The effects of improving low dietary protein utilization on the proteome of lamb tissues

Highlights

• Differences in muscle glycolysis derive mostly from increased glucose availability in lambs fed with lower dietary protein

• Lower levels of dietary protein affect the metabolism of sulphur amino acids and increase Fe abundance in the hepatic tissue

• Lower dietary protein, particularly with condensed tannin treatment, have higher adipocyte turn-over rate in comparison to control

Abstract

Cistus ladanifer L. is a common shrub endemic to the Mediterranean region with high levels of condensed tannins (CT). CT form complexes with dietary protein resisting microbial degradation in the rumen, which enhances dietary protein utilization in ruminant diets. The objective of this study was to evaluate the utilization of CT in the diet of lambs on the proteomes of muscle, hepatic and adipose tissues.

Twenty-four Merino Branco ram lambs were randomly allocated to three treatments (n = 8): C – control (160 g crude protein (CP)) per kg DM, RP – reduced protein (120 g CP/kg DM); and RPCT – reduced protein (120 g CP/kg DM) treated with CT extract. At the end of the trial, lambs were slaughtered and the longissimus lumborum muscle, hepatic and peri-renal adipose tissues sampled. A two-way approach was used for proteomic analysis: 2D-DIGE and nanoLC-MS.

In the muscle, C lambs had lower abundance proteins that partake in the glycolysis pathway than the lambs of other treatments. Control lambs had lower abundance of Fe-carrying proteins in the hepatic tissue than RP and RPCT lambs. The latter lambs had highest abundance of hepatic flavin reductase. In the adipose tissue, C lambs had lowest abundance of fatty-acid synthase.

Significance

soybean meal is an expensive feedstuff in which intensive animal production systems heavily rely on. It is a source of protein extensively degraded in the rumen, leading to efficiency losses on dietary protein utilization during digestion. Protection of dietary protein from extensive ruminal degradation throughout the use of plants or extracts rich in CT allow an increase in the digestive utilization of feed proteins. In addition to enhance the protein digestive utilization, dietary CT may induce other beneficial effects in ruminants such as the improvement of the antioxidant status.

Introduction

Cistus ladanifer L.(rockrose) is a shrub commonly found in the SW Iberian Peninsula [1]. It is a plant with high content of condensed tannins (CT). These phenolic compounds are part of the plants natural defences against herbivores. They have anti-nutritional properties, reducing the ingestion and digestibility of the protein and carbohydrate fractions of the feed, which are particularly serious in monogastric animals. They also have interesting antioxidant and antimicrobial effects [2]. It was additionally found that dietary CT reduce ruminal methanogenesis by directly inhibiting archaeal and protozoal methanogenesis [3].

In ruminants, CT provide an interesting approach to manipulate animal digestion and metabolism. Dietary CT are able to modulate ruminal biohydrogenation, promoting the increase of healthy fatty acids, such as cis9–trans11 conjugated linoleic acid (CLA), in ruminant fat [2,4]. Indeed, authors have reported that quebracho tannins, when present in a forage-based diet, increase stearoyl-CoA-desaturase activity in the longissimus dorsi muscle of lambs. This enzyme is responsible for the synthesis of mono unsaturated fatty acids and cis9–trans11 CLA [5]. Moreover, dietary CT enhanced hepatic antioxidative status in sheep fed quebracho tannins [6]. The mechanism by which CT enhance the antioxidative status of tissues is not fully characterized, although it is thought to be indirect since these are not absorbed in the intestine [2]. The salivary protein profile of sheep fed with Cistus ladanifer has been reported before [7], but to our knowledge, no studies have been published regarding the effect of dietary CT on the proteome of other sheep tissues or fluids. Of particular interest to the present study, is the complex formation of CT and dietary proteins. They form insoluble complexes with ingested protein in the rumen, at a pH ranging between 5.5 and 7. In the abomasum, when the pH lowers to acidic levels between 2.5 and 3.5, this complex is dissociated and dietary protein becomes available for intestinal digestion and absorption [2,8]. Treating soybean meal with a CT extract from Cistus ladanifer reduced the rumen protein degradability in an invitro experiment carried out by Dentinho et al. [8]. An in vivo experiment with rumen-cannulated rams aiming to confirm these results showed that a concentration of 15 g of CT/kg of dry matter (DM) reduces rumen degradable protein without compromising the dry matter degradability and the digestibility in the whole digestive tract [9].

The complex formation of CT and dietary protein, and consequent dissociation in the abomasum, could be a viable way of reducing dietary protein of lamb diets while achieving comparable results to untreated diets with higher protein content. This would reduce the need of imported protein feedstuffs namely soybean meal whilst using an abundant resource: Cistus ladanifer, overall leading to more sustainable animal feeding and production. Typically, in Portugal as in other Mediterranean countries, lambs are weaned at 1 to 2 months of age and are either slaughtered and consumed as light lambs or are fattened and finished [10]. The latter system uses chiefly concentrate rations. Lambs with around 20 kg require roughly 16–17% Crude Protein (CP) [11]. Hence, it would be interesting to compare if lambs fed CT-treated diets with 12% CP, which is the protein requirement at the end of fattening, could achieve the same results as those fed with untreated diets with 16% CP. In the present experiment, a reduced protein diet treated with Cistus ladanifer CT (RPCT, 120 g CP/kg DM) was formulated to assess if, compared to a control diet (160 g CP/kg DM), a similar lamb growth performance could be achieved. An untreated diet with 120 g CP/kg DM was additionally included in the design in order to study the effect of lower dietary protein. The results of this trial are reported in a companion paper [67]. Briefly, CP intake was higher in the control treatment, but the final live weight, average daily gain and feed conversion ratio was not different from the RPCT treatment. These results are quite promising; however, there are no reported studies, to our knowledge, that explain these differences at the molecular level.

Indeed, the effect of Cistus ladanifer CT on edible tissue proteome is currently unknown. Muscle, hepatic and adipose tissues are edible tissues whose quality is of paramount importance in the industry. When their proteomes are analysed together in proteomic experiments, they provide a wide picture of the metabolic status of the animal under a particular effect, as we have recently demonstrated [12]. The muscle is the edible part of the carcass and therefore of higher economic value. The liver provides interesting information on the major biological processes occurring and the adipose tissue is a site of reserves accumulation that also informs regarding animal performance. Thus, the objective of this study is to assess the effect of dietary CT and different levels of dietary protein on the muscle, hepatic and adipose tissue proteome of lambs fed diets with different levels of dietary protein, using two different proteomic approaches.