Deciphering Estrus Expression in Gilts: The Role of Alternative Polyadenylation and LincRNAs in Reproductive Transcriptomics.

The fertility rate and litter size of female pigs are critically affected by the expression of estrus. The objective of this study was to elucidate the regulatory mechanisms of estrus expression by analyzing the differential expression of genes and long intergenic non-coding RNAs (lincRNA), as well as the utilization of alternative polyadenylation (APA) sites, in the vulva and vagina during the estrus and diestrus stages of Large White and indigenous Chinese Mi gilts. Our study revealed that the number of differentially expressed genes (DEG) in the vulva was less than that in the vagina, and the DEGs in the vulva were enriched in pathways such as "neural" pathways and steroid hormone responses, including the "Calcium signaling pathway" and "Oxytocin signaling pathway". The DEGs in the vagina were enriched in the "Metabolic pathways" and "VEGF signaling pathway". Furthermore, 27 and 21 differentially expressed lincRNAs (DEL), whose target genes were enriched in the "Endocrine resistance" pathway, were identified in the vulva and vagina, respectively. Additionally, we observed that 63 and 618 transcripts of the 3'-untranslated region (3'-UTR) were lengthened during estrus in the vulva and vagina, respectively. Interestingly, the genes undergoing APA events in the vulva exhibited species-specific enrichment in neural or steroid-related pathways, whereas those in the vagina were enriched in apoptosis or autophagy-related pathways. Further bioinformatic analysis of these lengthened 3'-UTRs revealed the presence of multiple miRNAs binding sites and cytoplasmic polyadenylation element (CPE) regulatory aspects. In particular, we identified more than 10 CPEs in the validated lengthened 3'-UTRs of the NFIX, PCNX4, CEP162 and ABHD2 genes using RT-qPCR. These findings demonstrated the involvement of APA and lincRNAs in the regulation of estrus expression in female pigs, providing new insights into the molecular mechanisms underlying estrus expression in pigs.

Genomic background of biotypes related to growth, carcass and meat quality traits in Duroc pigs based on principal component analysis.

As the swine industry continues to explore pork quality traits alongside growth, feed efficiency and carcass leanness traits, it becomes imperative to understand their underlying genetic relationships. Due to this increase in the number of desirable traits, animal breeders must also consider methods to efficiently perform direct genetic changes for each trait and evaluate alternative selection indexes with different sets of phenotypic measurements. Principal component analysis (PCA) and genome-wide association studies (GWAS) can be combined to understand the genetic architecture and biological mechanisms by defining biological types (biotypes) that relate these valuable traits. Therefore, the main objectives of this study were to: (1) estimate genomic-based genetic parameters; (2) define animal biotypes utilizing PCA; and (3) utilize GWAS to link the biotypes to candidate genes and quantitative trait loci (QTL). The phenotypic dataset included 2583 phenotypic records from female Duroc pigs from a terminal sire line. The pedigree file contained 193,764 animals and the genotype file included 21,309 animals with 35,651 single nucleotide polymorphisms (SNPs). Eight principal components (PCs), accounting for a total of 99.7% of the population variation, were defined for three growth, eight conventional carcass, 10 pork quality and 18 novel carcass traits. The eight biotypes defined from the PCs were found to be related to growth rate, maturity, meat quality and body structure, which were then related to candidate genes. Of the 175 candidate genes found, six of them [LDHA (SSC1), PIK3C3 (SSC6), PRKAG3 (SSC15), VRTN (SSC7), DLST (SSC7) and PAPPA (SSC1)] related to four PCs were found to be associated with previously defined QTL, linking the biotypes with biological processes involved with muscle growth, fat deposition, glycogen levels and skeletal development. Further functional analyses helped to make connections between biotypes, relating them through common KEGG pathways and gene ontology (GO) terms. These findings contribute to a better understanding of the genetic relationships between growth, carcass and meat quality traits in Duroc pigs, enabling breeders to better understand the biological mechanisms underlying the phenotypic expression of these traits.

Characterization and analysis of transcriptomes of multiple tissues from estrus and diestrus in pigs.

