Changes in Productive Performance, Blood Metabolites and Hematological Parameters of Growing Lambs Supplemented with Two Sources of Choline

Choline has important functions in energy metabolism as a lipotropic factor and in DNA synthesis as a donor of methyl groups (Zhu et al., 2014; Roque-Jimenez et al., 2020). Biswas and Giri (2015) described antioxidant capacity and protective properties of choline against some toxic agents, while other studies (Tokes et al., 2015) have indicated that dietary choline phospholipids such as phosphatidylcholine may also have anti-inflammatory effects or to reduce fatty liver condition (Acharya et al., 2020).

Although the choline requirements of sheep are unknown (NRC, 2007), earlier studies have indicated that supplementation with ruminally-protected choline (RPC) improves lamb growth (Bryant et al., 1999). Recent studies have confirmed that weight gain can be improved in lambs (Li et al., 2015) with an estimated dose of 37 mg/kg BW0.75 of bypass choline. However, the use of commercial RPC requires knowing its choline content and the bypass fraction to establish the desired dose. In vitro ruminal incubations indicate that ruminal protection varies from 63 to 98% (Kung et al., 2003). Previous studies with lambs demonstrated that an herbal product could substitute RPC as a choline source (Godinez-Cruz et al., 2015). Information on herbal sources of choline in ruminants is scarce compared with choline chloride rumen-protected forms (Li et al., 2015) for which bypass has been estimated (Kung et al., 2003). Most of the choline in herbal sources is found in the lipid fraction as total conjugates, which prevent rumen degradation (Godinez-Cruz et al., 2015, Khose et al., 2019). The lipid fraction of choline is mainly in the form of phosphatidylcholine (Zeisel et al., 2003). Therefore, the objective of this experiment was to compare choline from an herbal source versus a ruminally-protected source on performance, carcass characteristics, meat quality, hematological and biochemical plasma values in finishing lambs.

All animal management procedures were conducted following the set of regulations and standards that are required by the Mexican government for the use of animals for diverse activities, as well as the guidelines of the Academic Committee, under the State Law on Animal Protection for the State of San Luis Potosi, Mexico (ONSSLP, 2014).
 
Animals
 
Twenty-four male lambs (Rambouillet 23.48 kg±3.17 kg initial BW) were randomly assigned to one of three dietary treatments (n = 8 lambs/treatment): a) control group; b) oral dose of ruminally-protected choline (4 g/d RPC, Balchem Reashure); c) oral dose of plant-based Biocholine (4 g/d Nuproxa Mexico; Indian Herbs). Supplementation of choline from the two sources was top dressed on the feed every day. Lambs were fed in individual pens with a basal diet (Table 1) (NRC, 2007).



The rations were ground (using a 1.25 cm screen) and mixed in a grinder-mixer (Vigusa, Mexico) and provided ad libitum in individual feeders (50 cm bunk space/lamb) at 08:00 and 15:00 h, ensuring 100 g feed refusal daily.
 
Feed samples collection
 
One sample of feed per week was collected, dried in forced-air oven at 60oC until constant weight was reached and stored for analysis. All samples were dried to calculate dry matter (DM) and crude protein (CP) according AOAC (1995) methodology. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) analyses were carried out following Van (Soest et al., 1991) using the filter bag technique with addition of sodium sulfite and heat-stable amylase to determine NDF.
 
Finishing and carcass data collection
 
Daily feed intake was calculated as feed offered minus feed refused, measured daily before the morning feeding. The lambs were weighed at the beginning and at the end of the experiment after an adaptation period to estimate average daily gain. Feed conversion was expressed as the ratio of feed intake to average daily gain.

After the 46-d feeding period, all lambs were slaughtered immediately in a commercial abattoir. After slaughter hot carcass was recorded. After chilling for 24 h at 4^oC, cold carcass weight was obtained.
 
