Evaluation of the use of Kolin plus™ FC (polyherbal formulation) as a natural replacer of choline chloride in weaned piglet diets: Effects on performance and blood variables

doi:10.21203/rs.3.rs-2967808/v1

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Research Article

Evaluation of the use of Kolin plus™ FC (polyherbal formulation) as a natural replacer of choline chloride in weaned piglet diets: Effects on performance and blood variables

https://doi.org/10.21203/rs.3.rs-2967808/v1

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Background

Choline is a vitamin-like nutrient that plays a crucial role in several biological processes. Choline is typically added to pig diets in the form of choline chloride. However, the use of this synthetic choline has some disadvantages such as high capacity to absorb moisture, oxidative loss of vitamins in feed and its metabolism generates a compound that can taint animal-origin products. Thus, we conducted a study to evaluated the effects of feeding diets containing natural choline (Kolin plus™ FC) as a replacement of choline chloride on performance and blood variables of weaned piglets.

Results

The use of Kolin plus™ FC tended (p = 0.09) to improve the feed conversion ratio in the pre-starter II phase. No statistical differences were observed for the remaining performance variables. The mortality did not differ among treatments. There were no differences among treatments for any blood variable evaluated.

Conclusion

The polyherbal formulation Kolin Plus™ FC successfully replaced choline chloride in weaned piglet diets without compromising the growth performance and health of nursery piglets.

choline

polyherbal formulation

nursery piglets

growth performance

health

Weaning is arguably one of the most stressful events in the life of the pig [1]. During this critical phase, piglets are exposed to a host of stressors, including removal from the sow, transportation, interaction with unacquainted pigs, and the transition from sow milk to a less digestible diet [2]. This can result in intestinal and immune system dysfunctions that lead to reduced pig health, feed intake and growth performance [3, 4]. Likewise, piglets at the time of weaning have reduced digestive enzymatic activity [5]. Pancreatic lipases, which are involved in lipid digestion and absorption, are markedly compromised after weaning [6]. Therefore, the digestion and absorption of dietary lipids in weaned piglets declines [7].

In recent years, in order to mitigate the biological stress of weaning, improve nutrient utilization and growth performance, several nutritional strategies have been evaluated [1, 8, 9]. These includes, but are not limited to, dietary inclusion of probiotics [10], phytochemicals [11], dietary amino acids [12], organic and inorganic acids [13], acidifiers, bacteriophages [14] and exogenous enzymes [15]. Micronutrients (i.e., trace minerals and vitamins) are critical factors affecting health, growth and development in pigs [16].

Choline (trimethyl-beta-hydroxyethylammonium) is a water-soluble nutrient which plays a vital role in neurotransmission, membrane integrity and methylation pathways [17, 18], influencing several biological process [19]. Of particular note, choline has a critical function in lipid regulation; it serves as a methyl group donor and a precursor of phosphatidylcholine, which is involved in very low-density lipoprotein (VLDL) assembly in the liver [20]. Further, as an important component of the mitochondrial membrane, choline plays a significant role preventing oxidative damage caused by mitochondrial dysfunction [19].

In poultry, a number of studies have demonstrated that supplemental choline exerted beneficial effects on growth performance. Broilers supplemented with choline had higher feed intake, improved feed conversion ratio and weight gain [21]. Likewise, Selvam et al. [22] reported improved growth performance and feed efficiency in broilers supplemented with choline. In pigs, studies evaluating supplemental choline are more limited. Choline supplementation has been shown to improved growth performance and prevented gut inflammation in weaned piglets by altering gut microbiota and lipid metabolism [7]. Further, dietary choline supplementation improved the birth weight, uniformity of neonatal piglets as well as litter performance during lactation; possibly because of better antioxidant capability and metabolic status [23]. Likewise, dietary choline supplementation improved fat deposition by enhancing the lipogenesis and reducing the lipolysis in intrauterine growth retardation piglets [18]. Choline is typically added to pig diets in the form of choline chloride. However the use of choline chloride has some drawbacks, including high hygroscopicity, acceleration of oxidative loss of vitamins, its corrosive nature is a practical concern in processing units and feed mills, and since less than half of the choline chloride is absorbed, the remaining is converted to trimethylamine (TMA) in the gastro intestinal tract [22, 24]. TMA is a compound that has an unpleasant odor and can taint animal-origin food products [22].

