1. Introduction
Approximately 17 million lambs die annually in Australia [
1] with twin lambs representing 35.3% of total reproductive loss between ovulation and weaning [
2]. This equates to an estimated loss of around
$540 million per annum [
3] and represents a significant economic and welfare concern. Lambs are most vulnerable during the first 72 h of their life, with 43 to 70% of perinatal deaths occurring during this period [
4,
5,
6]. Approximately 50% of deaths arise from complications occurring in utero and/or immediately following birth: stillbirth, dystocia and hypoxic birth injury [
5,
6]. Twin-born lambs are lighter at birth [
7] and often have impaired thermoregulatory ability due to poor accumulation of energy reserves [
8]. Together these factors decrease lamb vigour and consequently delay the standing and sucking responses, making them more likely to die from starvation and/or hypothermia [
9,
10].
Supplementing pregnant ewes with betaine has the potential to improve neonatal twin lamb survival. Betaine is a trimethyl derivative of the amino acid glycine and diffuses across the ovine placenta [
11,
12,
13]. Betaine increases the production of creatine via the donation of methyl groups in a transmethylation reaction [
11,
14,
15,
16]. Creatine is an energy-rich amino acid that facilitates the recycling of adenosine triphosphate (ATP); the main energy carrier molecule involved in tissue development and growth, particularly in the brain and muscle. Therefore, creatine acts as an endogenous neuroprotectant, protecting the neonatal brain from oxygen deprivation experienced during parturition [
17]. In sows, dietary betaine supplemented throughout gestation reduced the levels of plasma homocysteine [
18]; an amino acid that has shown to adversely affect embryonic development and increase prenatal mortality in several species [
19,
20]. Further, supplementary betaine during late gestation increased the number of piglets born alive [
18], as well as piglet energy synthesis and glycogen stores at birth [
21]. Supplementing creatine to pregnant spiny mice reduced the incidence of neonatal hypoxia due to higher ATP levels resulting from increased creatine supply and its neuroprotective capacity [
22,
23,
24].
Based on the literature described, we suggest that maternal betaine supplementation will increase creatine synthesis by the ovine fetus, and increase energy stores available to the newborn lamb. As a result, incidences of intrapartum deaths (stillbirths) and the adverse effects of hypoxic birth injury will be reduced, resulting in improved lamb vigour, thermoregulation, colostrum intake and, ultimately, survival. To the best of our knowledge, studies directly addressing the effects of maternal supplementation with betaine as a means of improving twin lamb survival have not been explored. Therefore, this experiment examined the effects of maternal supplementation with two different doses (2 or 4 g/ewe/day) and administration times (early or late) of dietary betaine during gestation on twin lamb weight, thermoregulatory capacity, time to stand and suck, colostrum intake and survival from birth to weaning.
4. Discussion
This study determined whether maternal supplementation with dietary betaine during pregnancy would improve twin lamb body weight, thermoregulatory capacity, time to stand and suck, colostrum intake and survival from birth to weaning. Supplementing ewes with 4 g/day of betaine during the second half of pregnancy increased twin lamb survival to Day 7 and shortened the time interval from birth to first suck. Whereas, feeding ewes 2 g/day of betaine for the duration of pregnancy increased post-natal growth at weaning but reduced twin lamb survival rates and increased the time to attain behavioural milestones.
Betaine supplementation during the second half of pregnancy improved early post-natal twin lamb survival rates. The current data indicate that cumulative survival for LB lambs was consistently higher to Day 7 post-partum. Betaine can enhance energy production and neuroprotective capacity of neonates by increasing creatine synthesis via the donation of methyl groups in a transmethylation reaction [
11], which in turn, reduces incidences of stillbirths and hypoxic birth injury [
17]. This is supported by van Wettere et al. [
18] and Cai et al. [
21] who demonstrated that betaine-supplemented sows had reduced stillbirth frequency and subsequent live-born piglets had increased energy synthesis at birth. It is, therefore, suggested that the increases in cumulative survival in LB lambs may be the result of increased creatine production. However, since this study did not determine circulatory levels of betaine and creatine in the lambs, future research is required to further elucidate this physiological mechanism.
