Stillbirth is defined as the birth of a dead fetus. Many factors such as maternal, piglet, environmental, and genetic factors have been associated with stillbirths [1]. According to the review by Vanderhaeghe et al [1], maternal factors are body condition, litter size, parity, gestation length and farrowing duration; piglet factors are birth interval, birth order, and birth weight; and environmental factors are nutrition, stress, insufficient ventilation and so on. In addition, there are differences in the incidence of stillbirth among the lines or breeds of pigs that may involve various complicated genetic factors [2,3]. Recently, genome wide association analyses were applied to studies of stillbirth events, and several candidate genes associated with increased stillbirth were mapped on a few chromosomal regions in maternal and terminal Landrace, Duroc, and Yorkshire lines [4]. However, the specific genes responsible for stillbirth have not been identified so far.

The major histocompatibility complex (MHC) molecules that present non-self and self-antigens to T cells and have crucial roles in inducing antigen-specific immune responses and preventing infectious diseases [5–7] might also have a direct or indirect role in stillbirth events. The MHC molecules influence various biological traits, such as immune recognition, autoimmunity, mating preferences, and pregnancy outcomes [8]. Furthermore, associations between specific swine leukocyte antigen (SLA) alleles or haplotypes and productive or reproductive traits such as ovulation rates, litter sizes, and gestation periods have been reported in several breeds of pigs [9–12].

Microminipigs (MMPs) were developed as miniature pigs with an extra-small body size for experimental use [13]. The population of MMPs are distinguished by eight different kinds of SLA class II haplotypes [14] that could be used as genetic markers for analyses of associations with specific phenotypes for various productive or reproductive traits and outcomes. In this regard, we previously reported associations of the SLA haplotypes with body weights [15] and some reproductive traits, such as gestation periods, fertility indices, litter sizes, and numbers of abnormal newborn piglets in MMPs [16]. Moreover, stillborn piglets in the MMP population are a serious recurring problem and the reduction of the risk factors is important for a more effective farrowing management of MMP and other commercial pigs. In the present study, to reduce stillbirths and the associated risk factors in the MMP population, we investigated relationships between stillbirth and factors such as litter sizes, parities, and gestation periods, as well as their genetic association with eight SLA class II haplotypes.

The incidences of stillbirths in domesticated pigs of commercial breeds vary between 3% and 8% [1,17,18]. In our population of MMPs, the incidence of stillbirths was 17.7%, which is more than double the rate of that in pigs of commercial breeds. The relatively high incidence of stillborn piglets in MMPs seems to be associated with their extra small body sizes [13]. The average litter size was 5.48±0.04 (mean±standard error) in a population of MMPs of our previous study [16], which is almost the same value as that in the AA group (5.36± 0.12) of the present study. On the other hand, the AS group had the smallest average litter size (3.87±0.39) compared to the PS (6.38±0.14) or AA group. In contrast, large litter sizes and small body weights at birth were suggested to be risk factors for stillbirth in domesticated pigs [3,19] and that might lead to the extension of farrowing duration and induce the stillbirth [2,18,20,21]. Although the occurrence of the stillbirth might be attributed to large litter sizes, the distribution patterns of litter sizes were normal in the PS and AA groups, but not in the AS group of MMPs. Also in domestic pigs, larger stillbirth numbers were commonly associated with higher parities [2,19] and/or in the first parity dams possibly due to a narrower birth canal at the first farrowing [2,18,19]. In MMPs, the percentage of parity in each group tended to decrease gradually as the parity increased in all three groups. Therefore, we could not conclude from our findings that there was a direct relationship between the occurrence of stillbirth and parity in the AS group.

Recently, genome wide association studies (GWAS) were applied for identification of affecting regions of average birth interval and stillbirth in swine [4]. Furthermore, to identify possible candidate genes affecting average birth interval and stillbirth, GWAS were also undertaken in Landrace-Duroc-Yorkshire females by Schneider et al [4]. ADAM metallopeptidase with thrombospondin type 1 motif, 9 (ADAMTS9), collagen 19 (COL19A1), nucleotide binding protein-like (NUBPL), LOC100518697 (a nostrin-like gene), and dipeptidyl-peptidase 10 (DPP10) were identified as the candidate genes that might help in improving marker assisted selection. However, it has not been revealed how these candidate genes might be involved in the average birth interval and occurrences of stillbirth. In a more recent study using GWAS, Sanglard et al [22] showed that a single nucleotide polymorphism (SNP, H3GA0020505) on the SLA class II region was associated strongly with reproductive performance including with the number of liveborn and stillborn piglets in PRRSV vaccinated commercial sows. However, no candidate genes involving reproductive performance were identified near the SNP.

In the present study, we analyzed some associations be tween the SLA class II haplotypes and stillbirth in MMPs. Comparison of SLA class II haplotype frequencies in piglets, sires, or dams among three groups, AS, PS, and AA groups, exhibited that Lr-0.7, Lr-0.16, and Lr-0.23 were associated with more stillbirths, whereas Lr-0.11 and Lr-0.13 were associated with fewer stillbirths. In a MMP population, body weights at birth and 50 days after birth were relatively low in pigs with Lr-0.7 and Lr-0.23, and relatively high in pigs with Lr-0.13 compared to those with the other five haplotypes [15]. We also found that the two haplotypes, Lr-0.7 or Lr-0.23, were associated with high stillbirth rates. Therefore, differences in body sizes associated with SLA class II haplotypes might be involved with occurrences of stillbirth in MMPs, however, Lr-0.7 was the lowest frequency haplotype in the MMP population [16]. The same SLA class II high resolution haplotype (Hp), Hp-0.7, also was identified previously in Yucatan miniature pigs, but associations between the haplotype and reproductive traits such as number of stillbirths per delivery were not reported for this breed [23]. On the other hand, Lr-0.7 was assigned with high frequencies in both the piglets and the dams in the AS group. Also, we observed in our previous study that the number of stillbirths per delivery tended to be larger in dams and sires with Lr-0.7, although the statistically significance could not be observed between Lr-0.7 and the other seven haplotypes due to the low frequency of MMPs with Lr-0.7 [16]. Thus, the SLA class II haplotype Lr-0.7 appears to be the most strongly associated with high stillbirth numbers and poor reproductive fitness. Furthermore, pigs with Lr-0.7 might disappear from the MMPs population in the near future if specific breeding programs are not performed to conserve them. Moreover, the SLA class II genes or haplotypes might not be directly responsible for occurrence of stillborn piglets. Although the polymorphic features of SLA genes or haplotypes might correlate only indirectly with stillbirth of MMPs, it nevertheless is evident from the present study that SLA class II haplotypes could be useful genetic markers for a more effective breeding management of MMPs to produce lower rates of stillbirths.

In this study, we focused our attention on an examination of the associations between SLA class II haplotypes and stillbirth rates. In future investigations, it would be relevant to extend our present study on SLA haplotype associations by including both the MHC class I and II genomic regions and molecules in relation to stillbirth rates in the MMPs population and other pig breeds with various SLA haplotypes.