Because of the critical function of the placenta for exchange of gases, nutrients, and wastes to developing fetuses, the porcine placenta plays a major role in uterine capacity, fetal growth and survival, which further influences postnatal piglet growth and survival. Uterine crowding also influences fetal growth and placental development, resulting in reduced fetal weights [27, 31] and altered placental size, structure, and function [4, 7]; thereby, providing a mechanism for increased within-litter birth weight variation and smaller piglets that result in increased preweaning piglet mortality. Unique pig breeds can serve as useful models of reproductive prolificacy and improved preweaning mortality, particularly the Chinese Meishan pig, which has decreased preweaning mortality rates compared to Western breeds despite having large litters with smaller average piglet weights [13, 14]. In the present study, we report breed differences in placental development between MS and WC gilts in response to intrauterine crowding, both on gross and histological levels. These differences may indicate that mechanisms for accommodating intrauterine crowding differ between MS and WC breeds.
In the current study, ovulation rate was greater for similar-aged MS gilts compared to WC gilts following the UHO procedure to induce intrauterine crowding. The corresponding ovulation rates from MS and WC gilts observed in this study were similar to the ovulation rates from intact MS and WC gilts . This first reported use of the UHO model in MS females illustrates that the UHO procedure caused compensatory ovarian hypertrophy, resulting in similar ovulation rates to intact reproductive tracts but with only one ovary and half of the uterine space; thereby, inducing uterine crowding . The breed differences in ovulation observed in the current study support previous literature from intact females illustrating that age-matched MS dams have greater ovulation rate compared with Western breed dams [12, 16, 32]. This difference in ovulation rate is likely due to the reduced age (~ 80–100 days) for MS females to undergo puberty compared to WC females [32, 33]; thereby, resulting in 3–5 more ovulations at a given age for MS females.
Despite differences in ovulation rates, there were no breed differences in uterine capacity or fetal survival in the current study following intrauterine crowding. Inconsistencies in litter size and fetal survival have been reported when comparing differences between MS and Western breed females with intact uteri. Studies evaluating litter size from purebred MS to purebred Western breeds (i.e., Large White or Yorkshire) of various ages illustrate increased litter size for MS females compared to purebred Western breeds [11, 34]. However, Haley et al. reported no differences in prenatal survival between Large White and MS females in their 1st and 2nd parities . In contrast, White et al. demonstrated that 2nd parity MS females had reduced embryo survival during mid-gestation (~ day 50) compared to Yorkshire . Furthermore, limited differences in litter size and fetal survival during early gestation (day 30) have been reported between purebred MS and White crossbreds from intact gilts of the same age . Several investigators have hypothesized improved uterine capacity from MS females as a mechanism from improved reproductive prolificacy [3, 18, 19]. However, all studies to date evaluating litter size between MS and Western breeds have involved intact females and did not fully assess uterine capacity due to the influence of ovulation rate on litter size, particularly in the young female . As a result, to fully assess uterine capacity, it is necessary to crowd the uterine environment either by superovulation, embryo transfer, or surgically using the UHO to gain a full measure of uterine capacity. Contrary to previous hypotheses, the results of the current study following induced intrauterine crowding do not seem to fully support that MS females have greater uterine capacity compared to their Western breed contemporaries. Although uterine capacity was not significantly different in this study, there was a numerical increase in uterine capacity observed in MS pregnancies with limited number of gilt observations in the current study. Therefore, a larger scale evaluation of uterine capacity may be necessary to fully evaluate this hypothesis.
