Low birth weight has profound implications for perinatal mortality and morbidity in lambs, causing higher mortality and lower growth potential. Low birth weight, as a consequence of fetal growth restriction, occurs in undernourished and multiple pregnancies, where hypoxia and oxidative stress could play a critical role. Our aim was to establish the effects of nutritional deprivation and pregnancy rank on fetal growth, oxygenation, and oxidative status in sheep pregnancies under extensive Patagonian conditions. At 30 days after mating, single- and twin-bearing ewes were offered only natural pasture (undernutrition group) or natural pasture plus concentrate supplementation (well-nourished group). At day 140 of gestation, blood gases and redox status were evaluated in venous cord blood, and fetal biometric characteristics were obtained after cesarean section. Both maternal undernutrition and twinning led to decreased oxygen supply to the fetuses (p = 0.016 and p = 0.050, respectively), which was associated with decreased intrauterine growth (r = 0.446, p < 0.01). Moreover, twinning increased oxidative stress in cord blood (p < 0.05), which might also contribute to fetal growth restriction. These results reinforce the importance of maternal nutrition, especially for those ewes bearing multiples, and opens new possibilities for nutritional or antioxidant interventions for preventing fetal hypoxia and oxidative stress.

Fetal hypoxia, resulting in oxidative stress in pregnancies contributes to reduced fetal growth. Melatonin, a potent antioxidant has been associated with improved oxidative status. Maternal oral melatonin supplementation in sheep from day 50 of gestation ameliorates the consequences of fetal growth restriction in sheep. In rats, melatonin supplementation increases fetal weight via improved placental efficiency and reduction of oxidative stress. The objective of this study was to evaluate whether melatonin supplementation of single (S)- or twin-bearing (T) ewes using either 0 (0MEL), 1 (18 mg MEL) or 2 (36 mg MEL) slow release 18-mg melatonin implants (Regulin®) from 100 to 140 days of pregnancy (n=8 per group) influenced fetal oxygen supply and fetal weight. Fetal umbilical vein blood samples were collected at P140 and partial pressure of oxygen (PO₂) and hemoglobin saturation by oxygen (SatHb) measured. The placenta from each fetus was excised and placentomes individually weighed and typed (A-D). Pregnancy rank, sex of the fetus, number of implants and their interaction on fetal weight, blood gases and placentome weight were analyzed using ANOVA. A 22% and 14% increase (P < 0.05) in body weight was obtained in 36 and 18 mg MEL compared to 0 MEL twin male fetuses, respectively but no treatment effects were observed in singletons or females from twin pregnancies. Fetuses from ewes receiving 36 mg MEL had an 18–20% increase in cord PO₂ (P < 0.05) compared to 18 mg MEL and 0MEL fetuses, which in turn did not differ. Fetal weight was positively correlated with PO₂ (r = 0.37; P = 0.02), SatHb (r = 0.26; P = 0.03) and O₂ content (r = 0.236; P = 0.048). No treatment effect on placentome average weight, total placentome weight per fetus or per ewe nor total number of placentomes per fetus was observed. However, placentae from 36 mg MEL fetuses had a greater proportion of Type C (P < 0.05) than 0MEL and 18 mg MEL ewes, and tended to have lower proportion of Type A (P = 0.1), and greater proportion of Type D (P = 0.06) placentomes, compared to 0MEL ewes. These results indicate that maternal melatonin implants, independently of sex, improve oxygen supply to the fetus, which could potentially improve lamb vigour at birth. In addition, melatonin can increase fetal weight of twin males, by improving placental adaptation and fetal blood oxygenation.

Twinning and maternal nutritional restriction leads to fetal hypoxia, oxidative stress, and intrauterine growth restriction (IUGR) in near-term sheep pregnancies. Our aim was to determine the effect of oral supplementation of vitamins C and E in pregnant sheep on maternal and umbilical cord blood concentrations of vitamins C and E and the effects on fetal antioxidant status, growth, and placental efficiency. Sixteen single- and sixteen twin-bearing ewes, grazing natural Patagonian prairies, were selected after transrectal ultrasound at day 30 after mating. Half of ewes from each pregnancy rank were supplemented daily with vitamins C and E, administered orally, from 30 to 140 days of gestation, when maternal jugular and fetal venous cord blood samples were obtained during cesarean section. Fetuses were weighed and sexed. Placental weight in each fetus was also obtained. Blood plasma was harvested for measurements of maternal and fetal vitamins concentration and fetal antioxidant capacity. Maternal administration of vitamin C and E was associated with increased fetal cord levels of both vitamins, improved antioxidant status, and enhanced fetal growth in both singleton and twin pregnancies associated with increased placental efficiency. These results highlight the potential of vitamin C and E supplementation to reduce the impact of IUGR in both livestock and humans.