Currently, in search of a more sustainable pig production, there has been great interest in the use of phytogenics [14]. In this context, Kolin Plus™ FC is a polyherbal formulation composed by a combination of Acacia nilotica and Curcuma longa [22].The extract of these plants was shown to have hepatoprotective, antioxidant, and lipotropic properties [25, 26]. Kolin plus™ FC has been demonstrated to successfully replace synthetic choline (choline chloride 60%) in broilers diets [22], which was represented by improved growth performance and feed efficiency. However, few studies in pigs have evaluated the use of natural choline sources in replacement of choline chloride.

Based on the foregoing, the objective of this study was to evaluate the effects of feeding diets containing choline from a polyherbal source (Kolin plus™ FC) a replacement of choline chloride 60% on performance and mortality of weaned piglets.

Locality, animals, housing and diets

The experiment was carried in the nursery unit of the Animalnutri & Auma Experimental Center of Swine (18.5962°S, 46.5150°W, Patos de Minas, Minas Gerais, Brazil). A total of 480 crossbred piglets (DB90 [Large white x Landrace] x LQ 1250 [Duroc]; DB Genética Suína, Patos de Minas, Brazil) with an initial average body weight (BW) of 6.33 ± 0.20 kg and 23 ± 1 days of age. The piglets were allocated in slatted pens equipped with semi-automatic feeders and drinking nipple. The temperature was controlled manually according to the age of the animals by opening or closing the windows. Temperature thermometers were used to indicate when the windows should be closed or opened. The room temperature was recorded daily. The piglets were submitted to the same sanitary management used in the farm. The diets were formulated to meet or exceed the nutritional requirements of each phase, according to the Brazilian Poultry and Swine Tables [27] (Table 1). Piglets had ad libitum access to feed (mash) and water until the end of the experiment.

Table 1

Composition of the experimental diets.
Ingredients (%)	Pre-starter I	Pre-starter II	Starter I	Starter II
Corn 7,88% CP	43.6	48.8	55.7	58.5
Soybean meal 46% CP	15.7	19.7	25.0	32.0.
Spray-dried plasma	5.0	3.0	1.5	-
Dried milk	9.0	6.0	-	-
Dried whey	20.0	12.5	7.5	-
Soybean oil	3.0	2.64	2.84	2.77
Sugar	-	2.5.0	2.5.0	2.5.0
Vitamin premix¹	0.05	0.05	0.05	0.05
Micromineral premix²	0.1	0.1	0.1	0.1
Phytase 10.000FTU	0.01	0.008	0.008	0.008
Novicid 18 CA³	0.5	0.4	0.3	0.25
Adimix Precision⁴	0.2	0.15	0.1	0.1
Antioxidant	0.02	0.02	0.02	0.02
Flavoring	0.05	0.05	-	0.05
Dicalcium phosphate 18.5% P	0.428	0.839	1.093	0.935
Limestone	0.658	1.22	1.143	0.877
Salt	-	0.118	0.30	0.5
Copper sulfate	0.03	0.05	0.05	0.05
Zinc oxide 80%	0.3	0.24	0.18	0.15
L-Lysine-HCL	0.444	0.546	0.59	0.437
DL-methionine	0.239	0.253	0.23	0.174
L-treonine	0.244	0.28	0.29	0.189
L-tryptophan	0.059	0.06	0.059	0.026
L-valine	0.085	0.135	0.156	-
Inert	0.2.	0.2	0.2	0.2
Total	100.	100	100	100
Calculated levels (%)
ME (kcal/kg)	3459.5	3400.0	3375.0	3350.0
Fat	5.30	5.10	5.55	5.62
Crude protein	20.6	19.6	18.8	19.7
SID Lysine	1.52	1.45	1.35	1.25
SID Methionine	0.52	0.52	0.47	0.43
SID Met + Cys	0.85	0.81	0.75	0.71
SID Threonine	1.02	0.97	0.90	0.81
SID Tryptophan	0.29	0.28	0.26	0.24
SID Arginine	1.01	1.01	1.06	1.20
SID Valine	1.05	1.00	0.93	0.81
SID Isoleucine )	0.78	0.73	0.68	0.74
SID Leucine	1.68	1.55	1.43	1.50
SID Histidine	0.52	0.48	0.44	0.47
SID Phenyalanine	0.89	0.84	0.80	0.87
Crude fibre	1.53	1.81	2.18	2.57
Lactose	17.6	11.15	5.25	-
Total Calcium	0.88	1.07	0.97	0.79
Available Phosphorus	0.55	0.53	0.48	0.39
Sodium	0.32	0.25	0.23	0.22
Copper (mg/kg)	98.2	147.3	146.4	146.4
Zinc (mg/kg)	2565.8	2076.8	1588.1	1343.3
Choline (mg/kg)	1230.0	1178.7	1177.8	1238.1
¹The vitamin premix provided the following quantities of vitamins per kg of complete diet: 30.000.000 IU vitamin A, 6.000.000 IU vitamin D3, 180.00 IU vitamin E, 6.700 mg vitamin K3, 5.250 mg, 11.88 g vitamin B2, 47.5 g pantothenic acid, 6.880 vitamin B6, 68.750 mcg vitamin B12, 75 g nicotinic acid, 6.880 mg folic acid, 810 mg biotin. ²The micro mineral premix provided the following quantities of micro minerals per kg of complete diet: 80 g iron, 15 g copper, 41.3 g manganese, 91.2 g zinc, 330 g cobalt, 1.209 mg iodine, 351 mg selenium.
³Acidifier.
⁴Encapsulated sodium butyrate.

