Reproductive fitness of gilts is tightly linked to metabolic status, and imbalance of metabolism can cause reproductive disorders and even infertility [3, 17]. A successful cultivation of replacement gilts not only require a sound growth performance, but also a suitable activation of HPG axis to activate the pubertal onset and normal maintenance of estrous cycle . Meal frequency played a critical role on metabolic status in pig, human and rodent [6–8]. Less MF can affect growth, and alleviate high-fat diet-induced lipid accumulation and inflammation in adipose tissue in pigs [12, 19]. In this study, we tested the hypothesis that whether less MF could influence both nutrient utilization and reproductive process of replacement gilts, and the current findings revealed that less meal frequency increased nutrient utilization, promoted the secretion of luteinizing hormones, and improved ovarian follicular development in gilts. To the best of our knowledge, the results of this study offered a novel linkage between dietary pattern and development of peripheral and reproductive tissues, which will provide new insights into the nutritional strategy for replacement gilts.
Currently, the replacement gilts were typically fed ad libitum, in which the gilts may visit the feeders up to 15 times per day . This eating pattern encourages the gilts extending the period of daily nutrient intake and thereby shortening the fasting period . However, growing evidence revealed that this free eating pattern has been associated with a negative effect on growth, inflammation of adipose tissues and systemic health in growing pigs [12, 19]. In this study, gilts were fed either with one, two, or six meals per day, but the eating windows of 3 hours were allowed for three dietary treatment groups, which eliminate the differences of energy expenditure (such as heat increment) induced by variations of feeding duration. Early results revealed that the gestating gilts or sows fed two or six meals per day during gestation had differential effects of reproductive outcomes , and evidence also revealed that growing pigs fed one meals per day, two meals per day, or free access to feed had different nutrient digestibilities and adipose tissue metabolism [22–24]. Based on those researches, the meal frequencies were set at one, two or six meals per day in the present study. In agreement with previous studies , pigs fed less MF increased the ADG and the final bodyweight (P < 0.05, Table 3). In this study, the feed intake was the similar among the three groups, and the increased the body weight observed in T1 gilts suggested that less MF increased nutrient utilization. In the present study, T1 gilts showed a faster glucose clearance with increased serum insulin concentration (P < 0.05, Fig. 2A and 2B) which agrees with studies in mouse and human [6, 8], showing that glucose was removed faster from the circulation when having a limited access to a single meal compared with ad libitum. Glucose is the most important fuel for metabolism, faster glucose clearance suggests a higher glucose utilization. Insulin augments glucose transport in skeletal muscle, adipose tissue, and other tissues, and this is also supported by enhanced body weight gain in gilts fed less meal frequency. In human and rodent, limited MF has beneficial effects on energy homeostasis, which increased energy utilization and decreased lipid accumulation [10, 25]. To our surprise, in this study, less MF had no effects on the backfat thickness in gilts (P > 0.05, Table 3), which suggested that less MF did not affect the fat deposition in gilts. The mainly reason for this inconsistency may be attributed to the difference of the age of the animals. Both in human and mouse researches, the models are adults which were easy to have greater fat deposition [6–8], while the period of gilts in this study were at growing phase which has a greater lean tissue accretion than adipose tissue before sexual maturity [26, 27]. In this study, gilts fed one meal per day had increased bodyweight gain but had no effect on backfat thickness, which suggested meal frequency influenced the lean deposition. In order to test whether less meal frequency can influence the lean mass metabolism, we tested the nitrogen balance in gilts. Indeed, the T1 gilts had decreased the urine excretion (T1 vs T6: 24.1 g/d vs 32.2 g/d), increased amount of retained nitrogen (T1 vs T6: 39.1 g/d vs 29.3 g/d) and the biological value of nitrogen (T1 vs T6: 61.3% vs 47.2%). In addition, the gilts fed one meal per day had lower serum α-amino nitrogen and urea levels