S.J. Mabjeesh

Casein-derived phosphopeptides disrupt tight junction integrity, and precipitously dry up milk secretion in goats

Mammary involution is triggered by local stimuli, but the precise mechanism has not been defined. Milk stasis accumulate local signals, which makes the tight junctions (TJ) leaky. The aim of the study was to check the hypothesis that casein hydrolyzates (CNH) compromise TJ integrity and dry up milk secretion. A single dose of CNH transiently (12 to 24 h) compromised TJ integrity in the treatedudder. This was associated by a transient (12 to 96 h) decline in milk secretion. No such changes were recorded in the contralateral gland that served as a control. Four repeated doses of CNH after each milking caused drastic changes in mammary secretion and composition, which were associated with irreversible cessation of milk secretion within 96 h. No such changes were recorded in goats treated with de-phosphorylated casein (control). We conclude that CNH are the milk-borne factors that cause the disruption of TJ integrity and induction of involution, and that the serine-Ps in the CNHs are essential for the excretion of biological activity.

Effect of feeding cows in early lactation with diets differing in roughage-neutral detergent fiber content on intake behavior, rumination, and milk production

This study measured the effects of including soyhulls as partial roughage replacement in total mixed rations (TMR) fed to 25 pairs of cows during early lactation, on the dry matter (DM) intake, particle kinetics, rumination, in vivo DM and NDF digestibility, milk and FCM yields, and BW changes. The 2 diets used in this study differed in the content of roughage and roughage NDF [23.5 vs. 35.0%, and 12.8 vs. 18.7% in the experimental (EXP) and control (CON) TMR, respectively]. The EXP TMR contained 20.5% less physically effective NDF than the CON TMR (11.7 vs. 14.1% of DM, respectively). These differences were expressed in a greater intake per meal (by 13.3%), a higher rate of meal intake (by 23.2%), a similar number of meals per day, a shorter daily eating duration (by 13%), and a higher total daily DMI (by 7.2%) in the EXP cows as compared with the CON cows. The in vivo DM and NDF digestibility was higher by 4.9 and 22.7%, respectively, in the EXP cows than in the CON cows. The rumination time for the TMR in the EXP cows was 12.7% (54.3 min/d) shorter than in the CON cows, and this was probably related to the difference of 12.4% in physically effective NDF intake between the 2 groups. Patterns of daily rumination and feed consumption throughout an average day showed a delay of approximately 1 to 2 h between the eating and rumination peaks. Particle flow from the rumen of the EXP cows was characterized by a longer rumen mean retention time (by 17.8%) and longer rumination time per kilogram of roughage ingested (by 23.5%) as compared with the CON cows. Thus, favorable conditions for NDF digestion were created in the rumen of the EXP cows, as reflected in their rumen pH values (6.67). The advantage of the EXP cows in intake and digestibility was reflected in a concomitant increase of 7.4% in milk production and of 9.2% in FCM yield as compared with the CON cows. No difference was found between the 2 groups with respect to efficiency of feed utilization for milk production and BW changes.

Release of Pleurotus ostreatus Versatile-Peroxidase from Mn2+ Repression Enhances Anthropogenic and Natural Substrate Degradation

The versatile-peroxidase (VP) encoded by mnp4 is one of the nine members of the manganese-peroxidase (MnP) gene family that constitutes part of the ligninolytic system of the white-rot basidiomycete Pleurotus ostreatus (oyster mushroom). VP enzymes exhibit dual activity on a wide range of substrates. As Mn2+ supplement to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds and lignin, we examined the effect of Mn2+ on the expression profile of the MnP gene family. In P. ostreatus (monokaryon PC9), mnp4 was found to be the predominantly expressed mnp in Mn2+-deficient media, whereas strongly repressed (to approximately 1%) in Mn2+-supplemented media. Accordingly, in-vitro Mn2+-independent activity was found to be negligible. We tested whether release of mnp4 from Mn2+ repression alters the activity of the ligninolytic system. A transformant over-expressing mnp4 (designated OEmnp4) under the control of the β-tubulin promoter was produced. Now, despite the presence of Mn2+ in the medium, OEmnp4 produced mnp4 transcript as well as VP activity as early as 4 days after inoculation. The level of expression was constant throughout 10 days of incubation (about 0.4-fold relative to β-tubulin) and the activity was comparable to the typical activity of PC9 in Mn2+-deficient media. In-vivo decolorization of the azo dyes Orange II, Reactive Black 5, and Amaranth by OEmnp4 preceded that of PC9. OEmnp4 and PC9 were grown for 2 weeks under solid-state fermentation conditions on cotton stalks as a lignocellulosic substrate. [14C]-lignin mineralization, in-vitro dry matter digestibility, and neutral detergent fiber digestibility were found to be significantly higher (about 25%) in OEmnp4-fermented substrate, relative to PC9. We conclude that releasing Mn2+ suppression of VP4 by over-expression of the mnp4 gene in P. ostreatus improved its ligninolytic functionality.

