Environmental factors such as temperature and altitude affect fetal and placental development. Nutrition is the most important among these factors (Wu et al., 2004). In the North American Midwest during times of drought, dietary energy requirements are often not met. Furthermore, ewes may need nutritional supplements either during the period of major placental development (early and mid-gestation) or during the period of major fetal development (last third of gestation).

The majority of placental growth happens in the first two-thirds of pregnancy, with the placenta reaching its maximum weight by day 90 in sheep (Stegeman, 1974). In sheep, abnormal Doppler velocimetry measurements from a variety of fetal blood vessels have been correlated with intrauterine growth restriction, fetal and neonatal mortality, and developmental abnormalities in the offspring (Galan et al., 1998; Rigano et al., 2001; Ferrazzi et al., 2002). Moreover, changes in umbilical blood flow are the earliest Doppler abnormalities detected in sheep intrauterine growth restriction (Rigano et al., 2001; Ferrazzi et al., 2002). Some studies in sheep have shown that a nutrient restriction from days 30 to 80 of gestation does not affect fetal or placental growth (Anthony et al., 2003); however, our laboratory has demonstrated that, in young ewes, a 40% nutrient restriction beginning on day 50 reduces umbilical blood flow (UBF) at day 70 and UBF does not recover to control values until day 130 in which ewes were euthanized (Lemley et al., 2012). Furthermore, impairing placental growth or uteroplacental blood supply affects fetal growth trajectory (Reynolds et al., 2005; Vonnahme et al., 2013). Late-gestation nutrient restriction results in decreased growth and development of the fetus (Redmer et al., 2004). Therefore, low birth weight is a common result of late-gestation nutrient restriction with several studies reporting intrauterine growth restriction (Ferrazzi et al., 2002; Redmer et al., 2004; Lemley et al., 2012). Few studies have been done analyzing the effects of realimentation in pregnant animals. In beef cows, a 40% nutrient restriction applied from days 30 to 140 of gestation did not change the uterine blood flow (Camacho et al., 2014). Upon realimentation (days 140 to 198 of gestation), ipsilateral blood flow was increased in the previously restricted cows, compared to cows that never experienced a nutrient restriction (Camacho et al., 2014).

In several species, parity can influence litter size as well as birth weight. The number of pigs born from multiparous sows is greater than that from primiparous sows (Mahan, 1998; Whitley et al., 2002). In sheep, the conception rate increases and fetal loss decreases as parity increases (Lafi et al., 2009). Similarly, the likelihood of twins increases as the number of parities increases in sheep (Lafi et al., 2009). In cattle, sheep, and mares, birth weight increases as parity increases (Kayisiz et al., 2011; Yakubu et al., 2014; Abdel-Mageed and El-Gawad, 2015; Klewitz et al., 2015; Lv et al., 2015). In mares, the diameter of the uterine artery increases more throughout pregnancy in multiparous (three to eight foalings) compared to first- and second-parity mares (Klewitz et al., 2015). Similarly, blood flow is increased in the uterine artery during the third period of gestation in multiparous mares when compared to first- and second-parity mares (Klewitz et al., 2015). In women, pulsatility index (PI) measurements taken on the uterine artery are greater in primiparous mothers compared to multiparous mothers during mid-gestation (17 to 18 weeks; Suzuki, 2006). Similarly, many studies show that nulliparous women have greater blood pressure, pregnancy-induced hypertension, and greater risk of preeclampsia than multiparous women (Duckitt and Harrington, 2005; Rurangirwa et al., 2012). The pulsatility index, an indirect measurement of blood vessel resistance, is correlated with the resistance index (RI; Suzuki, 2006), and both measurements are usually increased when blood flow is decreased.

A good proportion of the studies analyzing the influence of parity in reproductive traits do not report maternal age (Abdel-Magged and El-Gawad, 2015; Klewitz et al., 2015). To our knowledge, in sheep, only one study has analyzed the effect of maternal age, controlling for parity, in placental development (Borowicz et al., 2005). Several studies in humans and other mammals suggest that there could be an independent effect of parity in the maternal utero-placental physiology that could enhance the reproductive capacity of the multiparous mother (Kelly et al., 1992; Whitley et al., 2002; Wilsher and Allen, 2003; Elliott et al., 2009). Studies in animals and humans show no difference in the reproductive effects of parity after the second parturition (Wilsher and Allen, 2003; Zaborski et al., 2009), suggesting that possible adaptive physiological changes of parity happen after the first pregnancy (Wilsher and Allen, 2003; Zaborski et al., 2009).