Experimental design and treatments

The experimental design was a randomized blocks design, with three treatments and twelve replicates (six male and six female). The pen (10 piglets) was considered the experimental unit. The initial weight was used as a block factor. The experiment lasted 41 days and was divided in four periods: pre-starter I (0 to 7 days of the trial), pre-starter II (7 to 14 days of the trial), starter I (14 to 27 days of the trial) and starter II (27 to 41 days of the trial). The piglets were allotted to one of three treatments as follows: Negative control (NC, n = 120, basal diet without choline chloride); Choline Chloride 60% (CC60%, n = 120, choline chloride supplementation – 600 grams/ton) serving as positive control and Kolin Plus™ FC (n = 120, corresponding to 20% of choline chloride; Indukern, Brazil).

Growth Performance and mortality

The piglets were weighted at day 0, 7, 14, 27 and 41 of the experimental periods. The experimental diets were changed on the same days piglets were weighed, as described above. Piglets were individually weighed at day 0, 14, 27 and 41 of the experimental period to determine the coefficient of variation in body weight per pen. The feed provided and refusals were evaluated daily. Thus, the average daily gain (ADG), the average daily feed intake (ADFI) and feed conversion ratio (FCR) were calculated weekly in the aforementioned experimental periods. When a piglet died in the pen, the dead piglet was weighted as well as the litter mates, and the feed provided to the pen. The medication rate was calculated as the percentage of individual piglets treated with injectable antibiotics. Mortality was calculated as the percentage of piglets that died in each dietary treatment during the experiment. Apathy was diagnosed by direct observation. For this study, piglets were considered to have signs or apathy when they were in lateral or sternal recumbency for long periods of time, were not observed consuming feed and had no fever.

Blood collection and analysis

At the end of the experiment, blood samples were collected from all pigs via venipuncture. Blood was allowed to clot at room temperature and was stored at -20 until analysis. The serum levels of cholesterol and total triglycerides were determined using the enzymatic Trinder method. The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined by the U.V. Kinetic method. All samples were analyzed in the AU480 Chemistry Analyzer equipment (Beckman Coulter, Brea, USA).

