Effect of melatonin treatment of pregnant Sarda ewes on lactation and lamb development

Introduction

Melatonin, the main pineal hormone, is produced during the hours of darkness and nocturnal production levels vary according to the season, following the length of the night (Bittman et al. 1983; Carcangiu et al. 2013). Characterized by its characteristic circadian and circannual secretion rhythm, melatonin can be considered the organic informer of the changing seasons, preparing the body for modifications in environmental factors (Reiter 1980). The study of melatonin’s effects has long focused on regulating reproductive seasonality, and its administration in small ruminants has allowed for improved reproductive efficiency (Staples et al. 1992; Abecia et al. 2011). Today, slow-release melatonin subcutaneous implants are used all over the world to control the reproductive seasonality of small ruminants (Carcangiu et al. 2012; Luridiana et al. 2016)

Melatonin is produced in many bodily tissues and regulates various physiological processes, making it a molecule with pleiotropic activity (Reiter et al. 2010; Hardeland et al. 2011). Melatonin synthesis and its receptors have been identified in the placenta, suggesting that with autocrine and paracrine actions this hormone is essential for the functionality of this organ (Hobson et al. 2018; Genario et al. 2019). Furthermore, maternal melatonin production increases during pregnancy and this hormone can freely cross the placenta without modification, reaching the fetal circulation and influencing fetal development and its photoperiodic programming (McMillen et al. 1995; Nakamura et al. 2001; Cipolla-Neto and Do Amaral 2018).

The receptors for this indolamine are present in different areas of the fetal central nervous system (CNS), although the fetus produces melatonin only after birth. This suggests that maternal melatoninfetus plays a role not only in the transmission of photoperiodic information, but also in neuroprotection and fetal brain development (Gomes et al. 2021).

During pregnancy, melatonin influences maternal metabolism through insulin resistance and increases insulin synthesis, which supports glucose utilization by the fetus and therefore favours its development (Picinato et al. 2008). Melatonin administration during pregnancy has various effects on the fetus, ensuring its health and vitality at birth (Flinn et al. 2020). Furthermore, in meat ewes treated with melatonin, an effect on uterine blood flow, a reduction of fetal hypoxia during birth, and an increase in lamb birth weight and survival have been observed (Thakor et al. 2010; Flinn et al. 2020). In several dairy sheep breeds, treatment with melatonin implants during early lactation did not influence milk production and composition (Abecia et al. 2005; Cosso et al. 2021). However, treatment with melatonin at the end of pregnancy, in different breeds, influenced the composition of colostrum, the quality and quantity of milk produced and the somatic cell count (SCC) (Bouroutzika et al. 2020; Molik et al. 2020; Canto et al. 2022).

Previous studies have shown a lack of uniformity in the dose, timing (whether at the beginning or end of pregnancy) and condition of ewes (stressed or not stressed) used for melatonin administration during pregnancy (Bouroutzika et al. 2020; Cosso et al. 2021; Abecia et al. 2022). Therefore, our study aimed to investigate the effects of melatonin administration in non-stressed Sarda sheep, using doses similar to those used to control reproductive seasonality. Specifically, the objectives of our research were to administer melatonin to Sarda sheep throughout the spring pregnancy period and to verify whether it: (1) improved lamb weight at birth and at 7 and 21 days after birth, as well as the time to first colostrum intake and lamb survival; and (2) improved milk quantity and composition after the lambs were weaned.

Materials and methods

The animals were housed and managed in compliance with the procedures established by the Organization in charge of Animal Welfare and Experimentation (OPBSA) of the University of Sassari, Italy. All animals were also under the supervision of the National Veterinary Health Service, adhering to the animal welfare guidelines established by the Italian Ministry of Health.

