Invited review: Muscle protein breakdown and its assessment in periparturient dairy cows.

Mobilization of body reserves including fat, protein, and glycogen is necessary to overcome phases of negative nutrient balance typical for high-yielding dairy cows during the periparturient period. Skeletal muscle, the largest internal organ in mammals, plays a crucial role in maintaining metabolic homeostasis. However, unlike in liver and adipose tissue, the metabolic and regulatory role of skeletal muscle in the adaptation of dairy cows to the physiological needs of pregnancy and lactation has not been studied extensively. The functional integrity and quality of skeletal muscle are maintained through a constant turnover of protein, resulting from both protein breakdown and protein synthesis. Thus, muscle protein breakdown (MPB) and synthesis are intimately connected and tightly controlled to ensure proper protein homeostasis. Understanding the regulation of MPB, the catabolic component of muscle turnover, and its assessment are therefore important considerations to provide information about the timing and extent of tissue mobilization in periparturient dairy cows. Based on animal models and human studies, it is now evident that MPB occurs via the integration of 3 main systems: autophagy-lysosomal, calpain Ca2+-dependent cysteine proteases, and the ubiquitin-proteasome system. These 3 main systems are interconnected and do not work separately, and the regulation is complex. The ubiquitin-proteasomal system is the most well-known cellular proteolytic system and plays a fundamental role in muscle physiology. Complete degradation of a protein often requires a combination of the systems, depending on the physiological situation. Determination of MPB in dairy cows is technically challenging, resulting in a relative dearth of information. The methods for assessing MPB can be divided into either direct or indirect measurements, both having their strengths and limitations. Available information on the direct measures of MPB primarily comes from stable isotopic tracer methods and those of indirect measurements from assessing expression and activity measures of the components of the 3 MPB systems in muscle biopsy samples. Other indirect approaches (i.e., potential indicators of MPB), including ultrasound imaging and measuring metabolites from muscle degradation (i.e., 3-methylhistidine and creatinine), seem to be applicable methods and can provide useful information about the extent and timing of MPB. This review presents our current understanding, including methodological considerations, of the process of MPB in periparturient dairy cows.

Assessment of estrus cyclicity in the Asian elephant (Elephas maximus) by measurement of fecal progesterone metabolite 5α-P-3OH, using a non-invasive assay.

Reproductive management of the Asian elephant (Elephas maximus) is important for its conservation. To monitor its estrous cyclicity, we earlier used an indirect ELISA to show that levels of fecal progesterone (P(4))-metabolite (allopregnanolone: 5α-P-3OH) in semi-captive females sampled randomly positively correlated with serum P(4) levels [12]. In this longitudinal study (51 weeks), we measured levels of fecal 5α-P-3OH and serum P(4) in seven semi-captive female elephants. Females exhibited three types of hormonal profiles. Four females showed cyclical patterns of fecal 5α-P-3OH and serum P(4) typical of normal estrous cycles, two showed acyclic pattern while one showed high values indicative of a pregnant animal. Values for anestrous or follicular phases were ≤ 0.3 µg g(-1) (5α-P-3OH) and ≤ 0.3 ng mL(-1) (P(4)); for luteal phase 0.32-11.09 µg g(-1) (5α-P-3OH) and 0.32-1.48 ng mL(-1) (P(4)); for pregnancy 1.41-7.38 µg g(-1) (5α-P-3OH) and 0.39-1.6 ng mL(-1) (P(4)). A positive correlation (t = 8.8, p < 0.01, n = 321) between levels of fecal 5α-P-3OH and serum P(4) was observed. A random sample of 30 free-ranging female elephants showed fecal 5α-P-3OH values of 0.06-23.4 µg g(-1), indicating them to be in different stages of estrous cyclicity. This study is the first to assess the reproductive phases of female Asian elephants based on the correlative-patterns of both the fecal 5α-P-3OH and serum P(4) values over multiple estrous cycles. This has a potential application in the reproductive management and conservation of Asian elephants.

Effect of undernutrition in foetal life on energy expenditure during gestation in ewes.