Twin sheep fetuses have reduced skeletal muscle weight near birth relative to singles as a result of restricted muscle hypertrophy. Intracellular free amino acids (FAA) are reported to regulate metabolic pathways which control muscle protein accretion, whereby reduced intracellular content of specific FAA may reduce their activation and therefore, muscle hypertrophy. The aim of this study was to determine whether differences in muscle weight between singleton and twin fetuses, under different maternal conditions is associated with reduced concentration of specific FAA. The FAA content in the semitendinosus muscle (ST) in singleton and twin fetuses (rank) at 140 days of gestation from heavy (H) or light (L) ewes fed ad libitum (A) or maintenance (M) level of nutrition was measured. Muscle weight was reduced in twin fetuses compared to singletons in all groups. Reduced concentrations of leucine, threonine and valine, but higher concentrations of methionine, ornithine, lysine and serine were found in twin fetuses compared to singletons. Maternal size and nutrition interaction with rank resulted in reduced glutamine in twins from HM-ewes (H-ewes under M nutrition) compared to their singleton counterparts. Maternal weight interaction with pregnancy rank reduced the concentration of arginine in twins, with a larger effect on H-ewes compared with L-ewes. Maternal size interaction with pregnancy rank resulted in twins from M-ewes to have lower alanine, while twins from A-ewes had lower aspartic acid concentration compared to singletons. The ST muscle weight was positively correlated only with arginine concentration after taking into account rank, size and nutrition. The present results indicate that reduced concentrations of specific intracellular FAA, such as arginine, leucine, valine, glutamine, which are known to play a role in muscle growth, could be acting as limiting factors for muscle hypertrophy in twin fetuses during late gestation. Ewe size and nutrition can influence the concentration of specific FAA in muscle and should be considered in any intervention plan to improve twin fetal muscle growth.

The effects of a combined supplementation with herbal antioxidants during pregnancy on reproductive traits and piglet performance (number of live, dead, and mummified newborns and litter weight at birth and individual body weight at both birth and weaning) were assessed in a total of 1027 sows (504 treated and 523 control females) kept under commercial breeding conditions. The supplementation increased the number of live-born piglets (13.64 ± 0.11 vs. 12.96 ± 0.13 in the controls; p = 0.001) and the total litter weight, decreasing the incidence of low-weight piglets without affecting the number of stillbirths and mummified newborns. Such an effect was modulated by the number of parity and the supplementation, with supplementation increasing significantly the number of living newborns in the first, second, sixth, and seventh parities (0.87, 1.10, 1.49, and 2.51 additional piglets, respectively; p < 0.05). The evaluation of plasma vitamin concentration and biomarkers of oxidative stress (total antioxidant capacity, TAC, and malondialdehyde concentration, MDA) performed in a subset of farrowing sows and their lighter and heavier piglets showed that plasma levels of both vitamins were significantly higher in the piglets than in their mothers (p < 0.05 for vitamin C and p < 0.005 for vitamin E), with antioxidant supplementation increasing significantly such concentrations. Concomitantly, there were no differences in maternal TAC but significantly higher values in piglets from supplemented sows (p < 0.05). On the other hand, supplementation decreased plasma MDA levels both in the sows and their piglets (p < 0.05). Finally, the piglets from supplemented mothers showed a trend for a higher weaning weight (p = 0.066) and, specifically, piglets with birth weights above 1 kg showed a 7.4% higher weaning weight (p = 0.024). Hence, the results of the present study, with high robustness and translational value by offering data from more than 1000 pregnancies under standard breeding conditions, supports that maternal supplementation with herbal antioxidants during pregnancy significantly improves reproductive efficiency, litter traits, and piglet performance.

Twin-bearing pregnancies of sheep reared in harsh environmental conditions result in maternal undernutrition and feto-maternal oxidative stress, leading to intrauterine growth restriction (IUGR). We assessed the efficiency of supplementation with antioxidant herbal vitamins C and E alone or in combination with concentrate throughout gestation on pregnancy outcomes, pre-weaning growth, and survival of twin lambs from grazing ewes at the Magellan Steppe. Four groups (n = 30 each) of twin-bearing ewes received a base natural prairie (P) diet, supplemented with either herbal vitamins C 500 mg and E 350 IU per day (V) or concentrated food (S); groups were: P, P + V, P + S, and P + VS. Vitamins and concentrate were supplemented until parturition. At birth, lambs were weighed, and blood was drawn for total antioxidant capacity (TAC) evaluation. Lamb body weight (BW) and survival rate were evaluated at mid-lactation (60 days) and at weaning (120 days). Vitamin supplementation resulted in increased lamb birth weight and TAC, with a trend towards higher BW at weaning, while nutritional supplementation only had a positive effect on birth weight. Lamb survival was higher in both vitamin supplemented groups. In conclusion, supplementation with herbal vitamins C and E alone or in combination with concentrate food during pregnancy may constitute a good nutritional strategy for sheep reared in harsh environmental conditions.