Statistical Analysis

All residues were tested for normality and homogeneity using the Shapiro-Wilk test. When variables did not follow a normal distribution the PROC RANK (SAS INSTITUTE INC, 2009) procedure was used to produce a transformed variable. The data was analyzed as a randomized complete block design using the PROC GLIMMIX procedure in SAS 9.3 (SAS Inst. Inc., Cary, NC). The pen was used as the experimental unit. Dietary treatments were considered as fixed effects and blocks as random effects. All data were described as LSMEANS. The means were compared by Tukey test. Differences were considered significant if p < 0.05, with a trend toward significance at 0.05 ≤ p ≤ 0.10. The medication and mortality rates were analyzed using the chi-square test by the GMOD procedure of SAS.

Growth performance

The results of growth performance were displayed in the Tables 2 and 3. The use of Kolin plus™ FC only tended to improve (p = 0.09) FCR in the pre-starter II phase. In the starter II phase, the Kolin plus™ FC group had numerically better (p = 0.45) FCR compared to Choline Chloride 60%. In the pre-starter II and starter II phases the Kolin plus™ FC treatment had numerically greater (p = 0.12 and p = 0.35, respectively) ADG values.

Table 2

Effect of the experimental diets on piglet performance in the pre-starter II phase (7 to 14 days of the trial)
Variables	Treatments			SEM	P
Variables	NC (n = 120)	CC60% (n = 120)	Kolin plus™ FC (n = 120)	SEM	P
Initial BW, kg	6.33	6.33	6.33	0.26	0.72
BW at 7 days, kg	7.69	7.77	7.72	0.31	0.52
ADFI, kg	0.31	0.32	0.32	0.01	0.23
ADG, kg	0.19	0.20	0.19	0.01	0.53
FCR	1.59	1.58	1.64	0.05	0.45
BW at 14 days, kg	10.0	10.0	10.1	0.39	0.42
ADFI, kg	0.54	0.55	0.55	0.01	0.75
ADG, kg	0.33	0.32	0.35	0.01	0.12
FCR	1.65	1.70	1.58	0.03	0.09

NC: negative control; CC60%: choline chloride 60%: BW: bodyweight; ADG: average daily gain; ADFI: average daily feed intake; FCR: feed conversion ratio; SEM: standard error of the mean.

Table 3

Effect of the experimental diets on piglet performance in the starter II phase (27 to 41 days of the trial)
Variables	Treatments			SEM	P
Variables	NC (n = 120)	CC60% (n = 120)	Kolin plus™ FC (n = 120)	SEM	P
BW at 27 days, kg	16.6	16.5	16.7	0.59	0.43
ADFI, kg	0.77	0.75	0.78	0.02	0.26
ADG, kg	0.50	0.50	0.50	0.01	0.76
FCR	1.52	1.51	1.53	0.02	0.71
BW at 41 days, kg	26.3	25.9	26.7	0.71	0.13
ADFI, kg	1.14	1.11	1.14	0.04	0.38
ADG, kg	0.69	0.67	0.70	0.01	0.35
FCR	1.64	1.66	1.61	0.02	0.58
0 to 41 days
ADFI, kg	0.78	0.77	0.78	0.02	0.30
ADG, kg	0.48	0.47	0.49	0.01	0.19
FCR	1.60	1.60	1.58	0.02	0.70

NC: negative control; CC60%: choline chloride 60%: BW: bodyweight; ADG: average daily gain; ADFI: average daily feed intake; FCR: feed conversion ratio; SEM: standard error of the mean

Medication and mortality incidences

The incidence of medication is described in Table 4. In the pre-starter I phase, no piglets were medicated. In the pre-starter II phase, one piglet from the Kolin plus™ FC treatment were medicated for arthritis. In the starter I phase, a total of nine piglets were medicated. Of these, one piglet from the negative control treatment (due to apathy); four from the choline chloride 60% treatment (two for diarrhea, one due to apathy and one for arthritis) and four piglets from the Kolin plus™ FC treatment (two for diarrhea, one for encephalitis and one due to apathy). Regarding mortality, there were no deaths in the pre-starter II and starter II phases. In the pre-starter I phase, one animal from the Kolin plus™ FC treatment died, showing respiratory symptoms. In the starter I phase, one animal from the Kolin plus™ FC treatment died, with arthritis being the only alteration observed. In the same period, two piglets from the Kolin plus™ FC treatment were removed from the experiment. These piglets had already been medicated, notwithstanding, none of them showed signs of improvement.

