Effect of maternal supplementation with calcium and magnesium on immune response, energy metabolism and mineral profile of Merino ewes and their lambs

Ewes have high energy and mineral requirements to provide essential nutrients for foetal development through the placenta during gestation and to neonates through milk at lactation. The high nutritional demands of ewes at late gestation and early lactation and often also low content of energy and minerals in pasture result in severe metabolic stress on ewes which consequently affect the general health of ewe, fetal growth and later neonatal life. Clinical deficiency of calcium (Ca), magnesium (Mg) and energy resulting in metabolic diseases such as hypocalcaemia, hypomagnesemia and pregnancy toxaemia can cause negative impacts on health. In addition subclinical deficiencies have also shown to contribute to displaced abomasum, retained placenta, decreased probability of pregnancy after the first service, increased risk for culling, poor colostrum/milk production, low lamb birthweight and reduced lamb growth rate.Animals require Ca and Mg to support the developing fetus and growing lambs, and also they are critical for a number of physiological processes such as boosting the immune system and improving energy regulation. Therefore, it is hypothesised that supplementing with Ca and Mg during high demand states such as gestation and lactation could lead to an improvement in mineral status of the animal which in turn would have beneficial effects on the immune response in both ewes and their offspring and energy regulation in ewes. Thus, this PhD study aimed to evaluate the effect of Ca and Mg supplementation on immunity response, the mineral profile and energy regulation of ewes at late gestation to early lactation, and also lambs after birth. The first experiment for this PhD project was a pilot study which aimed to assess the effect of maternal supplementation (2 weeks pre-lambing to 4 weeks post-lambing) with Ca and Mg on immune response in lambs and energy regulation in ewes. Forty-eight twin-bearing ewes (Merino ewes -3-5 years old) were allocated between four dietary treatment groups: control (base pellet), Ca group (base pellet + 50 g Limestone/head.day), Mg group (base pellet + 50 g Causmag®/head.day) and Ca + Mg group (base pellet + 25 g Limestone/head.day + 25 g Causmag®/head.day). Samples including blood, urine and milk from ewes at four time points—two weeks before lambing (–2 W), 12 hours after lambing (+12 H), two weeks post lambing (+2 W) and four weeks post lambing (+4 W)—were collected and blood from lambs was collected at +12 H, +2 W and +4 W. Collected samples were analysed for mineral concentration (Ca, Mg, P), immunity responses (oxidative burst of leukocytes, total antioxidant capacity (TAC), reactive oxygen metabolites (ROM), oxidative stress index (OSi) - calculated as a ratio of ROM/TAC, colostrum Immunoglobulin G (IgG) concentration and plasma IgG concentration) and non-esterified fatty acids (NEFA) for energy balance. The results of this study showed that maternal supplementation with Ca improved oxidative burst response in lambs. A significant increase in plasma Mg was noted in ewes supplemented with Mg and supplementation of ewes with Ca, Mg and Ca + Mg decreased plasma NEFA concentration which suggests an improved energy regulation compared with the control group. To address the gaps in the first experiment and to investigate the effect of Ca and Mg further, ewes were supplemented with Ca and Mg for a longer duration (5 weeks prior to lambing to one-month post lambing) in the second study. Twin-bearing ewes (n=44, Merino ewes, 3-5 years old) were allocated to four groups fed with formulated pellet to provide different concentration of Ca and Mg: control (0.33% DM Ca and 0.28% DM Mg), high Ca (0.72% DM Ca and 0.29% DM Mg), high Mg (0.33% DM Ca and 0.48% DM Mg) and high Ca + Mg (0.66% DM Ca and 0.47% DM Mg), offered as part of a whole ration. Blood and urine samples were collected weekly starting at five weeks prior to lambing (–5 W, –4 W, –3 W, –2 W, –1 W), 12 hours post lambing (+12 H) and then at fortnightly intervals (+2 W, +4 W) post lambing. Colostrum/milk samples from ewes and blood samples from lambs were collected at +12 H, +2 W and +4 W. The body condition score of ewes and the live weight gain of lambs were measured at each time point. Collected samples were analysed for mineral concentration, immunity response and energy balance (factors measured same as in previous study). In addition, plasma hormone concentration (parathyroid hormone (PTH), 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and 25-hydroxyvitamin D3 (25 (OH)D3)), and immunity responses for oxidative burst and phagocytosis in ewes were measured as well. There was a significant effect of supplementation on plasma NEFA concentration suggesting an improvement in energy regulation. Hence, peripheral blood mononuclear cell (PMBC) were isolated to assess the effect of Ca and Mg supplementation on genes regulating energy balance (Pyruvate dehydrogenase kinase 4 (PDK4), Carnitine palmitoyltransferase 1a (CPT 1A), Fatty acid desaturate 1 (FADS1), β-hydroxybutyrate dehydrogenase (BDH1), 14α-sterol demethylases (CYP51A)) in the second study. Due to the vital role of Ca and Mg in uterine myometrial contraction, it was hypothesised that this could