In contrast, lambs born to ewes supplemented with 2 g/day throughout pregnancy experienced significant increases in losses to Day 7 post-partum. As reviewed by Eklund et al. [
11], there is minimal evidence to support betaine negatively impacting animal performance and welfare. Thus, the observed negative trend for cumulative survival in EB lambs is likely to be due to other physiological interactions. Interestingly, despite insignificance, the duration and difficulty of parturition were higher in EB ewes compared with CTL and LB ewes. In addition, the birthing interval between the first- and second-born EB lamb tended to be longer than CTL and LB lambs. This suggests that complications in utero and/or immediately following birth may have occurred for EB ewes and their second-born lamb. Prolonged or traumatic parturition often leads to poor cerebral blood flow and oxygen delivery to the fetus, a process known as hypoxic-ischaemic encephalopathy (HIE). HIE is characterized by metabolic acidosis that impairs brain function and causes long-term neurodevelopment morbidities and mortality, particularly for the second-born lamb [
33]. Blood lactate levels and degree of meconium staining are traditionally used as biomarkers of fetal distress and hypoxia [
34,
35,
36]. EB lambs did have higher blood lactate levels and meconium staining scores compared with CTL and LB lambs, although the differences were not significant. Further, while the data suggests that the second-born EB lamb was more likely affected by HIE than the first-born EB lamb, no significant differences were observed between birth order for blood lactate levels and meconium staining. Despite this, we cannot dismiss the possibility that EB lambs experienced some symptoms related to HIE, and thus, caused long-term neurodevelopment morbidities that ultimately resulted in the observed mortalities. However, this suggestion is inconsistent with the study by Refshauge et al. [
5] who found that since the majority of these losses did not occur around parturition, they are less likely to be caused by labour difficulties and more likely to be attributed to misadventure and exposure. This conclusion is supported by the findings from several lamb autopsies conducted in the current study, which indicated the cause of death was primarily due to hypothermia. Despite this, although the majority of EB lambs died between 24 h and Day 7 post-partum (approximately 17%), the negative impact of HIE on lamb vigour cannot be discounted. While not investigated in lambs, piglets which experience intrapartum hypoxia and asphyxiation typically possess poor neuro-motor and skeletal muscle activity, are less able to maintain thermogenesis and, as a result, typically ingest insufficient colostrum and are less vital [
34]. Taken collectively, it is plausible that intrapartum hypoxia was the underlying cause of the increased mortalities observed in EB lambs.
It was anticipated that betaine supplementation would improve lamb birth weight (BW) due to betaine’s osmolytic properties which improve animal growth and feed efficiency. More specifically, betaine increases intestinal cell proliferation and survival by stimulating epithelium in the gut wall to enlarge and thus increase surface area for nutrient absorption [
11,
37]. However, neither BW nor subsequent weight to marking were improved. Lamb BW is considered a critical determinant of neonatal survival [
38], with optimum BW for Merino lambs ranging between 3.5 to 6.0 kg [
39,
40]. In the current study, lambs were weighed four hours after birth to avoid disrupting standing and sucking behaviours, and thus, the four-hourly weight is assumed to be indicative of BW. While BW was similar between treatment groups, the lightest and heaviest average weight fell within the range identified as being optimal for survival. As such, it is unlikely that BW in this study would be a predisposing factor for lamb survival. Maintaining adequate ewe energy levels is critical due to the strong correlation between lamb BW and maternal nutrition, particularly during late gestation where fetal growth predominates [
10,
41]. The optimal nutritional management of ewes in this study may have limited the ability of betaine to influence BW, therefore, the potential for betaine to improve BW of lambs born to nutritionally stressed ewes needs to be examined.
Despite this, betaine supplemented to ewes at 2 g/day throughout pregnancy resulted in lambs which were heavier at weaning, with higher ADG noted from 4 h to both marking and weaning. These results are in agreement with Tsiplakou et al. [
42] who reported that lambs born to ewes supplemented with 0.6 g/day of rumen-protected betaine during late gestation were 8.9% heavier at weaning and gained 16.7% more weight from birth to weaning. In addition to improved feed efficiency, betaine can also positively influence osmoregulation by increasing water-binding capacity and retention due to improved movement of water across the epithelium via enhanced intraepithelial ion pumps and/or aquaporins [
43]. It is, therefore, possible that betaine could have improved nutrient digestibility and utilisation, water retention, or a combination of both in the neonatal gut, thus explaining the observed increases in body weight and ADG at weaning. On the other hand, it is also possible that these results may be related to the reduced survival rates for this treatment. Since more EB lambs died during the first 72 h of life, the ewes would have had to only feed one lamb thereby eliminating competition for teat access and thus, increasing colostrum intake of the surviving lamb. By removing the less viable lamb in the twin litter, it is not surprising that the remaining twin grew at a faster rate and were heavier when compared to a ewe with two viable twins in the CTL and LB groups. Therefore, further research is required to establish the mechanisms whereby maternal betaine supplementation improves post-natal offspring growth and performance.