A comprehensive understanding of the complex regulatory mechanisms governing estrus and ovulation across multiple tissues in mammals is imperative to improve the reproductive performance of livestock and mitigate ovulation-related disorders in humans. To comprehensively elucidate the regulatory landscape, we analyzed the transcriptome of protein-coding genes and long intergenic non-coding RNAs (lincRNAs) in 58 samples (including the hypothalamus, pituitary, ovary, vagina, and vulva) derived from European Large White gilts and Chinese Mi gilts during estrus and diestrus. We constructed an intricate regulatory network encompassing 358 hub genes across the five examined tissues. Furthermore, our investigation identified 85 differentially expressed lincRNAs that are predicted to target 230 genes associated with critical functions including behavior, receptors, and apoptosis. Importantly, we found that vital components of estrus and ovulation events involve "Apoptosis" pathway in the hypothalamus, "Autophagy" in the ovary, as well as "Hypoxia" and "Angiogenesis" in the vagina and vulva. We have identified several differentially expressed transcription factors (TFs), such as SPI1 and HES2, which regulate these pathways. SPI1 may suppress transcription in the autophagy pathway, promoting apoptosis and inhibiting the proliferation of ovarian granulosa cells. Our study provides the most comprehensive transcriptional profiling information related to estrus and ovulation events.

Genomic predictions and GWAS for heat tolerance in pigs based on reaction norm models with performance records and data from public weather stations considering alternative temperature thresholds.

Genetic improvement of livestock productivity has resulted in greater production of metabolic heat and potentially greater susceptibility to heat stress. Various studies have demonstrated that there is genetic variability for heat tolerance and genetic selection for more heat tolerant individuals is possible. The rate of genetic progress tends to be greater when genomic information is incorporated into the analyses as more accurate breeding values can be obtained for young individuals. Therefore, this study aimed (1) to evaluate the predictive ability of genomic breeding values for heat tolerance based on routinely recorded traits, and (2) to investigate the genetic background of heat tolerance based on single-step genome-wide association studies for economically important traits related to body composition, growth and reproduction in Large White pigs. Pedigree information was available for 265,943 animals and genotypes for 8686 animals. The studied traits included ultrasound backfat thickness (BFT), ultrasound muscle depth (MDP), piglet weaning weight (WW), off-test weight (OTW), interval between farrowing (IBF), total number of piglets born (TNB), number of piglets born alive (NBA), number of piglets born dead (NBD), number of piglets weaned (WN) and weaning-to-estrus interval (IWE). The number of phenotypic records ranged from 6059 (WN) to 172,984 (TNB). Single-step genomic reaction norm predictions were used to calculate the genomic estimated breeding values for each individual. Predictions of breeding values for the validation population individuals were compared between datasets containing phenotypic records measured in the whole range of temperatures (WR) and datasets containing only phenotypic records measured when the weather station temperature was above 10°C (10C) or 15°C (15C), to evaluate the usefulness of these datasets that may better reflect the within-barn temperature. The use of homogeneous or heterogeneous residual variance was found to be trait-dependent, where homogeneous variance presented the best fit for MDP, BFT, OTW, TNB, NBA, WN and IBF, while the other traits (WW and IWE) had better fit with heterogeneous variance. The average prediction accuracy, dispersion and bias values considering all traits for WR were 0.36 ± 0.05, -0.07 ± 0.13 and 0.76 ± 0.10, respectively; for 10C were 0.39 ± 0.02, -0.05 ± 0.07 and 0.81 ± 0.05, respectively; and for 15C were 0.32 ± 0.05, -0.05 ± 0.11 and 0.84 ± 0.10, respectively. Based on the studied traits, using phenotypic records collected when the outside temperature (from public weather stations) was above 10°C provided better predictions for most of the traits. Forty-three and 62 candidate genomic regions were associated with the intercept (overall performance level) and slope term (specific biological mechanisms related to environmental sensitivity), respectively. Our results contribute to improve genomic predictions using existing datasets and better understand the genetic background of heat tolerance in pigs. Furthermore, the genomic regions and candidate genes identified will contribute to future genomic studies and breeding applications.

Longitudinal genomic analyses of automatically-recorded vaginal temperature in lactating sows under heat stress conditions based on random regression models.