Table 4

Effect of the experimental diets on the incidence of medication in each experimental period
Period	Treatments					Total
Period	NC (n = 120)	CC60% (n = 120)			Kolin plus™ FC (n = 120)	Total
Pre-starter I	0		0		0	0
Pre-starter II	0		0		1	4
Starter I	1		4		4	13
Starter II	0		1		0	1
Total	1			5	5	18

NC: Negative control; CC60%; Choline Chloride 60%

Blood Analysis

The results of blood analysis are described in the Table 5. No statistical difference was observed for any blood parameter evaluated. The values of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were within normal range for piglets from all treatment groups.

Table 5

Effects of the experimental diets on serum metabolites (Mean).
Variables	Treatments			SEM	P
Variables	NC (n = 120)	CC60% (n = 120)	Kolin plus™ FC (n = 120)	SEM	P
Cholesterol (mg/dl)	70.3	66.0	69.5	2.44	0.34
Triglycerides (mgldl)	33.3	32.3	34.1	1.89	0.87
AST (U/L)	39.3	39.7	37.2	2.13	0.84
ALT (U/L)	39.3	40.8	41.8	1.74	0.76

NC: Negative control; CC60% = Choline Chloride 60%; SEM: standard error of the mean; AST: aspartate aminotrans-ferase; ALT: alanine aminotransferase.

Whilst no statistical differences were observed for most of the performance variables evaluated, the most important outcome of the present study is the fact that the polyherbal formulation Kollin plus™ FC successfully replaced choline chloride in weaned piglet diets without compromising the growth performance and health of nursery piglets. Similarly, Selvam et al. [22] reported that the use this polyherbal formulation replace the function of 1 kg/ton of synthetic choline (choline chloride 60%) at 400 g/ton inclusion rate in broiler diets. Moreover, in their study, the replacement of synthetic choline by the natural choline resulted in improved feed efficiency and growth performance. In the present study, however, the use of choline from a polyherbal source only tended to affect FCR in the pre-starter II phase. For the remaining performance variables only numerical difference was observed among treatments, including the negative control group (i.e., diet without the inclusion of choline). This is not in agreement with Qiu et al. [7], who reported that choline supplementation, in the form of choline chloride, significantly improved the growth performance of weaned piglets and resulted in a significantly higher ADG, FCR and greater BW gain compared to experimental diets without the inclusion of choline. Of particular interest, it has been reported that choline supplementation, may not affect the performance of pigs when the content of methionine and vitamin B in the diet is adequate [28], which is the case of the present study. Further research is warranted to investigate the effects of supplemental choline in methionine and vitamin deficient diets on growth performance of weaned piglets.

The incidence of medication and mortality rates observed in the present experiment demonstrates that the use of choline from a polyherbal source had no deleterious effect on piglet health. Further, only two piglets from the Kolin plus™ FC treatment died during the experimental period, one had respiratory symptoms and the other one had arthritis. Aside these alterations, none of the piglets that died had other signs indicating metabolic alterations or intoxication. Only one piglet from the Kolin Plus™ FC treatment was medicated due to apathy. The absence of deleterious effects is corroborated by the results of the blood analysis. Indeed, there was no difference in the levels of AST and ALT among treatments, indicating normal liver function. The absence of deleterious effects of Kolin Plus™ FC is not surprising given the fact that it is a polyherbal composed by A. nilotica and C. longa, a rich source of polyphenols and curcuminoids, which have proven hepatoprotective effects [26]. Further, it has been demonstrated that the supplementation of C. longa extracts had a beneficial impact on transmethylation pathways and osmotic regulation by increasing the liver betaine content, which plays a role in liver lipid metabolism [29].

