Melatonin administration during the first half of pregnancy improves physiological response and reproductive performance of rabbits under heat stress conditions

Introduction

Rabbits are among the most sensitive farm animals to the negative impacts of heat stress. They have few sweat glands and depend on respiratory panting for heat loss. This main heat tolerance adaptive mechanism is not only energy consuming but also is associated with other physiological and hormonal imbalances (El-Desoky et al. 2022). Therefore, maternal exposure to heat stress has been found to decrease fertility and embryo development, and increase the stillborn rate in rabbits (Marco-Jiménez et al. 2017).

Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine lipophilic hormone that is synthesised and secreted by the pineal gland. Studies have shown that the biological roles of melatonin are beyond synchronisation of circadian rhythms. It regulates several biological functions/pathways in mammals including oxidative and inflammatory reactions/pathways, cell differentiation/apoptotic pathways, and immune system functions. Moreover, at the level of reproductive processes, melatonin is distributed in various reproductive tissues and involved in folliculogenesis, oocyte maturation, luteal function, early embryonic development, and pregnancy maintenance in different mammalian species (Jiang et al. 2021). In pregnant animals, the release of melatonin in the ovary, placenta, uterus, embryo, and fetus is elevated, highlighting its pivotal role in pregnancy maintenance (Lanoix et al. 2008; Ejaz et al. 2021). Hashem et al. (2023) found that melatonin administration during the first half of pregnancy upregulated the expression of gonadotropin and steriod-related genes in the ovary and antioxidant-related genes in the ovary and placenta of day 18 pregnant rabbits, leading to better embryo survival and pregnancy outputs. Other studies on farm animals have highlighted the potential positive effects of melatonin on pregnant animals even under some environmental stresses such as heat stress (De Rensis et al. 2015). For example, heat-stressed ewes treated with melatonin expressed higher total antioxidant capacity and fertility rate compared to non-treated ewes (Bouroutzika et al. 2020). Similarly, heat-stressed dairy cows treated with melatonin implants before calving expressed reduced subsequent days open and fewer services per conception (artificial insemination semen doses; Garcia-Ispierto et al. 2013). Moreover, it has beneficial effects on blastocyst in vitro production from heat-stressed bovine oocytes (Cebrian-Serrano et al. 2013). As reported in these studies, the main mechanism by which melatonin can improve fertility in heat-stressed animals could be ascribed to its antioxidant effect. High ambient temperatures during summer increase oxidative stress in animals, and melatonin and its metabolites are considered indirect antioxidant agents and effective direct free radical scavengers (Bouroutzika et al. 2020). In this study, the effects of melatonin administration during the first half of pregnancy on the heat-tolerance capacity, hormone profile, ovarian structures, fetal development, and pregnancy outputs of heat-stressed rabbits were investigated.

Materials and methods

The present study was conducted in the Laboratory of Rabbit Physiology Research, Agricultural Experimental Station, Animal and Fish Production Department, Faculty of Agriculture, Alexandria University, Egypt (31°20′N, 30°E). The experimental protocols and procedures were checked and approved by the Institutional Animal Care and Use Committee (IACUC), Faculty of Veterinary Medicine, Cairo University (Ethical approval number: Vet CU 01122022584).

Animals, housing system, and experimental design

Forty sexually mature, nulliparous, V-line female rabbits (23 weeks old and 2.62 ± 0.13 kg of body weight, BW) were used. Each female rabbit was individually kept in a galvanised wire cage (60 cm × 55 cm × 40 cm, L × W × H) equipped with a feeder and automatic drinker. The female rabbits were kept under natural metrological conditions of the summer season, which is characterised by high ambient temperature and relative humidity. To monitor the meteorological data inside the rabbitry, an electronic digital thermo-hygrometer (Data logger-Log 100/110, Germany) was used to record ambient temperature (°C) and relative humidity (%) every hour during the entire experimental period. To calculate the temperature–humidity index (THI), temperature and relative humidity values were involved in the following equation: THI = db °C − [(0.31 − 0.31 × RH%) × (db °C − 14.4)], where db °C is dry bulb temperature in Celsius and RH% is the percentage of relative humidity. The THI values were categorised into four levels: <27.8 = absence of heat stress, 27.8–28.8 = moderate heat stress, 28.9–29.9 = severe heat stress, and >30.0 = very severe heat stress (Marai et al. 2002). The means of ambient temperature, relative humidity, and THI were 30.34 ± 1.29°C, 80.58 ± 3.75%, and 29.93 ± 1.33, respectively.

