Effect of inhibiting the lactogenic signal at calving on milk production and metabolic and immune perturbations in dairy cows.

During the periparturient period, the abrupt increase in energy demand for milk production often induces metabolic and immunological disturbances in dairy cows. Our previous work has shown that reducing milk output by milking once a day or incompletely in the first few days of lactation reduces these disturbances. The aim of this study was to reduce metabolic and immunological disturbances by limiting milk production during the first week of lactation by inhibiting the lactogenic signal driven by prolactin. Twenty-two fresh cows received 8 i.m. injections of the prolactin-release inhibitor quinagolide (QUIN; 2 mg) or water as a control (CTL). The first injection was given just after calving, and the subsequent 7 injections were given every 12 h. Milk production was measured until d 28 after calving. Blood samples were taken from d 1 (calving) to d 5 and then on d 7, 10, 14, 21, and 28 to measure concentrations of urea, phosphorus, calcium, glucose, nonesterified fatty acids (NEFA), ß-hydroxybutyrate, and prolactin. Other blood samples were taken on d 2, 5, 10, and 28 to analyze oxidative burst, phagocytosis, and the effect of the serum on the lymphoproliferation of peripheral blood mononuclear cells from donor cows. Blood prolactin concentration was lower from d 2 to 5 but higher from d 10 to 28 in the QUIN cows than in the CTL cows. Milk production was lower from d 2 to 6 in the QUIN cows than in the CTL cows (24.3 ± 6.4 and 34.8 ± 4.1 kg/d on average, respectively). We observed no residual effect of quinagolide on milk production after d 6. During the first week of lactation, blood glucose and calcium concentrations were higher and ß-hydroxybutyrate concentration was lower in the QUIN cows than in the CTL cows. Blood NEFA, urea, and phosphorus concentrations were not affected by the treatment. At d 2 and 5, the phagocytosis ability of polymorphonuclear leukocytes was not affected by treatment; however, quinagolide injection enhanced the proportion of cells that entered oxidative burst, The mitogen-induced proliferation of peripheral blood mononuclear cells was greater when they were incubated with serum harvested from the CTL cows and was negatively correlated with the NEFA concentration in the serum. Reducing the prolactin peak at calving was effective in reducing milk production during the first week of lactation without compromising the dairy cow's overall productivity. Slowing the increase in milk production allowed a more gradual transition from pregnancy to lactation and led to a reduction in metabolic stress and an improvement in some immune system aspects during this period.

Homocysteine metabolism, growth performance, and immune responses in suckling and weanling piglets.

Homocysteine (Hcy), an intermediary sulfur AA, is recognized as a powerful prooxidant with deleterious effects on physiological and immune functions. In piglets, there is an acute 10-fold increase of plasma concentrations of homocysteine (pHcy) during the first 2 wk of life. This project aimed to maximize pHcy variations within physiological ranges using typical supplies of folates and vitamin B12 (B12) to sows and piglets. Growth, immune response, and Hcy metabolism of piglets were studied until piglets reached 56 d of age. Third-parity sows were randomly assigned to a 2 × 2 split-plot design with 2 dietary treatments during gestation and lactation, S(-) (1 mg/kg folates and 20 µg/kg B12, n = 15) and S(+) (10-fold S(-) levels, n = 16), and 2 treatments to piglets within each half litter, intramuscular injections (150 µg) of B12 (P(+)) at d 1 and 21 (weaning) and saline (P(-)). Within each litter of 12 piglets, 3 P(+) and 3 P(-) piglets were studied for growth and Hcy metabolism, and the others were studied for immune responses. During lactation, plasma B12 decreased and was transiently greater in S(+) vs. S(-) piglets on d 1 and P(+) vs. P(-) piglets on d 7 (sow treatment × age and piglet treatment × age; P < 0.05). From 14 to 21 d of age, pHcy was 33% lower in S(+)P(+) vs. S(-)P(-) piglets (sow treatment × piglet treatment interaction; P < 0.05). At 56 d of age, hepatic B12 was greater and pHcy was lower for P(+) vs. P(-) piglets (P < 0.05). No treatment effect was observed on growth except for a lower postweaning G:F in S(+)P(-) piglets than in others (sow treatment × piglet treatment interaction; P < 0.05). Positive correlations were observed between pHcy and growth (r > 0.29, P < 0.05) before and after weaning. Antibody responses to ovalbumin and serum tumor necrosis factor-α were not affected by treatments, but postweaning serum IL-8 peaked earlier in S(-)P(-) vs. S(+)P(+) piglets (piglet treatment × age; sow treatment × piglet treatment interaction, P < 0.05). Proliferation of lymphocytes in response to the mitogen concanavalin A tended to be lower in culture media supplemented with sera from S(-) vs. S(+) piglets (P = 0.081) and P(-) vs. P(+) piglets (P = 0.098), and the reduction of response was more marked (P < 0.05) with high (>21 µM) compared to medium (17 to 21 µM) or low (<17 µM) pHcy. In conclusion, the present vitamin supplements to sows and/or piglets produced variations of pHcy that were not apparently harmful for growth performance of piglets. The greater pHcy, particularly prevalent in S(-) and/or P(-) piglets, had negative effects on some indicators of immune responses, suggesting that these young animals may be immunologically more fragile.