The study was carried out in a farm located in North Sardinia (40.7°N), which housed approximately 800 sheep. The farm featured modern facilities, including small pens for lambing monitoring, a feed distribution machine and a milking machine. During the day the animals grazed on legumes and grasses and at milking time, they received 300 g of commercial concentrated feed per head, daily (20.4% of raw protein and 12.5 MJ ME/kg of dry matter (DM)). At night the sheep were penned and provided with hay (11.1% raw protein and 7.2 MJ ME/kg DM) and water ad libitum. For this study, 200 clinically healthy, lactating ewes were selected. These ewes had lambed before December, had an average age of 4.1 ± 1.1 year and a body condition score (BCS) of 2.8 ± 0.5.

On 18 April, these 200 ewes were randomly assigned to two groups of 100 each: a treated (M) and an untreated (C) group. The grouping was based on the date of lambing, BCS, parity, age, and milk production levels. On 20 April (35 days before ram introduction), group M received one subcutaneous melatonin implant (18 mg) (Melovine, CEVA Salute Animale, Agrate Brianza, MB), while group C remained untreated. Consequently, in the treated ewes, estrus was induced by the combined effect of melatonin treatment and the male effect, whereas in group C, estrus was induced solely by the male effect. On 25 May (Day 0), 12 rams were introduced to each group, then removed after 40 days. Before their introduction, the rams were separated from the ewes for approximately 90 days, in order to trigger the onset of estrus in females. The melatonin treatment was repeated in the treated group on 4 July and 17 September (corresponding to 40 and 115 days after ram introduction, respectively). From 45 to 90 days after the introduction of the rams, the pregnancy was diagnosed using transdominal ultrasound examination with a Tringa Esaote instrument (Esaote Europe BV, Maastricht, Netherlands) equipped with a five multifrequency linear probe, 0–7.5 MHz.

Based on the diagnosis of pregnancy, and the clinical signs of preparation for lambing, the ewes close to delivery were isolated in lambing paddocks and closely monitored by cameras and operators. Cameras were placed to frame the entire paddock. Additionally, two operators were ready to assist the ewes, weigh the lambs and record the first suckling. A maximum of eight ewes were placed in each lambing paddock and grouped back together after the third day after lambing.

Each lamb was identified at birth with a collar bearing an identification label and weighed at birth and at days 7 and 21 after birth. During the birthing process, the ewes were closely monitored until the expulsion of the fetus through the vaginal canal. Operators provided assistance to the ewes only if an hour had passed since the fetus engaged in the birth canal or the mother released fluids. Furthermore, the time between birth and the lamb’s first suckling was recorded along with any lambing assistance required.

The lambs were weaned at 22 days old. From 30 to 90 days of lactation, the individual daily milk yield was registered every 15 days (five sampling sessions in total), and individual 50 mL milk samples were collected and analyzed to determine the percentages of fat, protein, lactose, and SCC. The daily milk yield (kg/day) was calculated as the sum of morning and afternoon milking yields collected by the milking machine (Delaval). Milk fat, protein and lactose were analyzed using a Milkoscan FT 6000 FOSS milk analyzer (Foss Electric A/S, Hillerød, Denmark), according to FIL-IDF recommendations (ISO 9622:2013; ISO-IDF, 2013). Somatic cell count was determined with a Fossomatic 5000 FOSS somatic cell counter according to ISO 13366/IDF148 guidelines (2006).

The statistical analyses were conducted using R statistical software (ver. 4.3.2; R Core Team 2023).

To compare the lamb survival rate between groups, a Chi-square test was used.

To compare the weight (at birth, at 7 and 21 days) and the time elapsed between birth and the first colostrum intake in the two groups, the following linear model was performed:

where y_ijklm is the trait measured for each animal, μ is the overall mean of the studied ewes, T_i is the fixed effect of the melatonin treatment (two levels, treated and untreated), R_j is the random effect of the ram (24 rams), P_k is the effect of parity number (three levels), F_l is the fixed effect of the number of fetus (two levels, single and twin), S_m is the fixed effect of the sex of the newborn (two levels), and e_ijklm is the random residual effect of each observation.