The long-term effect of early life undernutrition on late gestation energy expenditure (EEgest) was investigated in sheep. Ewes were fed either adequate (100%) or restricted (60%) energy and protein during late foetal life as well as during last trimester of gestation later in life, resulting in three groups: Adequate-Adequate (AA, n = 5), Adequate-Restricted (AR, n = 5) and Restricted-Restricted (RR, n = 5). At two weeks pre-partum, EEgest were calculated from respiratory gaseous exchange and nitrogen excreted in urine and further it was partitioned to energy expenditure for conceptus development (EEconceptus) and homeorhetic adaptations in maternal metabolism (EEhomeorhetic). Late gestational energy and protein restriction reduced the EEgest in the AR ewes (4.1 MJ x d(-1)) but not in the RR ewes (5.2 MJ x d(-1)) compared with the AA ewes (6.8 MJ x d(-1)). Based on conceptus-weight, no significant difference was found in EEhomeorhetic among the groups; 172, 175 and 169 kJ/kg x d(-1) in AA, AR and RR ewes, respectively. However, EEconceptus was significantly lower in AR (135 kJ/kg x d(-1)) in comparison with AA (298 kJ/kg x d(-1)) and RR (252 kJ/kg x d(-1)) ewes. In conclusion, exposure nutrient restriction in early life impairs the ability of ewes to respond to nutritional restriction in terms of energy expenditure of gestation.

Partitioning of late gestation energy expenditure in ewes using indirect calorimetry and a linear regression approach.

Late gestation energy expenditure (EE(gest)) originates from energy expenditure (EE) of development of conceptus (EE(conceptus)) and EE of homeorhetic adaptation of metabolism (EE(homeorhetic)). Even though EE(gest) is relatively easy to quantify, its partitioning is problematic. In the present study metabolizable energy (ME) intake ranges for twin-bearing ewes were 220-440, 350- 700, 350-900 kJ per metabolic body weight (W0.75) at week seven, five, two pre-partum respectively. Indirect calorimetry and a linear regression approach were used to quantify EE(gest) and then partition to EE(conceptus) and EE(homeorhetic). Energy expenditure of basal metabolism of the non-gravid tissues (EE(bmng)), derived from the intercept of the linear regression equation of retained energy [kJ/W0.75] and ME intake [kJ/W(0.75)], was 298 [kJ/ W0.75]. Values of the intercepts of the regression equations at week seven, five, and two pre-partum were 311, 398, and 451 [kJ/W0.75], respectively. The difference between the intercepts for different weeks was used to calculate EE(homeorhetic). The remaining part of EE(gest) was considered to be EE(conceptus). In conclusion, the good agreement between our values of EE(conceptus) and those in the literature indicates the method's validity.

Use of metabolic profiles for the assessment of dietary adequacy in UK dairy herds.

Between April 1999 and March 2004, metabolic profile analyses were performed on individual blood samples from 35,506 dairy cattle in the UK. Assessment of the cows' energy status by the analysis of plasma samples for beta-hydroxybutyrate, glucose and non-esterified fatty acids showed that 70.4 per cent of the cows in early lactation (10 to 20 days calved), 57.1 per cent of the cows in mid-lactation (51 to 120 days calved) and 57.7 per cent of the dry cows within 10 days of their predicted calving date had one or more energy metabolites outside the optimum range; in addition, 16 per cent of the cows in early lactation, 5.6 per cent of those in mid-lactation and 20.5 per cent of the dry cows within 10 days of their predicted calving date had a low plasma urea nitrogen concentration, indicating poor intakes of effective rumen-degradable protein. Abnormalities in the concentrations of magnesium, inorganic phosphate, copper, selenium and iodine were relatively uncommon. The transitional period, particularly in late pregnancy, was commonly identified as a constraint on productivity. Nutritional problems were most commonly associated with poor feed intakes and poor feed management, rather than with the formulation of the rations.

Costs of gestation in an Arctic ruminant: copper reserves in muskoxen.

The transfer of trace minerals between mother and fetus may be critical for survival of young ruminants especially among species at high latitudes, which gestate during a long winter and grow through a brief summer. We examined the distribution of copper and metalloproteins (ceruloplasmin and metallothionein) in muskoxen and their fetuses, three times during gestation. Hepatic levels of copper were high in mothers (179 microg g(-1) whole tissue) and did not change through gestation, whereas fetuses accumulated large reserves of Cu (>300 microg g(-1)), likely stored in proteins such as metallothionein, during the last third of gestation. The effect of fetal Cu demands on the pregnant female was tested by supplementation of Cu by subcutaneous injections of Cu gluconate (30 mg Cu/week) during pregnancy. Maternal copper supplementation did not significantly increase hepatic Cu in newborns (412 microg g(-1) for supplemented vs. 303 microg g(-1) for unsupplemented neonates), probably because the diet was already adequate in copper (14 microg g(-1) dry matter). Ceruloplasmin activity declined in pregnant muskoxen that had not received injections of Cu and suggested increased systemic demands for copper during late gestation. Supplies of Cu to the fetus could be limited either by low levels of Cu in the maternal liver, or in the maternal diet during late winter when fetal gains in mass and liver Cu are greatest.