Meat analysis
 
Color was measured 24 h after slaughter in fresh cuts of loin (Longissimus dorsi muscle) samples using a Minolta CM-2006d spectrophotometer (Konica Minolta Holdings, Inc, Osaka, Japan). Luminosity (L*), redness (a*) and yellowness (b*) were recorded (Ripoll et al., 2012). Muscle samples (approximately 5 g) were collected and stored in a freezer (-20^oC) until analysis. To measure meat tenderness a model 1132 Instron Universal Testing Machine (Instron, Canton, MA) with a Warner-Bratzler attachment (Wheeler et al., 1997) was used. Moisture, protein, ether extract and ash contents were analyzed in samples of Longissimus dorsi following AOAC (1995) procedures. 
 
Blood collection and analysis
 
Blood samples were collected from all lambs after fasting (2 h) by jugular vein puncture using tubes without anticoagulant on days 7, 14, 21, 28 and 35 for hematological assessment were measured with an electronic analyzer (ABXMicros 60, Horiba ABX, France). Serum samples from day 35 were used to analyze glucose (glucose oxidase and peroxidase method), total protein (biuret method), triglycerides (glycerol 3-phosphate oxidase-PAP method) and cholesterol (cholesterol ester hydrolase, cholesterol oxidase and peroxidase) using Sigma diagnostic kits and alanine transaminase activity (GPT (ALT) Spinreact México) and aspartate aminotransferase activity (GOT (AST) Spinreact México).
 
Statistical analysis
 
Results were analyzed according to a completely randomized design using initial body weight as a covariate to the performance variables (average daily ganin, dry matter intake, feed conversion hot carcass weight and cold carcass weight). An ANOVA was performed and means were compared with the Tukey test (Steel et al., 1997). Variables measured more than once (hematological values) were analyzed as repeated measures over time (Littell and Henry 1998). Data were analyzed using JMP7 software (Sall et al., 2012).

The plant-based choline improved (P<0.05) daily gain, relative to the control group and RPC group (Table 2). In this experiment, intake was highest (P<0.05) in lambs with Biocholine. An experiment by (Li et al., 2015) indicates that higher doses of bypass choline may have a detrimental effect on gain in lambs; same authors did not detect differences in daily feed intake.

​

Some authors evaluated RCP where they found that intake of growing steers was not affected (Pinotti et al., 2009; Hajilou et al., 2014). But, due to the metabolism changes during lactation, a significant effect of choline on dry matter intake could be observed in dairy cows (Sales et al., 2010).

Some RCP evaluations showed that intake of growing steers was not affected (Pinotti et al., 2009; Hajilou et al., 2014) and Sales et al., (2010) indicates that few studies have found any significant effect of dietary RCP on dry matter intake of dairy cows.

Cold carcass weight was highest (P<0.05) in the lambs that received Biocholine. Shear force of meat was not affected (P<0.05). Meat brightness increased (P<0.05) with the two choline sources. The mineral content in the meat was increased (P<0.05) with both sources of choline, but the protein level was reduced with Biocholine. Shear force of the meat in our study contrasts with what was reported by (Li et al., 2015), who found that shear force was reduced by 0.25% in lambs with RPC. However, (Li et al., 2015), did not observe changes in intramuscular ether extract in lambs fed with different levels of RPC. In the case of color enhancement in lambs fed the herbal product, (Jiao et al., 2019) found that Biocholine changed meat color of supplemented finishing pigs. Moreover, choline itself has antioxidant effects that have been observed in pork L* values from pigs fed supplemented choline (Li et al., 2015), similar that (Kumar et al., 2018) observed on Barandur rambs.

Factors that affect the mineral content in meat have received little attention. The values observed in our control group (1.96%) are like those reported (1.16%) in finishing diets (Vnučec et al., 2016). Methyl donors could have affected protein and lipid partitioning in animal bodies (Schrama et al., 2003), accelerated synthesis of myoglobin and increased fat deposit in longissimus muscle. (Puchala et al., 1995) reported that the amino acid composition in sheep serum was changed by the methyl donor and some changes in amino acid composition in longissimus muscle of pigs were reported by (Yu et al., 2004).