In addition to improved survival rates, lambs born to ewes supplemented with 4 g/day of dietary betaine were quicker to contact and suck the udder compared with those born to ewes from the EB treatment. Attaining these behavioural milestones are critical for neonatal success, as delays in these homeothermic strategies increase the risk of mortality due to absence or insufficient colostrum intake [
9,
40]. This apparent improvement in lamb vigour could be attributed to a betaine-induced increase in creatine, which in turn, could have enhanced ATP levels thus protecting the neonatal brain from intrapartum hypoxic damage. As a result, early post-natal locomotor activity was improved as evidenced by the reduced latency from birth to first suck. However, the behavioural data for EB lambs challenges this conclusion as these animals were slower to stand and suck from birth. Despite this, as aforementioned, the data suggest that this result is less likely to be attributed to the betaine treatment itself and more likely to be related to the negative impacts of intrapartum hypoxia. As a result, this ultimately compromised locomotor activity at birth for EB lambs and thus, would explain the observed behavioural retardations.
While not significant, other factors that may have positively influenced the aforementioned improvement in lamb vigour for LB lambs include BW and thermoregulatory capacity. It is well documented that lamb BW and thermoregulatory ability are inextricably linked, as lighter lambs typically elicit lower rectal temperatures leading to poor vigour and survival [
8]. To increase heat production following birth, newborn lambs stimulate non-shivering thermogenesis via brown adipose tissue (BAT) [
44]. In large newborn lambs, BAT typically constitutes 2.0–4.5% of body weight; however, smaller lambs have disproportionally lower levels of BAT which impairs their ability to generate heat immediately [
40]. Although BW was not significantly different between treatments, LB lambs were heavier than CTL and EB lambs. This suggests that LB lambs may have greater BAT for thermoregulation, which is somewhat supported by their rectal temperatures falling within a normal range of 38.3 to 39.9 °C [
45,
46]. Despite this, there were no significant differences observed in rectal temperature between treatment groups. Taken together, this may have consequently led to improved lamb vigour and thus, could explain why these animals were quicker to suck.
Although betaine supplementation did not significantly improve colostrum IgG and lamb serum IgG concentrations, no adverse effects were noted. Since the observed values for lamb serum IgG concentration at 24 h were higher than the normal levels of around 20 mg/mL [
47], it is concluded that lambs received adequate colostrum. However, given that the IgG levels for 0 h colostrum were well above the normal value of 50 mg/mL for ruminants [
48] potentially due to carefully managed ewe nutrition; and that the lambs were individually housed with their mothers and had close and constant access to the teats for sucking, this was not surprising. Despite this, there was no significant correlation between colostrum IgG and lamb serum IgG. Interestingly, both betaine treatments resulted in numerically higher (but not significantly) IgG levels in colostrum than CTL ewes. Our results are consistent with the findings of Tsiplakou et al. [
42] and Wang et al. [
49] who also reported that dietary betaine supplemented to sheep and cattle significantly improved colostrum quality parameters (i.e., milk yield, fat and protein composition) as well as neonatal immunity attributed to increased total protein and globulin concentrations. Therefore, future research is required to further elucidate this biological mechanism in sheep.
While the current data demonstrated that maternal betaine supplementation during late gestation improved various aspects of neonatal twin lamb survival, the impact of indoor housing on these improvements cannot be discounted. In this study, the environmental conditions at lambing were benign due to minimal exposure to extreme weather, predators and infectious agents. Taken together, these factors provided favourable conditions for lamb survival. This is consistent with the findings of Pollard [
50] and Hatcher et al. [
51] who demonstrated that twin lamb survival rates increased by 13 to 37% within a sheltered environment. However, despite being indoors, significantly fewer LB lambs (0%) died in the first three days post-partum compared with approximately 14 and 20% of CTL and EB lambs, respectively. It could, therefore, be suggested that the beneficial effects of dietary betaine supplemented at 4 g/day may be more profound when ewes lamb in more challenging conditions. As such, future studies examining the effects of maternal betaine supplementation during late gestation, in conjunction with lamb survival parameters, under outdoor extensive conditions are critical. In addition, the experimental design of the current study did not allow an analysis of the effects of varying betaine dose. Future studies including a 2 × 2 factorial design comparing the effect of 2 and 4 g/day of dietary betaine in late, and possibly early, gestation is required to address this limitation. As aforementioned, supplementing 4 g/day of dietary betaine during early pregnancy has demonstrated negative effects on the physiological state of sheep during summer [
13], and was the reason why this dose was not fed during early gestation. Therefore, determining the effect of betaine administration at 2 and 4 g/day from early pregnancy, in ewes joined for an autumn lambing period instead of spring, and examining the effects of 2 g/day in late pregnancy, need to be considered in future trials.