Fetal and placental weights were not different at day 100 of gestation from MS or WC pregnancies following intrauterine crowding in the current study. This finding was unexpected and contradictory to the literature comparing weights of the fetus, placenta, and corresponding piglet birth weight from intact dams illustrating significant reductions in fetal [35–37], placental [17, 32], and piglet birth [11, 12, 24] weight from MS pregnancies compared to Western breeds. Intrauterine crowding induced in dams using the UHO procedure results in decreased fetal and placental weights when compared to intact dams throughout gestation [38, 39]. Given this observation, one plausible explanation for the limited differences in fetal and placental weights between MS and WC pregnancies may be reduced sensitivity to intrauterine crowding on fetal and placental growth within MS pregnancies; thereby, resulting in limited reductions in fetal and placental weights in MS pregnancies as compared to WC pregnancies in response to intrauterine crowding following UHO. Intrauterine crowding highly influences fetal growth and subsequent within-litter birth weight variation [40–42]. However, previous reports indicate that MS conceptuses elongate to a lesser extent during early pregnancy, reducing negative interactions between adjacent conceptuses [17, 43]. A reduction in the sensitivity to intrauterine crowding in MS gilts is further supported by decreased within-litter fetal weight variation from MS pregnancies compared to WC observed in the current study. Finally, when comparing the allometric growth rate between MS and WC gilts following intrauterine crowding, there was a reduction in the allometric growth rate in MS pregnancies compared to WC. Allometric growth measures the sensitivity of deviations of growth between fetus and placenta , where a slope (i.e., allometric growth rate) of 1 in this relationship indicates proportional growth and slopes significantly less than 1 illustrates a fetal sparing effect unaffected by changes in placental growth . Taken together, these results illustrate that fetal and placental development from MS pregnancies are less sensitive to intrauterine crowding when compared to WC pregnancies. Although the exact mechanisms for limited sensitivity in fetal growth to intrauterine crowding observed in MS pregnancies are not clear, one likely mechanism involves the development and function of the placenta.
To evaluate breed difference in placental development following intrauterine crowding in the current study, an assessment of divergent-sized littermates based on fetal weight (i.e., smallest and largest littermates) was made for both gross and histological morphology of placentas between MS and WC pregnancies. As expected, there were significant differences in the weights of the fetus and placenta from the smallest and largest littermates. However, like the overall litter average, there were no differences in fetal or placental weights between MS and WC when evaluating fetal size, further supporting the hypothesis that MS fetuses are less sensitive to intrauterine crowding on a weight basis. Interestingly, placental efficiency was not different between divergent-sized littermates or between the two breeds and this lack of breed difference for placental efficiency was also observed for the litter average in the current study. This finding contradicts reports of increased placental efficiency in purebred MS compared to Yorkshire pregnancies from intact uteri [19–21], but is supportive of data from comparing divergent-sized littermates in a crowded uterine environment following UHO from WC gilts throughout pregnancy . Given that the allometric growth (i.e., natural log relationship between fetal and placental weight) was significantly less than 1 from both breeds and lowest in MS pregnancies in the current study, lack of breed and fetal size differences in placental efficiency indicates that fetal growth is not proportional to placental growth and further supports the hypothesis that fetal to placental weight ratio (i.e., placental efficiency) is not indicative of the entire function of the placenta on fetal growth, particular during late gestation and in a crowded uterine environment . Therefore, other aspects of placental development and function likely explain the breed differences in limiting reductions in fetal placental growth as observed in MS pregnancies.
When evaluating gross morphology of placentas from divergent-sized littermates following intrauterine crowding in MS or WC pregnancies, volume displacement, surface area, and length of placentas were decreased in the smallest littermates compared to largest contemporaries, regardless of breed. During the last few days of gestation (< day 110), Vonnahme et al. and Bienson et al. reported increased surface area from purebred Yorkshire placentas compared to purebred MS from intact or co-breed (i.e., MS and Yorkshire embryos into either MS or Yorkshire dams) reciprocal embryo transfer pregnancies, respectively [21, 45]. However, Bienson et. al. reported no breed differences in surface area of placentas at day 90 of gestation following co-breed reciprocal embryo transfer . As a result, lack of breed difference in surface area in the current study could be due to timing in which placentas were evaluated (day 100) intermediate of the previous sampling time points and therefore, breed difference may not appear until the very end of pregnancy. Alternatively, the limited breed differences observed in surface area in the current study could be the influence of intrauterine crowding as the previous studies were performed with intact females with litter size less than 10 fetal pigs [21, 45]. Interestingly, the length of MS placentas, irrespective of size, were longer than WC placentas; whereas, the width of the placentas for the largest littermate from WC pregnancies had the wider placentas compared to all other groups, indicating size difference in placental width from WC pregnancies, but not for MS pregnancies following intrauterine crowding. Increased placental length in MS pregnancies further supports reduced sensitivity to intrauterine crowding for MS pregnancies given that placental length decreases following intrauterine crowding compared to intact controls . Increased number of fetuses has been shown to increase uterine length particularly during later stages of gestation . Although uterine length was not recorded in the current study, one likely mechanism for increased length of placentas from MS pregnancies may have resulted from increased uterine length in MS pregnancies possibly driven by the numerical increase in uterine capacity observed in the current study.