The differences in milk yield, milk composition and SCC between the two groups were statistically analyzed using repeated measures ANOVA model, where the melatonin treatment (two levels), the period of the sampling (five levels), and parity number (three levels) were set as fixed effects, while ewes were set as random effects. Additionally, the interaction between treatment and time was considered. Statistical significance was determined at P < 0.05 for each analysis.

Results

The reproductive data are available as Supplementary Tables S1 and S2. Parity, the number of fetuses, and the sex of newborns did not affect any recorded variable. There were nine sets of twins in group M, and 10 in the group C, but no triplets or quadruplets. Moreover, the number of females and males was 35 vs 42 (total 77) in group C and 42 vs 50 in group M (total 92). Ewes treated with melatonin implants (group M) gave birth to lambs that were heavier at birth and at 7 and 21 days of age compared to those born to untreated ewes (group C) (P < 0.05) (Table 1). Mortality at 7 days was higher in group C compared to group M (4 vs 1, P < 0.05) (Table 1).

Among untreated animals, four cases of difficulty during lambing requiring assistance were recorded, while treated animals did not show any difficulties. Two of these four lambs died 5 days after birth. Moreover, the time from birth to first suckling was shorter for lambs born from treated compared to control ewes (55.5 ± 5.3 vs 69.4 ± 5.6 min, P < 0.05).

Furthermore, melatonin-treated ewes had significantly (P < 0.05) higher daily milk yield (DMY) in all samples compared to group C (Table 2). The milk fat concentration was higher in group M in the first four samplings compared to group C. Protein and lactose concentrations were similar between groups. Instead, the SCC showed a continuous decrease in the treated animals from the first to the fifth sampling (P < 0.05), while in untreated animals there was a continuous increase in SCC from the first to the fifth sampling (P < 0.05). Finally, regarding SCC, the comparison between groups showed a difference in the third, fourth and fifth sampling (P < 0.05).

Discussion

The first objective of the present research was to evaluate the effect of treating pregnant Sarda ewes with melatonin on lamb weight and survival. The results indicate that melatonin treatment influenced the weight of lambs at birth, 7 and 21 days postpartum. This aligns with previous research showing that varying melatonin doses affected the birth weight of lambs (Sales et al. 2019). However, the findings contradict other reports where melatonin treatment did not alter lamb birth weight (Flinn et al. 2020). The effects of melatonin on fetal and lamb development are complex, as melatonin exerts different influences on both the mother and the offspring, which can impact developmental outcomes.

Melatonin can freely cross the ovine placenta (Yellon and Longo 1987; Aly et al. 2015), allowing its administration to the mother to immediately supply melatonin to the developing fetus throughout pregnancy. Previous studies have shown that administering melatonin during pregnancy affects fetal development by regulating the chronobiological metabolism and gene expression involved in organ development (Gomes et al. 2021). Disruption or deprivation of the melatonin signal has been linked to various CNS pathologies and developmental deficits in rats (Motta-Teixeira et al. 2018). Furthermore, melatonin has been shown to protect fetal nervous tissue from the hypoxia that can occur around delivery (Mallard et al. 2003). The altered cerebral oxygenation during birth asphyxia can induce widespread neurological damage, such as meningeal haemorrhage and CNS lesions, impairing functionality and neuromotor activity (Ikeda et al. 2000). This may explain the greater vitality and improved postpartum survival observed in the lambs from our trial.