Blood triglycerides (Table 3) were increased by RPC compared with the effect of Biocholine and the control group. Cholesterol was reduced by Biocholine but was not affected by RPC. Cholesterol concentrations were associated with the lipotropic functions of phosphatidylcholine (Cole et al., 2012) because choline increases hepatic lipid transport (Zhu et al., 2014). (Bindel et al., 2000) showed that triglyceride blood concentrations in heifers increased in response to RPC, but only when tallow was included in the diet. In a metabolic experiment conducted by (Bindel et al., 2005) on steers where choline was infused abomasally (4 g/d), triglyceride levels were reduced by 15% (with or without tallow), whereas cholesterol was reduced by 8.8% only in steers fed a diet containing tallow. (Hajilou et al., 2014) used a low-fat diet and reported a reduction in triglycerides with RPC in young Holstein bulls. In growing lambs, (Bryant et al., 1999) reported increased triglyceride concentrations and a tendency toward decreased serum cholesterol concentrations when RPC was included in a diet that included yellow grease.

​

The levels of the liver enzyme AST differed among all treatments (P<0.05), as it was reduced by both sources of choline relative to the control group. The ALT was higher (P<0.05) for Biocholine. Choline has been demonstrated to have liver protective effects (Zhu et al., 2014) and it has been shown to improve the integrity and signaling functions of cell membranes (Fagone and Jackowski, 2009). Supplementation with choline has been shown to reduce some hepatic enzyme levels, indicating improved hepatic function (Rahamani et al., 2014), whereas choline-deficient diets increase liver enzyme activity (Getty and Dilger, 2015) and cause liver damage in several species (Guo et al., 2005).

Erythrocyte counts (Table 4) showed differences on days 14, 28 and 35 of the experiment. Comparing choline from the two sources, on day 14, erythrocyte counts were lower in the Biocholine group, but in the RPC group they were lower on day 28 (P<0.05) and similar on day 35. For lymphocyte counts, on day 14 the lowest (P<0.05) values were found in the RPC group. Regarding erythrocyte counts, non-clinical anemia is a common anomaly observed in ruminant blood profiles, with a hematocrit below 24%, erythrocyte count below 5x106 cells/mL or Hgb concentrations below 8 g/dL (Cole et al., 1997). For leukocytes, no differences (P<0.05) in their total count volume were found between treatments; all values were in the normal range from 4,000 to 12,000 (106 x µL). Monocytes alone showed an increase (P<0.05) in the control group on day 21. There is no biological explanation for these values, although the analysis shows differences. The values for lymphocytes (2,000 to 9,000/mL) were normal in all treatments since the lowest value was 4,150 and the highest was 7,190/mL.

​

No lymphocytosis or lymphopenia was observed (Jones et al., 2007). Due to the function of choline as a component of the platelet activating factor (Prescott et al., 2000; McIntyre et al., 2009), in experimental endotoxemia a positive response in terms of platelet count and a return to the initial WBC were demonstrated following intravenous administration of choline chloride or cytidine-5'-diphosphate choline (Prescot et al., 2000). However, in an experiment investigating choline deficient and sufficient diets, no changes were observed in erythrocytes in piglets (Schrama et al., 2003). The response in these cells may depend on the physical condition of the animal, as studies in dairy cattle indicate that mastitis and morbidity can be reduced with RPC (McIntyre et al., 2009).

Daily gain and cholesterol concentrations had better results with choline supplementation. In turn, the lowest levels of triglycerides were observed in the control group. Both sources of choline modified meat color and increased its mineral content. ALT and AST activity decreased by effect of both source of choline. Hematological parameters were affected by choline supplementation there was a reduction in their counts with choline from both sources in growing lambs.