BACKGROUND: Automatic and continuous recording of vaginal temperature (TV) using wearable sensors causes minimal disruptions to animal behavior and can generate data that enable the evaluation of temporal body temperature variation under heat stress (HS) conditions. However, the genetic basis of TV in lactating sows from a longitudinal perspective is still unknown. The objectives of this study were to define statistical models and estimate genetic parameters for TV in lactating sows using random regression models, and identify genomic regions and candidate genes associated with HS indicators derived from automatically-recorded TV. RESULTS: Heritability estimates for TV ranged from 0.14 to 0.20 over time (throughout the day and measurement period) and from 0.09 to 0.18 along environmental gradients (EG, - 3.5 to 2.2, which correspond to dew point values from 14.87 to 28.19 ËšC). Repeatability estimates of TV over time and along EG ranged from 0.57 to 0.66 and from 0.54 to 0.77, respectively. TV measured from 12h00 to 16h00 had moderately high estimates of heritability (0.20) and repeatability (0.64), indicating that this period might be the most suitable for recording TV for genetic selection purposes. Significant genotype-by-environment interactions (GxE) were observed and the moderately high estimates of genetic correlations between pairs of extreme EG indicate potential re-ranking of selection candidates across EG. Two important genomic regions on chromosomes 10 (59.370-59.998 Mb) and16 (21.548-21.966 Mb) were identified. These regions harbor the genes CDC123, CAMK1d, SEC61A2, and NUDT5 that are associated with immunity, protein transport, and energy metabolism. Across the four time-periods, respectively 12, 13, 16, and 10 associated genomic regions across 14 chromosomes were identified for TV. For the three EG classes, respectively 18, 15, and 14 associated genomic windows were identified for TV, respectively. Each time-period and EG class had uniquely enriched genes with identified specific biological functions, including regulation of the nervous system, metabolism and hormone production. CONCLUSIONS: TV is a heritable trait with substantial additive genetic variation and represents a promising indicator trait to select pigs for improved heat tolerance. Moderate GxE for TV exist, indicating potential re-ranking of selection candidates across EG. TV is a highly polygenic trait regulated by a complex interplay of physiological, cellular and behavioral mechanisms.

Genetic parameters for automatically-measured vaginal temperature, respiration efficiency, and other thermotolerance indicators measured on lactating sows under heat stress conditions.

BACKGROUND: Genetic selection based on direct indicators of heat stress could capture additional mechanisms that are involved in heat stress response and enable more accurate selection for more heat-tolerant individuals. Therefore, the main objectives of this study were to estimate genetic parameters for various heat stress indicators in a commercial population of Landrace × Large White lactating sows measured under heat stress conditions. The main indicators evaluated were: skin surface temperatures (SST), automatically-recorded vaginal temperature (TV), respiration rate (RR), panting score (PS), body condition score (BCS), hair density (HD), body size (BS), ear size, and respiration efficiency (Reff). RESULTS: Traits based on TV presented moderate heritability estimates, ranging from 0.15 ± 0.02 to 0.29 ± 0.05. Low heritability estimates were found for SST traits (from 0.04 ± 0.01 to 0.06 ± 0.01), RR (0.06 ± 0.01), PS (0.05 0.01), and Reff (0.03 ± 0.01). Moderate to high heritability values were estimated for BCS (0.29 ± 0.04 for caliper measurements and 0.25 ± 0.04 for visual assessments), HD (0.25 ± 0.05), BS (0.33 ± 0.05), ear area (EA; 0.40 ± 0.09), and ear length (EL; 0.32 ± 0.07). High genetic correlations were estimated among SST traits (> 0.78) and among TV traits (> 0.75). Similarly, high genetic correlations were also estimated for RR with PS (0.87 ± 0.02), with BCS measures (0.92 ± 0.04), and with ear measures (0.95 ± 0.03). Low to moderate positive genetic correlations were estimated between SST and TV (from 0.25 ± 0.04 to 0.76 ± 0.07). Low genetic correlations were estimated between TV and BCS (from - 0.01 ± 0.08 to 0.06 ± 0.07). Respiration efficiency was estimated to be positively and moderately correlated with RR (0.36 ± 0.04), PS (0.56 ± 0.03), and BCS (0.56 ± 0.05 for caliper measurements and 0.50 ± 0.05 for the visual assessments). All other trait combinations were lowly genetically correlated. CONCLUSIONS: A comprehensive landscape of heritabilities and genetic correlations for various thermotolerance indicators in lactating sows were estimated. All traits evaluated are under genetic control and heritable, with different magnitudes, indicating that genetic progress is possible for all of them. The genetic correlation estimates provide evidence for the complex relationships between these traits and confirm the importance of a sub-index of thermotolerance traits to improve heat tolerance in pigs.