Females were fed a commercial pelleted diet that met their nutritional requirements (18.24% crude protein and 67.96% total digestible nutrients) and had free access to fresh clean water during the experimental period. To manage the females as one flock during the experimental period, each female was subjected to oestrous synchronisation by intramuscular (i.m.) injection of 25 IU equine chorionic gonadotropin (eCG, Gonaser®, Hipra, Girona, Spain) followed by an i.m. injection of 0.8 μg of gonadotropin releasing hormone (GnRH, Receptal, Boxmeer, Holland) 48 h later, and artificially inseminated with 20 × 10⁶ fresh pooled sperm cells/female. Semen doses were collected with the aid of an artificial vagina from four proven-fertile rabbit males. Females were stratified equally into two experimental groups (n = 20/group) and daily received an oral dose of either 1 mL of corn oil (control, placebo) or 1 mL of corn oil containing 1 mg melatonin/kg BW (Puritan’s Pride Melatonin, Oakdale, New York, USA). The dose used in this study was based on the study of Mousa-Balabel and Mohamed (2011). The treatment started from the day of oestrous synchronisation to day 15 post-insemination. The schematic design of the study is depicted in Fig. 1.

Fig. 1.

Schematic framework of the study (ES, oestrous synchronisation; eCG, equine chorionic gonadotropin; GnRH, gonadotropin releasing hormone; AI, artificial insemination; BS, blood sampling; AP, abdominal palpation/pregnancy diagnosis).

Ovarian structures, feto-placental characteristics, and reproductive performance

On day 18 of pregnancy, five pregnant female rabbits from the 10 pregnant female rabbits in the control group and 16 pregnant female rabbits in the melatonin group were randomly chosen and euthanised. The ovaries and reproductive tract of each female were dissected. The numbers of the visible ovarian follicles and corpora lutea were counted. Along each uterine horn, the numbers of implantation sites were counted and classified according to the presence or absence of fetus into developing implantation site (containing a differentiated/developed fetus) or absorbed implantation site/absorbed fetus (containing atrophic placenta without fetus). Each implantation site was separated and weighed (including weights of fetus, amniotic fluid, and placenta), and then the amniotic fluid was aspirated with a syringe and the remaining parts were dissected to separate fetus and placenta. For each implantation site, the weight of fetus and the maternal and fetal sides of the placenta were recorded. Placenta efficiency was determined by dividing the weight of fetus on the weight of correspondent placenta tissue (maternal and fetal sides) at each implantation site (Hashem and Aboul-Ezz 2018). Different ovarian structures and types of implantation site, developed or absorbed, are shown in Fig. 2.

Fig. 2.

Ovarian structures (A: corpus luteum; B: antral ovarian follicle) and fetal implantation sites (C: regressed/absorbed implantation sites; D: developed implantation site; E, F: fetus with placenta; G: fetal side of placenta; H: maternal side of placenta; I: fetus observed on day 18 of pregnancy.

Reproductive performance of the females was determined. Conception rate ([number of pregnant females on day 10 post-insemination/total number of inseminated females (20 females)] × 100) was determined on day 10 post-insemination by checking females for pregnancy using abdominal palpation procedure. Additionally, kindling rate ([number of females having birth/total number of females kept live to complete pregnancy term (15 females)] × 100) and litter size at birth ([number of bunnies at birth/number of females giving birth]) were estimated.

Results

Discussion

In rabbits, heat stress can negatively impact maintenance of pregnancy either at early or late pregnancy by its negative effects on embryo survival, implantation, maternal recognition of pregnancy, uterine microenvironment, and reproductive hormone balance (mainly sex steroids and gonadotropins). These effects may increase the incidence of early embryonic loss and/or abortion rate, interfering with animal welfare aspects and goals of the intensive production systems in rabbit farms (Mutwedu et al. 2021; Ebeid et al. 2023). The optimal ambient temperature range of rabbits is 15–25°C, and the optimal humidity is 55–65%. Heat stress occurs when the ambient temperature elevates above 30°C (Liang et al. 2022). Marco-Jiménez et al. (2017) reported that rabbits exposed to ambient temperatures between 25°C and 36°C compared to those kept between 14°C and 20°C during pregnancy and lactation had smaller litter sizes and kit weights at birth and had higher stillborn rates.

