Síntesis endógena de ácidos grasos en la glándula mamaria y síndrome de baja grasa en la leche en ovejas

Abstract

Fat is the most variable component of milk in dairy ruminants, its amount and composition being affected basically by the consumed diet. Thus, its fatty acid (FA) profile is the result of a complex interaction between nutrients and ruminal and mammary metabolism. Among FA present in ewe milk fat, it is worth mentioning the conjugated linoleic acid (CLA) because of its potential beneficial effects on human health. The cis-9, trans-11 CLA present in milk has a double origin: on the one side, ruminal through the action of local microbiota, and on the other side, endogenous in the mammary gland, through the ∆9 -desaturase enzyme (SCD). In the ewe, as well as in other dairy ruminants, the ∆9 -desaturase would be the main responsible for the synthesis of milk long-chain FA with a double bound in the cis-9 position (e.g., cis-9 18:1 from 18:0, and cis-9, trans-11 18:2 from trans-11 18:1). However, information on this topic is still scant. With the aim of trying to determine the endogenous synthesis of those FA in the ovine mammary gland, it was carried out a trial in which sterculic acid, a powerful and specific ∆9 -desaturase inhibitor, was administered to a group of 6 lactating ewes. Animals were monitored for a 15-day experimental period, which included a 5-day pretreatment period, a 5-day treatment period, and a 5-day posttreatment period. During the treatment period, ewes received 0.5 g/day of sterculic acid, delivered intravenously in 4 equal doses at 6-hour intervals. Throughout the whole experiment, animals were fed pasture to supply mainly α-linolenic acid and minimize the amount of milk cis-9, trans-11 18:2 of ruminal origin. Results showed a decrease in the milk content of those FA arising from SCD action (e.g., cis-9 10:1, cis-9 14:1, cis-9 16:1, cis-9 18:1, and cis-9, trans-11 18:2) together with an increase in the enzyme substrates (e.g., 14:0, 18:0, and trans-11 18:1). Concentration of some other milk FA, further to previously reported substrates of SCD (e.g., cis-15 18:1, and trans-11, trans-15 18:2) were also increased by sterculic acid administration, whereas concentration of its products were decreased (e.g., cis-9, cis-15 18:2, and cis-9, trans- 11, trans-15 18:3). The inhibition of the ∆9 -desaturase reached 70% and allowed to estimate that 63% of cis-9 18:1 and 74% of cis-9, trans-11 18:2 present in milk fat arise from endogenous synthesis. Another interesting aspect related to the cis-9 18:1 is the role that this FA plays in the maintenance of milk fat fluidity. In fact, the lack of 18:0 for the endogenous synthesis of cis-9 18:1 has been indicated to be able to explain the milk fat depression (MFD) observed in animals fed marine lipids, because the decrease of this 18:1 isomer would increase the melting point, making milk fat secretion more complicated. Very long-chain n-3 FA, abundant in marine lipids, are well known inhibitors of the last step of ruminal biohydrogenation, which causes a decrease in the amount of 18:0 leaving the rumen that is the substrate for cis-9 18:1 synthesis in the mammary gland. Furthermore, based on previous results in lactating ewes receiving a diet that was supplemented with marine microalgae (MA), it was hypothesized the possibility of an adaptation of the rumen microbiota to the consumption of marine lipids that allowed a gradual recovery of milk fat percentage. Based on these points, a second experimental trial was conducted to study the persistency of the response of 36 lactating ewes to the supplementation of their diet with MA. This response was measured in terms of animal performance and milk composition, with especial focus on milk FA profile in general and on those FA related to MFD in particular. Animals were distributed in 6 lots of 6 ewes/lot and allocated to 2 treatments: 3 lots were fed the control diet, consisting of a total mixed ration (40:60 forage:concentrate ratio) supplemented with 25 g of sunflower oil/kg of dry matter, and the remaining 3 lots were fed the same diet but supplemented with 8 g of MA/kg of dry matter. Milk production and composition, including FA profile, were analyzed before (day 0) and after 6, 12, 18, 24, 34, 44, and 54 days of treatment. Diet supplementation with MA did not affect milk yield, but decreased milk fat content. Differences in the latter were detected from day 18 onward and reached -17% at the end of the experiment (i.e., on day 54). Compared with the control diet, MA consumption caused a reduction in milk 18:0 and its desaturation product, cis-9 18:1, that lasted for the whole experimental period. Additionally, inclusion of MA in the diet enhanced the milk content of trans FA, particularly trans-10 18:1 (which was related to the persistency of MFD), and trans-11 18:1, cis-9, trans-11 18:2, trans-10, cis-12 18:2, trans-9, cis-11 18:2, and C20-22 n-3 polyunsaturated FA, mainly 22:6 n- 3. Overall, the persistency of the responses did not allow to accept the original hypothesis of the ruminal microbiota adaptation to the dietary supply of very longchain n-3 polyunsaturated FA. Even though nutrition represents the main factor affecting milk fat yield, there is still very scant available information on the potential relationship between diet and regulation of genes involved in lipid metabolism in dairy ewes. This relationship has been studied in the bovine during MFD, but is still unknown in the ovine, especially when the MFD has been induced by marine lipid consumption. Therefore, using the animals involved in the second trial, a study was conducted to investigate changes in the mRNA abundance of candidate genes involved in lipid metabolism in the mammary gland, the subcutaneous adipose tissue and the liver in response to long-term (8 weeks) MFD induced by MA. To this end, 11 animals, 5 from the control and 6 from the MA treatments, were euthanized at the end of the study (day 54), and samples of mammary gland, adipose tissue, and liver were harvested and analyzed by quantitative reverse transcription-PCR. There was no effect of MA on mammary and adipose tissue expression of genes encoding proteins required for FA uptake and activation (ACSS2, LPL), intracellular FA transport (FABP3, FABP4), de novo FA synthesis (ACACA, FASN), esterification (DGAT1, DGAT2, LPIN1), desaturation (SCD), elongation (ELOVL6), lipid droplet formation (ADFP, BTN1A1, XDH), and transcriptional regulation (INSIG1, MED1, PPARG, RXRA, SCAP, SREBF1, THRSP). In the hepatic tissue, addition of MA did not affect the expression of ß-oxidation- and lipoprotein-related genes (ACOX1, APOB, CPT1A, PPARA, RXRA), but it up-regulated hepatic HMGCS2, which controls ketogenesis. The concentration of plasma ß-hydroxybutyrate, NEFA, glucose, triacylglycerol, growth hormone, insulin-like growth factor 1, insulin, and leptin was not different between groups at d 54. However, it cannot be discarded that transcriptional changes were established during earlier stages of the feeding treatment, similarly to what occurred in the milk FA profile (Trial II), and it was more complicated to detect them in the long-term. Overall, the results show the complexity of the key factors involved in the development of MFD and highlight the need for further research in dairy ewes, because the topic is still full of uncertainties.