allotted to one of six dietary treatments (16 pigs for each dietary treatment) and were housed individually. Diets were formulated to provide 0.58, 0.62, 0.66, 0.70, 0.74 and 0.78 SID Val : Lys by adding graded levels of crystalline l-Val to the 0.58 SID Val : Lys diet. Lysine was sub-limiting and supplied 90% of the recommendation (10.95 g SID Lys/kg equal to 11.8 g/kg total Lys). Average daily feed intake (ADFI), average daily gain (ADG) and gain to feed ratio (G : F) were determined during a 14-day period of ad libitum feeding. Blood and urine samples were taken at the end of each week (day 7 and 14 of the experiment) 3 h after feeding the experimental diets. The maximum ADFI and ADG were obtained in pigs fed the 0.78 SID Val : Lys diet; it was not different from the results of pigs fed 0.70 SID Val : Lys diet. The highest G : F was obtained in pigs fed 0.70 SID Val : Lys. The plasma concentration of Val increased linearly (P<0.001) as the dietary SID Val : Lys increased. The increasing dietary Val : Lys also resulted in a linear increase in Cys (P<0.001) and a quadratic increase in Arg (P=0.003), Lys (P=0.05) and Phe (P=0.009). The plasma Gly showed a quadratic decrease (P=0.05) as the dietary Val : Lys increased. Neither plasma nor urinary urea to creatinine ratio was affected by treatment. The minimum SID Val : Lys required to maximize ADFI, ADG and G : F was estimated at 0.67 SID Val : Lys by a broken-line model, and at 0.71 SID Val : Lys by a curvilinear plateau model. The Val deficiency caused a reduction in ADFI, and Val supplementation above the requirement did not impair animal performance. In conclusion, 0.70 SID Val : Lys is suggested as the Val requirement for 8 to 14 kg individually housed pigs.

The optimum ratio of standardized ileal digestible leucine to lysine for 8 to 12 kg female pigs

The objective of the study was to estimate Leu requirement for weaned piglets to balance indispensable AA in reduced CP diets. A dose-response experiment was conducted to estimate the standardized ileal digestible (SID) Leu to Lys ratio required for the maximum growth of young pigs after weaning. In this study, 96 female pigs (initial BW of 8 kg) were allotted to 1 of 6 dietary treatments with 16 individually penned pigs per treatment. Graded levels of crystalline L-Leu were added to a basal diet to provide diets containing 0.70, 0.80, 0.90, 1.00, 1.10, and 1.20 SID Leu:Lys. Lysine was limiting and fulfilled 90% of the current recommendations. The ADFI, ADG, and G:F were determined during a 2 wk experimental period. Blood and urine samples were taken at the end of each wk. The ADFI increased linearly (P < 0.001) from 0.70 to 0.80 SID Leu:Lys and then remained constant from 0.90 to 1.20 SID Leu:Lys. The ADG showed a quadratic increase ( P= 0.02), as the SID Leu:Lys level increased from 0.70 to 0.90 SID Leu:Lys and did not change further from 0.90 to 1.20 SID Leu:Lys. The G:F increased quadratically (P < 0.001) with increasing SID Leu:Lys level, and the greatest G:F was achieved with pigs receiving the diet with 0.80 SID Leu:Lys. Increasing the dietary SID Leu:Lys resulted in a linear increase in plasma Leu concentration (P < 0.001) and quadratic increases (P < 0.001) in plasma Cys concentration. The plasma concentration of most of the other AA was lowest in pigs receiving the diets with 0.90 to 1.00 SID Leu:Lys. The plasma urea nitrogen concentration tended (P = 0.08) to be lowest in pigs receiving 1.00 SID Leu:Lys, suggesting a more balanced AA profile at this level. Using a curvilinear-plateau model, the SID Leu:Lys requirement was estimated at 0.93 to maximize growth in female pigs weighing 8 to 12 kg.

Ileal digestibility of sunflower meal, pea, rapeseed cake, and lupine in pigs

The standardized ileal digestibility (SID) of CP and AA was evaluated in soybean (Glycine max) meal, sunflower (Helianthus annuus) meal, rapeseed cake, and field pea (Pisum sativum) using 10 pigs and in lupine (Lupinus angustifolius) using 7 pigs. Pigs were fitted with either a T-cannula or a steered ileocecal valve-cannula. Diets contained 170 to 186 g CP/kg DM. Endogenous losses of CP and AA were estimated by feeding a N-free diet. The SID was calculated using the average of Cr(2)O(3) and TiO(2) as indigestible markers and corrected for type of cannula. The SID of CP was greater (P < 0.05) for soybean meal and pea compared to sunflower meal, rapeseed cake, and lupine. The SID of Lys and His were lowest (P < 0.05) in sunflower meal, and the SID of Met and Val were lowest (P < 0.05) in lupine. These results imply soybean meal and pea to be a high-digestible protein source relative to sunflower meal, rapeseed cake, and especially lupine, although all tested feedstuffs seem appropriate for inclusion in diets for organic pigs.

Mammary remodeling in primiparous and multiparous dairy goats during lactation

Milk production is generally lower but lactation persistency higher in primiparous (PP) than in multiparous (MP) goats. This may be related to differences in development and maintenance of mammary gland function, but the underlying mechanisms are not well understood. The present study aimed to elucidate whether differences in lactational performance between PP and MP mammary glands are related to the time course of development and maintenance, not only of the mammary epithelial cell (MEC) population, but also of the mammary vasculature that sustains synthetic activity. Mammary biopsies were obtained from both mammary glands of 3 PP and 6 MP (>or=2 parity) dairy goats at parturition (d 1), d 10, 60, and 180 of lactation. Gene transcription relating to MEC turnover and vascular function was quantified by real-time reverse transcription-PCR, mammary morphology was characterized (quantitative histology), and cell turnover was determined (terminal deoxynucleotidyl transferase dUTP nick end labeling assay and Ki-67). Primiparous glands showed higher expression for the genes involved in angiogenesis; namely, vascular endothelial growth factor receptor 2, and angiopoietin 1 and 2 and their receptor, a few days after parturition (d 10). Primiparous glands also had higher rates of MEC proliferation in early lactation. It therefore appears that initiation of lactation is associated with development and growth of the mammary gland into early lactation, which continues for a longer period in PP compared with MP glands. In addition, MEC survival was found to be higher in PP glands throughout lactation, and MEC in PP glands underwent more extensive differentiation. This could explain the reported flatter lactation curve and higher lactation persistency in PP glands. Although some of the genes included in this study were differentially expressed in PP and MP glands during the course of lactation, it was not possible to identify any specific genomic factor(s) that could account for the differences between PP and MP glands with respect to mammary development and MEC survival during lactation. It remains to be established why parity number affects MEC and vascular development and survival during lactation, and, in particular, which regulatory mechanisms are involved.