In the current study, there was a breed by fetal size interaction for placental areolae density illustrating increased areolae density from MS pregnancies compared to WC pregnancies. Although largest littermate fetuses in MS pregnancies had increased areolae densities compared to their smaller littermate contemporaries, smaller MS placentas had greater areolae density compared to either sized fetus of WC placentas. Furthermore, visual evaluation of placental areolae between the breeds, irrespective of fetal size (Fig. 1), illustrated that MS placentas had more pronounced and distinct areolae compared to WC placentas. Placental areolae form over the uterine glands and play a critical role in uptake of glandular secretions (i.e., histotrophic exchange) into the placenta [47, 48]. Knight et al. has demonstrated that intrauterine crowding using the UHO model results in a reduction in both the density and total number of placental areolae throughout gestation compared to non-crowded intact controls . Although histotrophic exchange is primarily thought to provide primary nutrient exchange during pre- and peri-implantation stages of gestation , histological and biochemical analysis of glandular secretions at the maternal gland and placental areolae interface illustrate the importance of these glandular secretions throughout gestation, particularly during late gestation when fetal growth is maximal [8, 49]. Given the importance of the placental areolae for the uptake of glandular secretion, the increased density and development of the areolae in MS pregnancies compared to WC pregnancies following intrauterine crowding provides a mechanism by which MS pregnancies have reduced effect of intrauterine crowding on fetal and placental growth observed in the current study.
On a histological level, the maternal-placental interface of the pig placenta consists of a folded bilayer of intact uterine epithelium and trophectoderm embedded in loose placental stroma (Fig. 2; [6, 7]). In the current study, the widths of the total placental interface and folded bilayer were reduced in MS placentas compared to WC placentas, irrespective of fetal size. Hong et. al. also reported decreased folded bilayer width from MS and Yorkshire placentas during late gestation . Conversely, these authors reported an assessment of fold complexity based on the fold length per unit area of placenta illustrating greater complexity in folded bilayer from MS gilts compared to Yorkshire and suggested that this complexity was due to greater expression and levels of heparanase in MS placentas resulting in modification of the folded bilayer . Although these results suggest compensatory mechanisms within the MS placentas to improve surface area at the maternal-placental interface, it is not clear as to what type of influence litter size and fetal size plays in this model as no information of fetal size nor litter size of sampled placentas were reported in this study .
The folded bilayer width was also greater from the smallest littermate fetus within both breeds in the current study. Conversely, placental stroma weight was reduced from placentas of the smallest littermate fetus, again regardless of breed. Vallet and Freking previously demonstrated that smallest fetal littermates have deeper folded bilayer, but decreased placental stromal depth compared to the larger contemporaries following intrauterine crowding in gilts . These investigators hypothesized that differences in placental histology between divergent-sized littermates following intrauterine crowding result in a compensatory response due to reductions in uterine surface access from smallest littermates to increase maternal-fetal interface by increasing the surface area; thereby, resulting in deeper folded bilayer at the expense of the placental stroma. However, at a certain point the placental stroma becomes limited and thus provides a mechanism that limits growth and development of smaller littermates resulting in greater susceptibility for pre- and post-natal mortality . Although the current study findings support the compensatory hypothesis that small littermates have increased folded bilayer width but decreased stromal width, regardless of breed, the breed difference in divergent-sized littermates for placental stromal width was significantly reduced from MS pregnancies compared to WC (Fig. 4). This illustrates that MS placentas might be less sensitive to limitations in placental stroma development between divergent-sized littermates, or alternatively they may have mechanisms for improved maintenance of placental stroma between littermates. The placental stroma is composed of many glycoproteins and glycosaminoglycans like hyaluronan and heparan sulfate . Biochemical analysis of the composition within the placental areolae illustrate many glycoproteins, proteoglycans and glycosaminoglycans are present in these structures . Furthermore, uterine glands juxtaposed to placental areolae produce and secrete many growth factors, proteases, enzymes, transport proteins, and adhesion proteins, which likely play a role in the development and modification of placenta . Therefore, maintenance of placental stroma between littermates maybe improved in MS placentas due to enhanced development and function of the placental areolae.