There is limited information on how realimentation and parity impact UBF in the ewe. The objective of experiment 1 was to determine if realimenting previously restricted multiparous pregnant ewes would restore UBF to control levels during mid-gestation. The objective of experiment 2 was to investigate if parity, independent of maternal age, influences the effect of nutrition on umbilical blood flow in sheep. We investigated the effects of parity and nutrient availability during mid-gestation on the UBF, PI, RI, body, and placental measurements of the fetus. We hypothesized that during dietary restriction, UBF, and fetal and placentome measurements, would be lower as compared to adequately fed ewes, but upon realimentation, ewes would have similar UBF as ewes that were not restricted. We also hypothesized that the negative effects of nutrient restriction would be worse in nulliparous ewes.

Animal care and use for both experiments were according to protocols approved by the North Dakota State University Animal Care and Use Committee (#A15076).

The placenta is essential for endocrine production and nutrient exchange between the dam and fetus. In ruminants, including sheep, the fetal–maternal exchange occurs in structures called the placentome. Placentome size has been previously used as an indirect measurement of nutrient delivery to the fetus (Redmer et al., 2004). The umbilical cord is the structure that transports the nutrients and oxygen from the placentomes to the fetal circulatory system and transports carbon dioxide and waste products from the fetus to the placentomes. Assessment of UBF is used as an index of nutrient delivery (Kiserud, 2005; Lemley et al., 2012). In experiment 1, on day 80, RES ewes had smaller placentomes, reduced UBF, and increased measurements of resistance (i.e., PI and RI). These measurements suggest that UBF and nutrient exchange might have been compromised on day 80. However, these measurements were similar prior to realimentation; therefore, the recovery in blood flow parameters was not an effect of realimentation.

A previous study performed in our laboratory reported that in primiparous young ewes a 40% nutrient restriction from days 50 to 130 of gestation resulted in decreased UBF starting on day 80 of gestation (Lemley et al., 2012). Experiment 1 was done with multiparous ewes, and we preserved the same housing conditions, breed, and experimental protocol of Lemley et al. (2012). Therefore, we hypothesized that in Lemley et al. (2012) parity had an aggravating effect, additionally to nutrient restriction, on blood flow. The effects of parity on uterine microanatomy and UBF in sheep are not well characterized. Therefore, we tested the effects of nutrient restriction and parity during mid pregnancy in ewes in experiment 2.

In experiment 2, we hypothesized that parity could have a protective effect on blood flow parameters and that nulliparous ewes would experience the detrimental effects of nutrient restriction on umbilical blood flow. However, none of the effects of nutrient restriction were observed in this experiment. Moreover, NUL-RES animals tended to have and had a greater UBF on days 90 and 110, respectively, when compared to MUL-RES. We also found that PI, RI, and UBF did not respond to treatment similarly. On days 50 and 100, in which PI and RI were greater and tended to be greater in MUL-CON vs. NUL-CON, UBF was not different between them. Similarly, on day 70, RI and PI, respectively, were greater and tended to be greater when comparing NUL-RES vs. MUL-RES and had a similar UBF. The only day in which PI and RI values responded the same as UBF was on day 90, in the comparison between NUL-RES vs. MUL-RES. These findings suggest that the generalized idea of PI and RI being inversely related to UBF is not always true (Elmetwally et al., 2016; Beltrame et al., 2017; Elmetwally and Meinecke-Tillmann, 2018). There could be other factors such as tissue-specific vessel properties that influence these results.