Immunohistochemical assessment of progesterone, oestrogen and glucocorticoid receptors in bovine placentomes during pregnancy, induced parturition, and after birth with or without retention of fetal membranes.

Steroid hormones play an important role in placental development. However, the exact cellular site of hormone action has not been evaluated in bovine placentomes. Thus, the present immunohistochemical study was designed to assess the distribution of progesterone receptors, oestrogen receptors and glucocorticoid receptors in bovine placentomes. Tissue specimens were obtained from cows at slaughter and from cattle during pre-term Caesarean section 27 h after prostaglandin administration, immediately after spontaneous parturition and from cattle that had retained the fetal membranes. Specific antibodies were used for receptor demonstration in tissue sections. Progesterone receptors were only detected in maternal connective tissue cells, whereas oestrogen receptors were also present in maternal crypt epithelium. At specific sites, both receptor immunoreactivities remained constant or changed significantly during pregnancy, were generally higher during Caesarean section and decreased post partum, but were less pronounced in cattle that released the fetal membranes than in those that retained the fetal membranes. Glucocorticoid receptors were evident in fetal connective tissue cells as well as in fetal and maternal blood vessels. Maternal crypt epithelial cells showed increasing immunoreactivities for glucocorticoid receptors during pregnancy. Receptor immunoreactivities tended to be lower after spontaneous parturition than during Caesarean section; these results were significant for progesterone and oestrogen receptors in animals that released the fetal membranes but not for those that retained the fetal membranes. The results indicate that in bovine placentome steroid hormone receptors are distributed in patterns that are specific to the type of cell, the stage of pregnancy and the tissue location, implying highly specific modulation of placental metabolism. Retention of the fetal membranes is reflected by altered placental receptor states at parturition.

Heterogenous response of knee cartilage to pregnancy in the rabbit: assessment of specific mRNA levels.

OBJECTIVE: The purpose of this study was to evaluate the effect of pregnancy on mRNA levels for several relevant molecules between five articular cartilage surfaces of the rabbit knee joint. DESIGN: Total RNA was extracted from the following five knee joint articular surfaces: the lateral and medial femoral condyles (LFC and MFC); the lateral and medial tibial plateau (LTP and MTP); and the femoral groove (G) from pregnant and age-matched non-pregnant skeletally immature New Zealand White rabbits. The RNA was analysed by the sensitive molecular technique of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using rabbit specific primer sets. Two types of comparisons were performed: (i) comparison of mRNA levels between cartilage surfaces and (ii) comparison of mRNA levels between pregnant and non-pregnant rabbits within the same articular surfaces. RESULTS: (i) Total RNA yield from the MFC and G represented 53 and 58% of the total RNA amount from the five cartilage surfaces in both non-pregnant and pregnant rabbits, respectively. Transcript levels for progesterone receptor (PR), aggrecan and biglycan were similar in all of the cartilage surfaces. In contrast, the cartilage surfaces exhibited significantly different transcript levels with a similar pattern for the estrogen receptor (ER), collagenase and urokinase (i.e., MTP

Thyroid hormone economy in pregnant rats near term: a "physiological" animal model of nonthyroidal illness?

We have studied the changes in thyroid hormone economy that occur in normal pregnant rats between 17-22 days of gestation. T4 and T3 decreased in all extrathyroidal tissues studied, namely plasma, liver, kidney, lung, heart, and skeletal muscle. The exception is the concentration of T3 in cerebral cortex, which remains unchanged, possibly as a consequence of an increase in type II 5'-iodothyronine deiodinase activity. The marked decrease observed in most T4 and T3 pools was not accompanied by a commensurate increase in circulating TSH levels, which at 21 days gestation were either unchanged or actually decreased. The TSH response to TRH appeared to be prolonged. alpha-Glycerophosphate dehydrogenase activity was decreased in the liver, in accordance with its thyroid hormone deficiency. Hepatic type I 5'-iodothyronine deiodinase activity, however, did not decrease, but was slightly increased. Thus, thyroid hormone economy in the pregnant rat near term shows striking similarities with several (but not all) of the changes described in patients with nonthyroidal illness and in several animal models used to study this condition. It is suggested that attenuation of the negative feedback response to the decrease in thyroid hormone pools, leading to low levels of thyroid hormones in most tissues, is the normal physiological response to situations where preservation of energy (and protein) represents a distinct adaptive advantage, as in the case of the pregnant rat and her conceptus.