High Rates of Mammary Tissue Protein Turnover in Lactating Goats Are Energetically Costly

The high energetic demands and metabolism of amino acids (AA) within the lactating mammary gland have been ascribed to the requirements for milk component synthesis and tissue maintenance. Our objective in this work was to assess rates of protein synthesis from several AA so that the energetic costs of tissue maintenance could be better reflected. Lactating goats (n = 4) were given staggered infusions of 5 labeled forms of phenylalanine (Phe) initiated at 30, 12, 9, 6, and 3 h before goats were killed. [5-(13)CH(3)] Methionine (Met), [1-(13)C] leucine, and [1-(13)C] valine were also infused for 30 h, during which time, the glands were milked hourly and arteriovenous flux measurements were performed the last 6 h. A dynamic, compartmental model capable of simulating fluxes of AA through extracellular and intracellular free, slow and fast turnover tissue-bound, and milk protein pools was developed and fitted to the observed data. The udder removed 81% of the Phe present in plasma using 31% for milk protein synthesis and releasing 66% back into plasma. Transamination accounted for 40% of Phe flux in the mammary and transmethylation accounted for a portion of mammary Met flux. Mammary tissue protein synthesis was >300% the value of milk protein synthesis with fractional protein synthesis rates >130%/d. Assuming 4 mol of ATP/mol of peptide bond formed, we estimate that approximately 50% of ATP generated by the lactating mammary glands is used for synthesis of tissue (nonmilk) protein.

Mammary Fat Can Adjust Prolactin Effect on Mammary Epithelial Cells via Leptin and Estrogen.

Leptin, like estrogen, is one of the endo/paracrine factors, which are synthesized in and secreted from mature adipocytes. The roles of the mammary fat pad and mammary adipocytes in the initiation of lactation are not clear. In this study, we showed that combination of prolactin, leptin and estrogen elevated the expression of the milk protein beta-lactoglobulin. We also showed that after prolactin stimulate the secretion of leptin from the mammary fat, leptin upregulated the expression of estrogen receptor alpha in the mammary epithelial cells. Also, prolactin affected aromatase mRNA expression in the bovine mammary fat and we demonstrated that leptin and prolactin can affect cholesterol secretion from explants in culture to the medium. Therefore, we suggest that prolactin initiates estrogen expression (as represented by aromatase mRNA) in the mammary fat pad, whereas leptin stimulates estrogen receptor alpha expression in the mammary epithelial cells. We hypothesize that leptin and estrogen, secreted from the mammary fat regulate lactation after stimulation of prolactin.

Assessment of duodenal amino acid profile in dairy cows by the in situ method

Abstract In situ evaluation of the duodenal amino acid (AA) profile was attempted in a 4 × 4 Latin square study using four lactating Holstein cows fitted with ruminal and duodenal cannulas. Dietary supplemental crude protein (CP) sources, making up 40% of the dietary CP, were: soybean meal, cottonseed meal, corn gluten meal or urea. Duodenal flow of organic matter (OM) and CP was determined in vivo by means of constant infusion of ytterbium and chromium, and predicted in situ from rumen degradation. Purines were used as microbial markers. In situ calculation of the AA profile, expressed as g AA in 100 g of total AAs, was based on the composition of AA in feed and in isolated bacteria as well as on in situ rumen degradation of OM and CP. The AA profile in the duodenum when assessed in vivo was affected by the nature of the supplemental protein; in situ assessment of the AA profile resulted in comparable profiles. The data are interpreted to suggest that the in situ method enables prediction of the profile of AA (g AA/100 g total AA) flowing to the duodenum, and that the undegraded portion of supplemented protein affects that profile.

Inactivation of a Pleurotus ostreatus versatile peroxidase-encoding gene (mnp2) results in reduced lignin degradation.

Lignin biodegradation by white-rot fungi is pivotal to the earths carbon cycle. Manganese peroxidases (MnPs), the most common extracellular ligninolytic peroxidases produced by white-rot fungi, are considered key in ligninolysis. Pleurotus ostreatus, the oyster mushroom, is a preferential lignin degrader occupying niches rich in lignocellulose such as decaying trees. Here, we provide direct, genetically based proof for the functional significance of MnP to P. ostreatus ligninolytic capacity under conditions mimicking its natural habitat. When grown on a natural lignocellulosic substrate of cotton stalks under solid-state culture conditions, gene and isoenzyme expression profiles of its short MnP and versatile peroxidase (VP)-encoding gene family revealed that mnp2 was predominately expressed. mnp2, encoding the versatile short MnP isoenzyme 2 was disrupted. Inactivation of mnp2 resulted in three interrelated phenotypes, relative to the wild-type strain: (i) reduction of 14% and 36% in lignin mineralization of stalks non-amended and amended with Mn(2+), respectively; (ii) marked reduction of the bioconverted lignocellulose sensitivity to subsequent bacterial hydrolyses; and (iii) decrease in fungal respiration rate. These results may serve as the basis to clarify the roles of the various types of fungal MnPs and VPs in their contribution to white-rot decay of wood and lignocellulose in various ecosystems.