Experiment 1 showed that a 40% nutrient restriction in mid and mid-to-late gestation does not influence kidney measurements. These results were corroborated in experiment 2; therefore, we conclude that there was no effect of any of the treatments in the kidney size of the developing fetus. The kidneys, unlike other organs of the developing fetus (e.g., small intestine, spleen), seem not to be affected by a lower nutrient availability in mid to late gestation (50 to 130 days of gestation; Osgerby et al., 2002; Lemley et al., 2012). In experiment 2, we saw a three-way interaction in biparietal distance. This interaction effect was probably driven by the lesser biparietal distance in NUL-RES ewes on day 70 of gestation. Placental growth occurs during the first two-thirds of pregnancy (Redmer et al., 2004), with fetal growth being exponential during the last third of pregnancy (Redmer et al., 2004). Nevertheless, some studies have shown that when a severe nutrient restriction is applied during mid-gestation, and the fetuses are collected at the end of the restriction period (before the last period of gestation; no realimentation), fetal weight can be affected (Murdoch et al., 2003; Zhou et al., 2008; Ma et al., 2011). Abdominal width, however, as well as the remaining days of biparietal width measurements were not affected by nutritional treatment. Similarly, these results are analogous to what we saw in experiment 1 and seems to demonstrate that fetal growth in adult white face ewes seems to be protected against a 40% nutrient restriction during mid-gestation.

In both experiments, we found that a 40% nutrient restriction during mid-gestation did not affect birth weights. Furthermore, others have demonstrated that moderate to severe nutrient restriction applied during early-to-mid and mid-to-late gestation periods have also shown no effects on birth weight (Gilbert et al., 2005; Sebert et al., 2008; Kotsampasi et al., 2009; Sharkey et al., 2009; Lekatz et al., 2013). These results corroborate that the majority of the fetal development occurs during the last third of pregnancy (Redmer et al., 2004). Studies in cows, mares, and sheep have shown an influence of parity in birth weight (Kayisiz et al., 2011; Yakubu et al., 2014; Abdel-Mageed and El-Gawad, 2015; Klewitz et al., 2015; Lv et al., 2015). In our study, parity did not have an effect on birth weight. This is opposite to another study done in cross-bred sheep in which nulliparous dam lambs had lower birth weights than multiparous animals (Yakubu et al., 2007). Breed differences could be influencing the dissimilar results.

Although the number of animals in experiment 2 is not enough to make inferences between birth-related problems and treatments, it is interesting that NUL animals seemed, at least numerically, to have more parturition problems than MUL animals and that NUL-RES animals seemed to be more prone to these problems than any of the other treatment groups. In sheep, some studies show higher lambing difficulty and early lamb mortality in nulliparous ewes (Kelly et al., 1992; Southey et al., 2004; McHugh et al., 2016), while others show no difference between nulliparous and multiparous animals (Leontides et al., 2000).

Nutrient restriction during mid-gestation in multiparous cows showed that uterine blood flow did not decrease during the restriction period; however, it increased upon realimentation (Camacho et al., 2014). In this study, the detrimental effects of a 40% nutrient restriction on ewe body weights were seen in both experiments. However, our findings from both experiments suggest that UBF in adult white-face sheep is resistant to changes with a 40% mid-gestation nutrient restriction, and there is not an increase in UBF upon realimentation. The decreased UBF observed in restricted adolescent nulliparous ewes (Lemley et al., 2012) might be therefore an effect of age at first gestation. In the pregnant adult sheep, nutrient partitioning prioritizes the placenta and fetus (Redmer et al., 2004). However, the hierarchy of nutrient partitioning in adolescent pregnancy has a higher priority for maternal tissue growth and fat deposition (Redmer et al., 2004). Adolescent pregnant ewes compete for nutrient resources with the developing fetus (Redmer et al., 2004). Additionally, nulliparous adult animals have greater fetal, cotyledonary, and caruncular weights as well as a greater cotyledonary angiogenic factor expression than nulliparous adolescent ewes (Borowicz et al., 2005). Nutrient restriction during mid-pregnancy could exacerbate these differences. Furthermore, mid-gestation nutrient-restriction effects seen in other studies in sheep vary in breed, period of restriction, and severity of the restriction (Kelly et al., 1992; Reynolds et al., 2005). More research is needed to further examine the effects of age and nutrient restriction on fetal development.

The raw data supporting the conclusions of this article will be made available by corresponding author under reasonable request.

Animal care and use for both experiments were according to protocols approved by the North Dakota State University Animal Care and Use Committee (#A15076).

MV-H performed and supervised the experiment, collected the samples, and analyzed and interpreted the data. KS assisted with the data and tissue collection, conceptualization, and supervision. KV supervised the data and tissue collection, conceptualization, funding acquisition, formal analysis, project administration, and writing—review and editing. All authors contributed to the article and approved the submitted version.

This project was supported in part by the Agriculture and Food Research Initiative Competitive Grant no. 2016-67016-24884 from the USDA National Institute of Food and Agriculture.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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