with time after meal, which suggested less meal frequency improved the nitrogen utilization, and the increased of bodyweight gain was probably attributed to lean mass accretion. Indeed, in neonates’ pig, intermittent bolus feeding enhanced muscle protein synthesis to a greater extent than continuous feeding via mTOR pathway . Meanwhile, no effects of meal frequency on the digestibility of DM, CP, gross energy, EE, and ash were observed, but the digestibility of NDF in gilts fed one meal per day were greater compared with gilts fed six meals per day, which suggested that less meal frequency increased the fiber fermentation. Gut microbiota has been recognized as an important factor in regulating host metabolism through SCFAs [10, 29, 30], which stimulated us to test the effect of MF on SCFAs production in feces. Our study showed that time-course changes of SCFAs concentrations in feces at different time points were different among different treatment groups (P < 0.05, Fig. 1G). In agree with that change the interval between meals results in a shift in the gut microbiota composition [10, 11]. The gilts fed one meal per day showed a cyclic fluctuation in feces SCFAs concentration but not in gilts fed six meals per day (P < 0.05, Fig. 1G). In this study, T1 feeding regimen increased the acetate and total SCFAs production (P < 0.05, Fig. 1A and 1G). Acetate is utilized for lipogenesis in the liver and as a fuel source once it enters the peripheral circulation  and the increased SCFAs production promoted energy availability for both microbiota and host intestine [32, 33] that may contribute to the health of gastrointestinal tract and peripheral tissues of host. Taken together, one meal per day increased the nutrient utilization and the growth performance of gilts.
Pubertal onset is the marks of sexual maturation of replacement gilts, which can be affected by the age of growth performance of gilts. In the present study, there was no exhibited marked delay of first observed estrous among the three groups (P > 0.05, Table 6), possibly attributed to two reasons. Firstly, the dietary treatment began at the age of 150 d and bodyweight at 77 kg, which was too late to be affected by dietary treatment at this time. Secondly, although gilts fed one meal per day had a greater bodyweight gain and a greater bodyweight than the gilts fed six meals per day at the 14th week of experiment, the ADG of the gilts fed six meals per day already exceeded the minimum growth rate (600 g/d) required for pubertal maturation. The exposure of female to sexually mature male pheromones leads to the initiation of proestrus, including increased hypothalamic secretion of GnRH and a preovulatory LH surge [2, 34], thus the gonadotropin concentrations were measured the 18th day of the 3rd estrus cycle. In the present study, we found the gilts fed one meal per day exhibited a substantially greater LH concentrations compared to gilts in the other two groups at different time points (P < 0.05, Fig. 3A), but there were no marked differences in FSH levels in gilts (P > 0.05, Fig. 3B), which suggested less MF promoted GnRH secretion. The concentrations of E2 in circulation were also greater in gilts fed one meal per day (Fig. 3C), and this was associated with the genes related to E2 biosynthesis, such as (CYP11a1, STAR, and 3βHSD) (P < 0.05, Fig. 3H – 3J). The gene expressions of GDF9 and BMP15, indicators of oocyte quality [35, 36], were also greater in gilts fed one meal per day and were lower in gilts fed six meals per day (P < 0.05, Fig. 3F and 3G). The release of reproductive hormones is closely associated with ovarian follicular development. In agreement with the increases of LH and E2 concentrations, the gilts fed one meal per day had greater number of corpora lutea, an indicator of recently ovulated matured follicles, compared with the gilts fed six meals per day (P < 0.05, Table 7). Interestingly, the gilts fed one meal also had greater number of growing follicles, suggesting that those gilts may have greater number of ovarian reservations. Most studies conducted in human, mouse, and pigs revealed that a larger ovarian pool would resulted in a greater reproductive potential and a longer reproductive span [37, 38]. Given that the reproductive process is controlled by muti-level regulators [18, 34], and metabolic status was known to be the important factors influencing the function of hypothalamus-pituitary-gonadal axis [3, 17, 39]. It will be interesting to examine which metabolic signals could mediated the effects of meal frequency on reproductive function of gilts.