In this study, we aimed to explore the potential roles of melatonin administration during the first half of pregnancy in mitigating heat stress negative effects on pregnancy in rabbits. Melatonin is a potent antioxidant and an anti-stress agent, in addition to its roles in maintaining circadian rhythm of different physiological processes, particularly under stressful environmental conditions (Adah et al. 2020; Ayo and Ake 2022). Environmental factors such as ambient temperature, photoperiod, stress, and food availability are known to influence circadian rhythms to allow animals to adapt to environmental changes (Contreras-Correa et al. 2023). In a thermoneutral zone, the physiological rectal temperature of rabbit ranges from 38.5 to 39.5°C (Liang et al. 2022) and respiratory rate ranges from 32 to 60 breaths/min (Nowland et al. 2015). In contrast, under heat stress conditions (ambient temperature around 30.7°C), the average of rectal temperature ranges from 39.8 to 40.2°C (Marai et al. 2002). Consequently, rabbits have to increase respiratory panting by increasing respiration rates as a physiological thermoregulatory adaptive mechanism to lose excess body heat (El-Desoky et al. 2022; El-Raghi et al. 2023). In this study, melatonin treatment improved heat-tolerance capacity of pregnant females, as melatonin-treated females had respiration rates and rectal temperatures approaching normal physiological ranges compared to control females. These findings are in accordance with those obtained by Bouroutzika et al. (2020) who found that melatonin administration to heat-stressed pregnant ewes lowered their rectal temperatures and respiration rates compared to control ewes.

Melatonin is known as one of the potent antioxidant candidates that can efficiently maintain redox homeostasis by scavenging free radicals and preserving functional integrity of antioxidant enzymes (Contreras-Correa et al. 2023). Hashem et al. (2023) found that melatonin administration to rabbits during the first half of pregnancy upregulated the expression of antioxidant-related genes in the ovary and placenta. In this study, the antioxidant role of melatonin was not clearly evidenced as none of the antioxidant indicators, total antioxidant capacity or glutathion preroxidase enzyme, were affected by the treatment. The lack of the antioxidant effects of melatonin observed in this study may be related to the inadequacy of the melatonin dose. Higher doses of melatonin may be required to scavenge produced free radicals released due to heat stress and pregnancy, as both factors can increase the rate of free radical production. However, it is important to note that melatonin-treated rabbits tended to have lower concentrations of the free radical malondialdehyde, indicating reduced lipid peroxidation, which is associated with increased high density lipoprotein. Melatonin is a small lipophilic indoleamine; this chemical nature may enable melatonin to easily incorporate in lipid metabolism (Jin et al. 2017).

Melatonin treatment significantly increased concentrations of nitric oxide. Thakor et al. (2010) reported that, in sheep, treatment with melatonin during pregnancy increases umbilical blood flow via nitric oxide-dependent mechanisms, suggesting melatonin as a useful clinical tool for increasing or maintaining umbilical blood flow in complicated pregnancy. Although nitric oxide is classified as a free radical, the physiological level of nitric oxide plays a vital role in the regulation of blood vessel vasodilation and permeability, and has multiple regulatory functions within the reproductive system. It is involved in many cycle-dependent ovarian events (such as ovulation and luteal function modulation) and fetoplacental vascularisation (Abdelnaby and Abo El-Maaty 2021). During pregnancy, it acts locally on the vascular smooth muscle of the fetoplacental vessels and systemically by inhibiting the production of vasoconstriction in the maternal systemic circulation, resulting in increased uterine and fetoplacental blood flow (Tarrade et al. 2014).