[The energy requirements of Holstein-Friesian heifers during rearing and pregnancy under the tropical conditions of Cuba. 1. Determination of dry matter and energy assimilation and of the live weight development and energy expenditure]. / Untersuchungen zum Energiebedarf bei Holstein-Frisian-Färsen während der Aufzucht und Trächtigkeit unter den tropischen Bedingungen Cubas.

In an individual feeding experiment with 48 Holstein-Friesian heifers divided into 3 groups, the effect of various energy levels on live weight development and energy expenditure in the age bracket of 97 days (78 kg live weight) to conception (350 kg LW) and from conception to calving (500 kg LW) was investigated. Rations with differing concentrate--roughage ratios were used. The differentiation of energy intake per day between groups 1 and 2 and group 3 amounted to 7.5% in the 1st development period and to 20% between group 1 and groups 2 and 3 in the 2nd development period. There were significant differences in the live weight gain of 95 g/animal and day in the first development period (up to conception) and of 81 g/animal and day in the second period of development (from conception to calving). Under the tropical climatic conditions the energy expenditure in these studies was approximately 10% under the energy expenditure recommended by the NRC (1978). While there was a clear reciprocal relation between energy expenditure and live weight gain in the first period of development, this relation was less distinct in the second period of development.

[Dietary calcium deprivation in the pregnant rat: evaluation of the repercussions on the maternal and fetal skeleton]. / Deprivación del calcio dietario a la rata gestante: valoración de la repercusión en el esqueleto materno y fetal.

Authors have studied: calcemia, bone ashes calcium content and bone turnover by histofluorescence in 44 Wistar female adult rats. Control group (A) received for 21 days (equivalent to pregnancy) a normocalcemic diet, and group B an hypocalcemic diet. Both received demethyl-chlortetracycline intra-peritoneally at the beginning and end of the experience. Fetuses were fully calcinated and ash weight and calcium content determined. Group B shows a significative decrease in calcemia (F = 284.66, p less than 0.01), bone ash calcium content (F = 47.2, p less than 0.01) and an increase in endosteal bone resorption (F = 32.89, p less than 0.01), while bone formation including synthesis of bone matrix and calcium deposition is diminished (total bone formation, F = 20.0, p less than 0.01). No significative differences have been found among rats of the same group for the fact of being pregnant. No differences have been detected among fetuses of both groups. Fetuses seem to act as parasites and authors support a possible explanation for neonatal hypocalcemia.

[Study on insulin resistance in rats treated with estrogen and progesterone--assessment with the euglycemic glucose clamp technic].

To clarify the mechanism of insulin resistance in pregnancy, we have used the euglycemic glucose clamp technique in estradiol(E) treatment(n = 6), progesterone(P) treatment (n = 28), and Control(n = 29) female rats. E(10 micrograms/day) and P(10 mg/day) were injected subcutaneously into female rats for 14 days, to increase E and P concentrations to pregnant levels. Glucose production and glucose utilization were measured by using [3-3H]-glucose. The results were as follows, 1) Glucose production was almost suppressed at hyperinsulinemia(11,000 microU/ml) both Control and P treatment rats. Then at hyperinsulinemia, glucose utilization rate was almost equal to glucose infusion rate. 2) In P treatment rats glucose utilization was significantly lower (p less than 0.05) than in Control rats at hyperinsulinemia (11,000 microU/ml). 3) In P treatment rats glucose infusion rate was significantly lower than in Control rats at plasma insulin concentrations of 1,000 microU/ml(p less than 0.02), and 11,000 microU/ml(p less than 0.01), and lower than in E treatment rats at plasma insulin concentrations of 11,000 microU/ml(p less than 0.05). 4) In a dose-response curve for the effects of four different concentrations of insulin on glucose infusion rate, the insulin resistance induced by progesterone is characterized by a decreased responsiveness to insulin. The results suggest that progesterone may play an important role in inducing insulin resistance in pregnancy.