Effect of Short-Term Supplementation of N-3 PUFA on the Acute Phase Response of Holstein Calves

 To date, there is not any accurate estimation of calf mortality in the world; however, annual pre-weaning calves’ mortality was estimated to be around 7.8, 6.5, 5.5, and 2.6% in the United States, Iran, China, and Sweden, respectively. Raboisson et al. (2013) represented that most neonatal calf mortality happens at age under one month. Hill et al. (2011) reported that nutritional factors could modulate the calf immune system's functions. Studies on non-ruminants confirm that the consumption of polyunsaturated fatty acids (PUFA) relating to the n-3 FA can affect the immune response. In calves’ nutrition, using PUFA in milk or milk replacer (MR) had a pleasant effect on immune responses and antioxidant status. Supplementation n-3 FA, especially EPA and DHA, would increase the proportion of PUFA in the membrane phospholipids, which might change the performance of the immune system. The n-3 PUFA plays a critical role in influencing the immune system through various mechanisms described in detail by Calder (2012). Previous studies showed that adding n-3 PUFA to milk or MR decreases the symptoms of diarrhea and inflammatory diseases caused by viral or bacterial infections. So far, there are not enough reports regarding dietary n-3 PUFA on the APR in neonatal calves. Nevertheless, most research regarding FO supplementation and its anti-inflammatory effects on neonatal calves' health has been done on long-term consumption. As earlier mentioned, most calf mortality occurs at the first 30 days of age; consequently, long-term (more than one month) consumption of FO might not provide clear evidence to evaluate the anti-inflammatory effect of FO on the status of neonatal calves’ health. Therefore, the purpose of this study was the first evaluation of short-term supplementation of n-3 PUFA on the APR of neonatal calves.

Twenty-four bull calves, with a mean age of 34.5  3.7 days, were housed outdoors in individual pens bedded with wheat straw at the dairy farm facilities of Astan Quds Razavi Animal Husbandry and Agriculture Co. (Mashhad, Iran) in February 2019. The criteria for calf selection were, namely, the type of calf delivery (without any difficulty) and no history of disease or diarrhea. To achieve a quantitative similarity between calves, we used age and body weight as further criteria. The experiment's duration was 11 days (a week before LPS challenge and three days after LPS challenge) with an adaptation period (seven days). After the adaptation period, calves were weighed (57.5 ± 4.4 kg) and randomly assigned to 1 of 4 groups (six calves/group). Randomized calves received treatments during the study period according to the group they were already allocated: 1. negative control group (NC), 2. Positive control group (LPS challenge, PC), 3. Tallow 350 mg/kg BW group + LPS (TA), 4. Fish oil 350 mg/kg BW group + LPS (FO). All calves were fed the same diet, 5 L/d of whole milk, and had free access to freshwater during the experiment. The PC, FO, and TA groups were intravenously challenged with 0.5 μg/kg BW ultrapure LPS from E. coli serotype O111:B4 (Sigma–Aldrich: registered; product NO. L2630) on day eight. Treatments FO and TA were mixed with whole milk and were offered two times a day (at 0800 and 1700). FO and TA groups were isocaloric to compare the effect of manipulating fatty acid intake in the same level of energy intake on the APR of neonatal calves. The blood samples were collected at 1, 2, 3, 4, 6, 12, 24, 48, 72 h, post LPS challenge (p.c.) to evaluate inflammatory condition. The clinical signs (RT, RR, and HR) were recorded at 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 18, 24 h p.c. According to Plessers et al.'s (2015) model, the appearance of behavioral phases (respiratory, depression, and recovery phase) was assessed. Data were analyzed as a completely randomized design by using JMP (13.2) software.

The results of this study confirm previous experiments that showed a significant increase of cytokines level by the LPS administration (26, 29). As expected, the IL-6 increased when the TNF-α decreased (Maximal level at 3 and 1 h p.c., respectively). There was no significant difference in cytokines and APPs between PC, FO, and TA, while the FO had the minimum level. The typical sickness behavior of LPS-challenged calves was distinguished as respiratory, depression, and recovery phases according to Plessers et al.'s (2015) model. In this study, there was no significant effect of decreasing n-6/n-3 FA ratio on sickness behavior. Besides, the level of inflammatory cytokines and acute-phase proteins were not affected by experimental groups. These results were in line with McDonnell et al., (2019) reported no FO effect on immune function during the pre-weaning period. Although the level of DHA + EPA requirement for calves has not been well known, studies represented that their highest level in humans is 5 g/d. Stanley et al. (2007) concluded that the n-6/n-3 FA ratio might not be a helpful concept and distracts attention from increasing absolute intakes of long-chain n-3 FA. In this regard, Flaga et al. (2019) represented that DHA-rich algae supplementation in milk replacer could decrease cytokines' mRNA expression. They suggested that 3 g/d DHA might be the maximum level in neonatal calves’ diet with an appropriate effect on the immune system. In the current study, NC, PC, and TA received 2 mg/d, and FO received 3 g/d DHA. It might be worthwhile considering the amount of DHA + EPA when FO is used as an n-3 PUFA source in calves’ diet.

The results showed that decreasing the n6/n3 FA ratio in diets by supplementing FO could not affect acute phase response in calves. Besides, short-term supplementation of FO could not improve calves' immune systems as no differences in cytokines and APP between PC and FO were observed. Although sickness behavior in FO finished sooner than PC, there was no significant difference between them. In this study, increasing MUFA intake could not affect APR in calve. It seems that more studies are needed to evaluate the effect of EPA and DHA on the performance and health status of calves.