In rabbits, heat stress results in many deleterious effects on reproductive organs and their functions. Exposure of female rabbits to heat stress weakens the responsiveness of ovarian granulosa cells to follicle-stimulating hormone, reduces ovarian weight, lowers plasma progesterone concentrations, and destroys the ovarian cell nucleoli (Mutwedu et al. 2021; Tang et al. 2022). As evidenced from our results, rabbits treated with melatonin had higher concentrations of blood serum oestradiol and progesterone. The elevation in steroid concentrations due to melatonin treatment may be related to the ability of melatonin to regulate the expression of several steroid-regulatory genes and related pathways. Melatonin has been found to upregulate the expression of the steroidogenic acute segulatory protein (STAR) gene in the ovaries of pregnant rabbits (Hashem et al. 2023). The protein encoded by the STAR gene plays a key role in the acute regulation of steroid hormone synthesis by permitting the cleavage of cholesterol into pregnenolone and by mediating the transport of cholesterol from the outer to the inner mitochondrial membranes. The improved concentrations of sex steroids observed in this study may be also due to the increased concentrations of high density lipoprotein, which is considered one of the sex steroid hormones precursors (Chang et al. 2017). Moreover, melatonin has been found to stimulate progesterone synthesis in bovine and ovine theca cells by activating the phosphatidylinositol 3-kinase and Akt (protein kinase B) pathway (Wang et al. 2019). It also stimulates the secretion of luteal and placental progesterone (Tamura et al. 2008), which reduces uterine contractility and prevents immunological rejection of the trophoblast.

In this study, the control rabbits had a significantly lower conception rate than melatonin-treated rabbits. Keeping in mind that conception rate was determined on day 10 of pregnancy, it can be said that heat stress can evoke early embryonic loss and hinder embryo implantation, leading to full lose of conceptus. Moreover, the results confirmed the continuous negative effects of heat stress on fetal growth and development as indicated by higher atrophic/absorbed implantation sites with dead/absorbed fetuses on day 18 of pregnancy in the control rabbits than those treated with melatonin. Thus, heat stress can lead to both complete and partial pregnancy losses in rabbits. The improved conception rate and number of developed/live fetuses due to melatonin treatment were associated with elevated plasma concentrations of oestradiol and progesterone. In rabbits, both oestradiol and progesterone play crucial roles in the maintenance of pregnancy and embryo/fetus survival and development. During the pre-implantation period, embryo survival is affected by the uterine micro-environment and the composition of its fluid. In this term, luteal progesterone is necessary to prepare the endometrium for embryo implantation, prevent uterine contractions, and stimulate the secretion of uterine proteins to maintain pregnancy. Interestingly, ovarian follicle-synthesised oestradiol plays a crucial role in the maintenance of luteal function and thus pregnancy via its luteotropic action (Hashem and Aboul-Ezz 2018).

The other reason for improved conception rate in melatonin-treated rabbits could be due to the ability of melatonin to improve placental functioning. Hashem et al. (2023) found that melatonin treatment during the first half of pregnancy upregulated the expression of several placental genes related to cell differentiation, functioning, and synthesis in rabbits. Melatonin can upregulate the expression of placental cyclin B1, cortexin 1, fibronectin 1, and Proliferation-Associated 2G4 genes which are involved in the cell invasion and cell adhesion process and provide structural support to tissues. It can also upregulate the placental expression of IMP metallopeptidase inhibitor 1, which plays a crucial role in the extracellular matrix remodelling process during implantation. Melatonin may also improve interactions between the conceptus and uterus by regulating sirtuin 1 during the early stages of pregnancy, which is important for decidualisation (Bae et al. 2020).

Improved placental functioning can be suggested in our study, as melatonin treatment significantly increased the weight of amniotic fluid. This effect may provide protection to the developing fetuses against the negative impacts of heat stress. It is now well established that the majority of in utero heat stress is transferred through the placenta via the fetal–placental circulation, and up to 20% of transferred heat can be dissipated via amniotic fluid (Kasiteropoulou et al. 2020). Amniotic fluid insulates the fetus and maintains a regular temperature. In addition, it contains antibodies against various infectious diseases, supports the development of fetal muscles and bones, and prevents the collapse of the umbilical cord, allowing efficient oxygen and nutrient transport to the fetuses (Khan et al. 2018). However, more studies are required to directly examine placental functioning (blood flow, nutrient or water transport, and vessel development) due to melatonin treatment under heat stress conditions for better elucidation of the mechanisms by which melatonin might affect placental functioning.

Conclusion

Heat stress has negative impacts on pregnancy outcomes in rabbits. High temperatures increased rectal temperatures of pregnant rabbits and reduced oestradiol and progesterone concentrations, which were associated with reduced numbers of live fetuses, and thus conception rates. Melatonin administration during the first half of pregnancy countered the negative effects of heat stress, leading to better fetal vitality and pregnancy outcomes. The positive effects of melatonin could be related to its thermoregulatory role and steroidogenesis-stimulating activity. Further studies are required to understand more clearly the mechanisms by which melatonin can act as a thermoprotective agent in rabbits during different stages of pregnancy.