affect parturition behaviour in ewes. Hence, in the second study the behaviour of the ewes was continuously recorded from three weeks before the expected date of lambing by using CCTV cameras. The analysis of parturition behaviour of each ewe started from first visible contraction until two hours after delivery of the first lamb and the behaviour of the lambs after birth was recorded until two hours of age. The findings of the second study was similar to that of the first study which is Ca and Mg supplementation improved immune status in lambs and Mg supplementation improved plasma Mg concentration in ewes. Maternal supplementation with Ca boosted the immune response in lambs via improving leukocyte function and also plasma IgG concentration in Ca group tended to be higher than the other groups. The mechanism by which Mg supplementation improved immune responses was through enhancing the total antioxidant capacity (TAC) in lambs. Supplementation with minerals improved energy balance (as assessed via plasma NEFA concentration), but genes associated with energy regulation (BDH1, CPT1A, FADS1 and CYP51A) were not significantly different between treatments. The expression of PDK4 tended to be higher in Mg group compared to Ca + Mg group. However, the energy regulatory genes were up-regulated at lambing. One of the important findings of the study was that longer duration of maternal supplementation with Ca and Mg increased live weight gain (204 g/head.day for Ca, 207 g/head.day for Mg and 245 g/head.day for Ca + Mg) of lambs compared to the control group (148 g/head.day). The findings from the behavioural analysis showed that the parturition length (P = 0.057) for the second lamb (16.9 seconds for Ca group, 16.1 seconds for Mg group and 21.9 seconds for Ca+Mg group) tended to be shorter in supplemented groups compared to the control group (49.2 seconds). It was also noted that for all groups the first born lambs of one twin pair (14.1 minutes) received longer grooming (P < 0.001) than the second lambs (8.9 minutes), causing the first born lamb to stand (20.1 minutes) quickly (P < 0.001) compared to the second lamb (22.1 minutes) that received shorter grooming.Both short and long duration of supplementation with Ca and Mg had significant positive impact on both ewe’s health during late gestation and early lactation and on lamb’s health from birth to lamb marking (4 weeks of age). Hence, the third and final experiment for this PhD assessed the effect of Ca and Mg supplementation on the general health of ewes and lambs from lamb marking (4 weeks of age) to weaning (8 weeks age) under field condition. A single 10 ha field sown to barley (Hordeum vulgare cv. Moby) was divided into eight paddocks and grazed by a flock of 104 Merino ewes (13 ewes/plot, 3-5 years old) and their progeny. The Ca and Mg concentration in barley forage was 0.48% DM and 0.17% DM, respectively, and ewes in four paddocks had ad libitum access to a mineral supplement supplying 12 g/ewe.day of calcium carbonate from limestone, 12 g/ewe.day magnesium oxide from Causmag® and 6 g/ewe.day sodium chloride as coarse salt in a ratio of 2:2:1 by weight (as fed). Control groups were not provided any mineral supplement. Blood, milk and urine samples from ewes and blood samples from lambs were collected at 30 days after lambing (Day 0), on 14 days after introduction to the grazing crop (Day 14) and at weaning (Day 28). Live weight of lambs was measured at the same time points. It was found in the third study that maternal supplementation with Ca and Mg significantly increased the growth rate of twin raised lambs at weaning by 31% and improved TAC in lambs.The results of this PhD study support the hypothesis that Ca and Mg supplementation improves the immune status as well as live weight gain in lambs, which significantly has a positive impact on both their production and welfare and could improve lamb survival. It should also be noted that the aim of this study is to evaluate the effect of Ca and Mg when the concentration of these minerals is adequate in the feed on offer. Thus, the Ca and Mg content in all of the above study for all groups were at NRC requirements for the control group and above NRC requirements for the supplemented groups. There was no incidence of metabolic disorders such as hypocalcaemia or pregnancy toxaemia in ewes in these studies. However, Ca and Mg supplementation improved energy regulation, and mineral status, both of which might reduce the risk of metabolic disorders. It is concluded that an important role of Ca and Mg in regulating energy balance and boosting immune system in both ewes and lambs have many implications on reproductive performance of ewes, prevention of peripartum metabolic disorders in ewes, lamb strength, lamb vigour and also lamb production. Thus, it is recommended that mineral supplementation of ewes raising multiple lambs should be considered, even when the mineral content in feed on offer appears adequate. In addition, further studies could be conducted to evaluate the effect of Ca and Mg supplementation on lamb survival and lamb performance. Future studies on analysing the financial benefits of Ca and Mg supplementation over the expense spent for supplying these minerals could provide more insights for farmer to manage pregnant and lactating ewes and their offspring more efficiently.