Evaluation of protein flow to the duodenum in dairy cattle by the in sacco method

Abstract In situ evaluation of the amount and composition of duodenal crude protein flow was examined in a 4×4 Latin square study using four lactating Holstein cows fitted with ruminal and duodenal cannulas. Dietary supplemental CP sources, making up 40% of the dietary CP, were: soybean meal, cottonseed meal, corn gluten meal or urea. Duodenal flow of organic matter (OM) and CP was determined in vivo by means of constant infusion of Yb and Cr, and was predicted in situ from rumen degradation. Purines were used as microbial markers. The two methods of estimating ruminally undegraded protein ranked the diets similarly. Microbial CP (1.9 to 2.1 kg/d) was estimated to make up 57% to 68% of the duodenal CP flow in the experimental diets. Efficiency of microbial protein synthesis were 201 (SEM 16) g CP/kg OM truly digested in the rumen, and 224 (SEM 18) g CP/kg OM disappearing in the rumen. Data are interpreted to suggest that the in situ method enables a prediction of total protein flow to the duodenum which is comparable to that obtained using the in vivo method. The added practicality of the former method renders it advantageous.

Heat-treated whole cottonseed versus maize gluten meal as a rumen undegradable protein supplement for lactating dairy cows

Abstract The effect of heat treatment on the nutritive value of rumen undegradable protein (UDP) fraction of whole cottonseed (WCS) was studied in this experiment. Forty two multiparous Israeli Holstein cows were used in a multiple double 3×3 Latin square design. Milk production averaged 38±4.1 kg at the beginning of the experiment. Three different diets were formulated to contain 160 g/kg crude protein (CP), 365 g/kg neutral-detergent fibre (NDF) on a dry matter (DM) basis, 7.24 MJ net energy for lactation (NE L )/kg DM and 170 g/kg WCS. 30% of dietary CP was of cotton origin. Diets were (1) whole cottonseed (WCS), (2) heated whole cottonseed (HWCS) and (3) whole cotton seeds plus 3.1% maize gluten meal (WCS+MG). Diets with HWCS and WCS+MG contained 60 g UDP/kg DM compared with 54 g UDP/kg DM in the WCS treatment. Heat treatment decreased ruminal CP degradability of WCS by 22% and ruminal organic matter (OM) degradability by 5%. Intakes of DM, OM and CP were similar across diets. Higher apparent total tract digestibilities of DM and OM were determined in HWCS and WCS+MG diets (0.62 and 0.64 and 0.63 and 0.65, respectively) compared to WCS diet (0.59 and 0.62, respectively). Higher ruminal propionate proportion was determined in diets containing WCS and WCS+MG over the sampling hours. A higher ruminal ratio of acetate:propionate was measured in the diet containing HWCS. Ammonia N concentration was similar in all treatments, however, blood urea N concentration was the lowest at 3 and 6 h after feeding in the HWCS diet compared to the other diets. Milk yield and composition were similar for all treatments and averaged 34.2±0.45 kg/d with 34.2 g fat/kg, 30.1 g CP/kg and 47 g lactose/kg. It was suggested that inclusion of HWCS in diets for high-yielding dairy cows decreased the endogenous catabolism of amino acids. In this study the effect of heat treatment on WCS on the performance of cows was not apparent because the cows consumed surplus of dietary CP.

Hyperinsulinemic clamp modulates milk fat globule lipid composition in goats

We determined the effect of insulin on milk fatty acid (FA) and lipid composition in goats. Four dairy goats, 150 d in milk, were subjected to hyperinsulinemic clamp (treatment) or saline (control) infusion for 4d in a crossover design study. Composition and concentration of plasma and milk FA, triglycerides, phospholipids, sphingolipids, and cholesterol were determined. Mammary gland biopsies were taken at the end of each experimental period and lipogenic gene expression was determined. Plasma insulin was elevated 3.5-fold, whereas plasma glucose remained constant during the treatment period. Feed intake decreased by 26% and fat yield decreased by 17% relative to controls. No change in nonesterified FA concentration was found between controls and treatment. Compared with controls, insulin decreased yield of long-chain saturated FA by 14%. Milk concentration of long-chain FA was reduced by 3%, whereas that of medium-chain FA increased by 5% during the treatment compared with controls. Hyperinsulinemic clamps increased the yields of milk phospholipids by 9% and cholesterol by 16%, whereas it only tended to decrease triglyceride yields (by 11%). Hyperinsulinemic treatment resulted in compositional changes in the milk fat globule membrane, as reflected by 15 and 9% decreases in phosphatidylethanolamine and phosphatidylcholine concentrations, respectively. Lipogenic gene expression of acyl coenzyme A carboxylase, stearoyl coenzyme A desaturase, and FA synthase did not change, whereas lipoprotein lipase gene expression tended toward an increase in the treatment period compared with controls. Hyperinsulinemic clamps reduce the availability of long-chain FA, which are considered to originate from the diet and adipose lipolysis for milk lipid synthesis by the mammary gland of goats. Under these conditions, long-chain FA might be preferentially channeled to phospholipid rather than triglyceride synthesis, hence increasing phospholipid yields. Mechanisms determining FA distribution among milk lipid components and the consequences of altered milk fat globule membrane lipid composition remained to be elucidated.