Choline plays a crucial role in lipid metabolism. Research efforts have demonstrated that choline supplementation improves lipid metabolism by decreasing circulating levels of free fatty acids and triglycerides [7, 30]. In this study, supplementing weaned-piglet diets with choline, regardless of source (i.e., natural or synthetic) had no effect on cholesterol and triglycerides levels. Interestingly, cholesterol and triglycerides levels were similar between piglets from the negative control group and the choline-supplemented groups. According to Li et al. [30], the choline oxidase activity in pig livers is relatively lower when compared to broilers chickens, therefore choline or its metabolites might take more time to show significant effects in pigs. This might be a possible explanation for the absence of effects on lipid metabolism observed in the present study. However, it should be noted that similarly to AST and ALT levels, the cholesterol and triglycerides levels of piglets from all treatment groups were within normal range.

A reduced digestive enzymatic activity of young piglets is important factor that contributes to the occurrence of diarrhea in the nursery phase [7]. For instance, lipase activity is dramatic reduced after weaning [6]. Choline has been shown to efficiently emulsify dietary fat, accelerating lipid metabolism in the small intestine [31]. It should be pointed out that piglets were kept under good sanitary conditions and no challenge was impose. The low incidence of medication and mortality observed in this study corroborates this assumption. Future trials should evaluate the use of natural choline in diets of challenged-piglets.

The supplementation of Kolin Plus™ FC in replacement of choline chloride 60% proved to be feasible and safe, with no statistical difference between the two treatments for the majority of the performance variables evaluated. Additionally, the supplementation of nursery piglet diets with Kolin Plus™ FC in replacement of choline chloride 60% tended to improve FCR in the pre-starter II phase. Further, the serum determination of AST and ALT demonstrated that no liver injury occurred in piglets supplemented with Kollin Plus™ FC. Although Choline is involved in lipid metabolism, no effect of choline, regardless of source was observed for serum cholesterol and triglycerides. Further studies are needed to evaluate the use of Kolin Plus™ FC in replacement of synthetic choline in nursery piglet diets during a more extended period of time and under challenging conditions.

Acknowledgements

The authors thank National Council for Scientific and Technological Development (CNPq) for the fellowships: 303750/2021-9 and 312659/2019-9.

Authors’ contributions

Conceptualization, A.V.B., A.T., S.L.F., R.C., V.C. and C.G..; methodology, A.V.B., A.T., S.L.F., R.C., R.C., V.C., and C.G..; formal analysis, R.C.; investigation, R.C., D.L., Y.P.; resources, R.C. and V.C.; data curation, R.C. and Y.P.; writing-original draft preparation, R.C., D.L., and Y.P.; writing-review and editing, A.V.B., A.T., S.L.F., R.C., V.C., and C.G.; supervision, R.C., V.C., and C.G.; project administration, R.C.; funding acquisition, R.C. and V.C. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by Natural Remedies Private Limited, Bangalore, India.

Availability of data and materials

The data sets and materials are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

The animal study was approved by Animalnutri animal care and use committee (CEUA protocol number: 008/22).

Consent for publication

Not applicable

Competing interests

The authors A.V.B., A.T., S.L.F. are affiliated with the company that distributes the technology, and it may influence the way in which the technology is presented and discussed in this publication. However, we want to assure our readers that we have taken measures to minimize any potential biases. We have followed rigorous scientific methods and have reported our findings in an objective and transparent manner. We have also enlisted the help of independent reviewers to assess the quality and validity of our work.

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Competing interest reported. The authors A.V.B., A.T., S.L.F. are affiliated with the company that distributes the technology, and it may influence the way in which the technology is presented and discussed in this publication. However, we want to assure our readers that we have taken measures to minimize any potential biases. We have followed rigorous scientific methods and have reported our findings in an objective and transparent manner. We have also enlisted the help of independent reviewers to assess the quality and validity of our work.

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Evaluation of the use of Kolin plus™ FC (polyherbal formulation) as a natural replacer of choline chloride in weaned piglet diets: Effects on performance and blood variables

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