Effect of Maternal Betaine Supplementation on Growth, Plane of Nutrition, Blood Biochemical Profile and Antioxidant Status of Progeny Pigs

Reactive oxygen species (ROS), including superoxide, hydrogen peroxide and hydroxyl radical are cytotoxic agents that lead to significant oxidative damage. High levels of ROS during embryonic, fetal and placental development are a feature of pregnancy (Kais et al., 2010). Consequently, an imbalance between antioxidants and ROS production has been reported as a key factor in early fetal loss (Hempstock et al., 2003) and is responsible for affecting female reproductive processes (Agarwal and Allamaneni, 2004). Additionally, oxidative damage causes abnormal intrauterine development of an embryo and may increase the risk of developing metabolic diseases later in life (Taylor and Poston, 2007).

Lin et al. (2012) reported that an improvement in maternal, placental and fetal antioxidant capacities benefit fetal development and growth. Thus, fetal development and growth processes are susceptible to oxidative stress and may continuously affect the offspring, whereas nutritional factors may contribute to adverse pregnancy outcomes and increase the susceptibility of offspring to diseases. Homocysteine, a thiol-containing derivative of methionine metabolism, undergoes auto-oxidation in vivo and generates ROS which causes vascular endothelial damage (Szasz et al., 2007). High maternal homocysteine concentration has been associated with low birth weight in offspring, preeclampsia, preterm birth and intrauterine fetal death (Murphy et al., 2004; Tamura and Picciano, 2006). The dietary supplementation of vitamins B₉, B₁₂, choline and betaine have been reported to reduce homocysteine levels by reducing oxidative stress in sows (Atkinson et al., 2009; Van-Wettere et al., 2013). Betaine is a quaternary ammonium compound (Yancy et al., 1982; Lokhande et al., 2019) and used widely as a feed additive in pig ration because it is a chemically stable and non-toxic substance having osmo-protective and methyl donor characteristics. Maternal nutrition affects metabolic homeostasis in the offspring of most animals (Grove and Smith, 2003) but it is evident from the literature that maternal supplementation of betaine can cause epigenetic changes in offspring (Bestor, 2000). However, it was hypothesized that the progeny antioxidant status in response to maternal betaine supplementation would also depend on the duration of betaine supplementation during pregnancy. Thus, the objective of the present study was to assess the response of progeny to the dietary betaine supplementation of sows in terms of growth, nutritional and antioxidant status.

Source of betaine
 
Betaine was purchased from Indian Trading Bureau Private Limited, Kolkata, West Bengal, India.
 
Experimental animals and housing
 
Eighteen adults healthy crossbred (Landrace × Desi) sows of same age reared under similar conditions were selected from the Piggery Farm, LPM Section, ICAR-IVRI, Izatnagar for the present investigation. This experiment was during November, 2017 and June, 2018. All the sows were ear-tagged and kept in well ventilated and clean pens under standard managemental conditions. The sows were vaccinated and dewormed for both endo- and ectoparasites as per the schedule before the start of the experiment.
 
Experimental design and dietary treatments
 
Immediately after the artificial insemination, the sows were divided into three groups (T₀, T₁ and T₂) with six sows in each as per a completely randomized design (CRD). The basal diet (conventional concentrate mixture) was formulated using crushed maize, wheat bran, deoiled soybean meal, mineral mixture and common salt as per the specifications of NRC (1998) (Table 1). The group T₀ fed basal diet served as control. The basal diet was supplemented with betaine @ 3 g/kg diet and fed to group T₁ during the second half of the gestation period and group T₂ during the whole gestation period. Betaine was purchased from Indian Trading Bureau Private Limited, Kolkata West Bengal, India.
 


Feeding schedule of the sows
 
The sows were offered weighed quantity of a mash feed as a single meal at 09:30 AM to meet the nutrient requirements (restricted feeding) with ad lib fresh and clean drinking water. The mash feed was provided @ 2.0 kg/day/sow up to farrowing, 2.5 kg/day/sow from farrowing to 4 days post-farrowing and 3.5 kg/day/sow thereafter till the weaning of piglets.
 
Measurement and analysis
 
The individual body weights of progeny piglets were recorded at weekly intervals to arrive at mean weekly body weight under each treatment group. The samples of feed offered, residue and faeces were analyzed for proximate principles following the standard procedures (AOAC, 2000) to arrive at dry matter intake (DMI), protein intake, energy intake and nutritive value of feed under respective treatment groups. The blood samples from the progeny piglets of each experimental group were collected before feeding and watering at 90 days (grower phase) and 180 days (finisher phase) of age in commercially available clot activating tube. The tubes were kept in a slanting position for 30-40 minutes for clot formation. The tubes were then centrifuged at 1500 rpm for 10 minutes in a refrigerated centrifuge. The harvested serum was subjected to the analysis of serum glucose total protein, albumin, creatinine, cholesterol and triglyceride using commercial diagnostic kits of Coral Clinical Systems, Tulip Diagnostics, India.