In vivo targeting of a sunflower oil body protein in yeast secretory (sec) mutants.

A sunflower oleosin was expressed in yeast to study the in vivo insertion of the protein into the endoplasmic reticulum (ER) and subsequent transfer to lipid bodies. The oleosin cDNA was expressed in a range of yeast secretory (sec) mutants to determine the precise targeting pathway of the oleosin to the ER. Subcellular fractionation experiments indicated that the signal recognition particle (SRP) is required for oleosin targeting to the ER and hence subsequent deposition on the lipid bodies in vivo. The expression of oleosin in a range of sec61 mutant alleles confirmed the role of the SEC61 translocon in insertion of oleosin into the ER membrane, as well as indicating an unusual substrate/translocon interaction for one particular allele (sec61-3). Mistargeting of the oleosin due to impaired SRP function resulted in enhanced proteolysis of the plant protein in the transformed yeast, as determined by pulse-chase analysis. These data therefore provide the first in vivo evidence for the SRP-dependent targeting of the oleosin to the ER, and the subsequent requirement for a functional SEC61 translocon to mediate the correct insertion of the protein into the membrane.

The targeting and accumulation of ectopically expressed oleosin in non-seed tissues of Arabidopsis thaliana.

Full-length and N-terminal deletions of a sunflower (Helianthus annuus L.) oleosin protein were expressed ectopically in transgenic Arabidopsis thaliana (L.) Heynh. Immunological detection of the sunflower protein revealed that it accumulated in a range of non-oil-storing tissues, including leaves, roots and petals. This accumulation was shown to result from deposition in the microsomal membrane fraction. Expression in oil-storing tissues (such as seeds) of oleosin N-terminal deletions revealed impaired transfer from the endoplasmic reticulum to the oil body. In non-oil-storing tissues, accumulation in the microsomal membrane fraction was progressively reduced by N-terminal deletion. These data confirm the role of the endomembrane system in the targeting of the oleosin and its intimate relationship with oil-body biogenesis.

Secondary structure of oleosins in oil bodies isolated from seeds of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.).

Oil bodies were isolated from mature seeds of sunflower (Helianthus annuus L.) and safflower (Carthamus tinctorius L.). Oil body preparations containing only oleosin proteins could be obtained from safflower seeds by salt-washing followed by centrifugation on discontinuous sucrose density gradients. However, it was necessary to treat sunflower oil bodies with urea to obtain preparations of similar purity. Incubation of the oil bodies with proteinases gave two fragments with molecular masses of 6 and 8 kDa which were protected from digestion. These fragments represented the hydrophobic domain of the oleosins, as determined by N-terminal sequencing. Intact and proteinase-treated oil bodies of both species were analysed by Fourier-transform infrared spectroscopy, as dry films and in aqueous medium, the spectra being compared with those obtained for pure oil samples in order to identify the bands resulting from the oleosin proteins and protected peptides. This investigation showed that the hydrophobic domain of the oleosins in intact oil bodies is predominantly alpha-helical in structure and that the conformation was not greatly affected by washing the oil bodies with urea during preparation.