Melatonin production and its receptors are also found in the placenta (Iwasaki et al. 2005) highlighting the importance of melatonin during pregnancy for maintaining placental functionality and fetal development. Furthermore, studies have shown that in various animals subjected to stress, blood melatonin levels decrease, despite constant production at the epiphyseal level (Tan et al. 2007). Thus, melatonin plays a role in neutralizing reactive oxygen species (ROS) and reactive nitrogen species (RON) both directly and through its metabolites. These effects of melatonin may have improved the well-being and health of the mother and the fetus, and mitigated the potential damage caused by environmental insults during pregnancy. This could have influenced better fetal development in the melatonin-treated ewes leading to the higher birth weight of their lambs compared to those untreated. As reported by Bouroutzika et al. (2021), melatonin administration throughout pregnancy likely helps newborn lambs overcome oxidative stress by supporting antioxidant defences and reducing plasma ROS production. The higher birth weight and better survival of the lambs in this study could also be due to the improved quality of the colostrum, as several authors have found a higher quantity of IgG in the colostrum of ewes treated with melatonin 40 days before giving birth (Sales et al. 2019; Bouroutzika et al. 2021; Canto et al. 2022).

The second objective of our study was to investigate the impact of melatonin treatment on milk quantity and composition after weaning the lambs. Previous studies have explored the influence of melatonin on milk quality in sheep, but the results have been quite variable, presumably due to the differences in the timing of melatonin administration. In fact, in a prior study on Sarda sheep, Cosso et al. (2021) administered melatonin with the aim of improving reproductive efficiency, but found no effect on milk quantity or quality. In contrast, in the current study, melatonin was administered throughout the course of pregnancy and the effects of this hormone on milk quantity and quality are evident, aligning with the findings reported in Assaf sheep by Canto et al. (2022).

Additionally, in goats, melatonin implants inserted 7 weeks before lambing showed a positive effect on milk production in the subsequent lactation and improved the daily weight gain of suckled goat kids (Avilés et al. 2019). This data agrees with our findings, as the greater weight of the lambs at weaning could be attributed to the increased quantity and improved quality of the milk produced by the ewes treated with melatonin. However, the increased milk fat content observed in the present study is difficult to interpret since other studies have found melatonin to decrease fat, proteins and lactose in sheep milk (Molik et al. 2011). In cows, however, melatonin administration has been shown to increase milk fat and protein contents while decreasing the quantity of milk produced and lactose levels (Auldist et al. 2007). Certainly, further research is necessary to understand the precise physiological mechanism of melatonin at the mammary level, particularly in light of the identification of MT1 and MT2 melatonin receptors in the mammary gland during lactation in goats (Zhang et al. 2019), suggesting a direct role in regulating mammary physiology.

This study found that melatonin affected SCC in milk, consistent with previous findings in sheep and cattle (Yang et al. 2017; Cosso et al. 2021; Canto et al. 2022). Our data showing a reduction of SCC in sheep treated with melatonin are highly relevant for improving milk quality. High SCC levels can lead to a depreciation in milk quality, indicating possible mastitis affecting the mammary parenchyma (Raynal-Ljutovac et al. 2007; Albenzio et al. 2012) Although the mammary glands may not show clinical signs of infection and the milk appears normal, the udder may still be inflamed and/or infected. Bacterial infections of the mammary gland remain the primary cause of variation in SCC. Sheep with subclinical mammary parenchyma infections can act as a reservoir for the bacteria, potentially infecting healthy sheep. Furthermore, subclinical intramammary infections can also damage the mammary parenchyma, compromising subsequent lactations in these ewes. As observed in our study, the administration of melatonin decreased SCC, suggesting its potential use as a therapy against subclinical intramammary infection. Melatonin may improve udder health through its control of mediators in local and systemic immune responses as well as through its antioxidant effect (Yang et al. 2017; Bouroutzika et al. 2021; Canto et al. 2022).

Conclusion

In conclusion, our data show that administering melatonin throughout the gestation period can influence lamb weight at birth and weaning. The lambs born from treated ewes showed a shorter latency in ingesting colostrum for the first time and likely had an improved survival rate. Sheep treated with melatonin produced more milk with a higher fat content and lower SCC. Therefore, administering melatonin during pregnancy in sheep could be an advantageous management practice for both pregnant females and their offspring. These potential effects warrant further research to clarify the mechanisms by which melatonin influences pregnancy and lactation.

Supplementary material

Supplementary material is available online.