The resulting supernatants were transferred into a clean polypropylene tube with the help of Pasteur pipette and stored under cold conditions (-20°C) until further analysis. The antioxidant status of the piglets assessed by the measurement of SOD and catalase (CAT) levels by using Cayman Diagnostic Biont Assay ELISA Kit which was based on the biotin double antibody sandwich technology.
 
Statistical analysis
 
Data obtained from the study were subjected to analysis of variance. Treatment means were separated by Duncan’s multiple range test (Duncan 1955) and was considered significant at P<0.05. All analysis were performed using statistical package SPSS (20.0).

There was no significant difference among the groups in weekly body weight changes in pigs during the whole experimental period (Fig 1). Similar findings were found by Cai et al., (2014) where maternal betaine supplementation did not affect the body weight of piglets, whereas Liu et al., (2017) showed that maternal methyl donor supplementation during gestation affected average body weight of piglets and was not significantly differed from that in the methyl donor group at birth, but tended to be lower at weaning. The variation in response could be due to level of inclusion (Eklund et al., 2005) and the ratio of lysine: calorie in the basal diet (Matthews et al., 1998).



Average body weight (kg), DMI (g/d) and (/ kgW0.75), CPI (g/d) and (g/ kgW0.75), energy utilization (GE intake, DE intake, ME intake) and nutritive value (%DCP andTDN) were comparable among the groups (Table 2 and 3). Similar findings were observed by Schrama et al., (2003), where they studied the effect of betaine supplementation on energy metabolism in growing pigs. It had earlier been reported that due to its osmolytic properties; betaine can reduce energy expenditure for the ion pump, particularly in the cells of the gastrointestinal tract (Simon, 1999). However, in this experiment, betaine supplementation in maternal diet during gestation showed no influence on energy utilization in piglets during the grower/finisher phase. The discrepancy in the response could be attributed to level of inclusion (Eklund et al., 2005) and dietary lysine: calorie ratio (Matthews et al., 1998). DMI, dry matter intake; CPI, crude protein intake; DCPI, digestible crude protein intake; GE, gross energy; DE, digestible energy; ME, metabolic energy; DCP, digestible crude protein; TDN, total digestible nutrient.



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There was no significant difference in serum concentration of glucose (mg/dl), albumin (g/dl), globulin (g/dl), total protein (g/dl), A/G ratio, creatinine (mg/dl), cholesterol (mg/dl) and triglyceride (mg/dl) among the groups (Table 4). All the values were within normal range reported for pigs. Thus, it is evident that maternal supplementation of betaine during gestation showed no adverse effect on general metabolism of the progeny at later stages of life. This corroborates well with the findings of Cai et al., (2014).

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The serum concentration of SOD (ng/ml) was comparable (p>0.05) among the groups, whereas serum concentration of catalase (ng/ml) was significantly higher (p<0.05) in piglets born to dam exposed to supplementary betaine during gestation (Table 5). Similarly, supplementation of betaine in broiler diets improved the antioxidant status (Jamina et al., 2020). This finding is also in accordance with that of Mou et al., (2018), where they reported that supplementation with methyl donors increased the antioxidant capacity of sows as well as newborn piglets by increasing the activities of catalase and glutathione peroxidase. In another study, supplementation of antioxidant improved the antioxidant status and the ability of pigs to combat stress (Chakraborty et al., 2018). Homocysteine (Hcy) concentrations in pigs are typically several folds higher than in other species, which results in increased levels of ROS. The betaine-homocysteine S-methyltransferase (BHMT) enzyme helps in the conversion of Hcy to Methionine by facilitating the transfer of methyl group from betaine.

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The maternal nutrition might reduce oxidative stress in the placenta and thus affect the redox status of the offspring, which is in agreement with the results of Lin et al., (2012), who reported that diets supplemented with methyl donors appear to decrease the homocysteine concentration in sows by the remethylation or trans-sulfuration pathways and increase maternal and offspring antioxidant capacity. This is in agreement with previous results; the present study showed that prepartum supplementation with betaine improved the antioxidant capacity of piglets.

In conclusion, the findings of the present study showed that dietary betaine supplementation @ 3 g/kg throughout the length of gestation reduced oxidative stress and improved the antioxidant capacity of piglets.

The authors are thankful to Director ICAR-India Veterinary Research Institute, Izatnagar and Head, Animal Nutrition Division ICAR-IVRI for providing necessary facilities and support for the successful completion of the project.