Postmortem Metabolism and Pork Quality Development Are Affected by Electrical Stimulation across Three Genetic Lines.

Variations in postmortem metabolism in muscle impact pork quality development. Curiously, some genetic lines are more refractile to adverse pork quality development than others and may regulate energy metabolism differently. The aim of this study was to challenge pork carcasses from different genetic populations with electrical stimulation (ES) to determine how postmortem metabolism varies with genetic line and explore control points that reside in glycolysis in dying muscle. Three genetic populations (GP) were subjected to ES (100 V or 200 V, 13 pulses, 2 s on/2 s off) at 15- or 25-min post-exsanguination, or no stimulation (NS). Genetic population affected relative muscle relative abundance of different myosin heavy chains, glycogen, G6P, and lactate concentrations. Genetic lines responded similarly to ES, but a comparison of ES treatment groups revealed a trend for an interaction between voltage, time of ES, and time postmortem. Higher voltage accelerated pH decline at 20 min up to 60 min postmortem. Trends in color and firmness scores and L* values were consistent with pH and metabolite data. These data show that genetic populations respond differently to postmortem perturbation by altering glycolytic flux and suggest differences in postmortem glycolysis may be partially responsible for differences in meat quality between genetic populations, though not entirely.

Evaluating phenotypes associated with heat tolerance and identifying moderate and severe heat stress thresholds in lactating sows housed in mechanically or naturally ventilated barns during the summer under commercial conditions.

An accurate understanding of heat stress (HS) temperatures and phenotypes that indicate HS tolerance is necessary to improve swine HS resilience. Therefore, the study objectives were 1) to identify phenotypes indicative of HS tolerance, and 2) to determine moderate and severe HS threshold temperatures in lactating sows. Multiparous (4.10 ± 1.48) lactating sows and their litters (11.10 ± 2.33 piglets/litter) were housed in naturally ventilated (n = 1,015) or mechanically ventilated (n = 630) barns at a commercial sow farm in Maple Hill, NC, USA between June 9 and July 24, 2021. In-barn dry bulb temperatures (TDB) and relative humidity were continuously recorded for naturally ventilated (26.38 ± 1.21 °C and 83.38 ± 5.40%, respectively) and mechanically ventilated (26.91 ± 1.80 °C and 77.13 ± 7.06%, respectively) barns using data recorders. Sows were phenotyped between lactation days 11.28 ± 3.08 and 14.25 ± 3.26. Thermoregulatory measures were obtained daily at 0800, 1200, 1600, and 2000 h and included respiration rate, and ear, shoulder, rump, and tail skin temperatures. Vaginal temperatures (TV) were recorded in 10 min intervals using data recorders. Anatomical characteristics were recorded, including ear area and length, visual and caliper-assessed body condition scores, and a visually assessed and subjective hair density score. Data were analyzed using PROC MIXED to evaluate the temporal pattern of thermoregulatory responses, phenotype correlations were based on mixed model analyses, and moderate and severe HS inflection points were established by fitting TV as the dependent variable in a cubic function against TDB. Statistical analyses were conducted separately for sows housed in mechanically or naturally ventilated barns because the sow groups were not housed in each facility type simultaneously. The temporal pattern of thermoregulatory responses was similar for naturally and mechanically ventilated barns and several thermoregulatory and anatomical measures were significantly correlated with one another (P < 0.05), including all anatomical measures as well as skin temperatures, respiration rates, and TV. For sows housed in naturally and mechanically ventilated facilities, moderate HS threshold TDB were 27.36 and 26.69 °C, respectively, and severe HS threshold TDB were 29.45 and 30.60 °C, respectively. In summary, this study provides new information on the variability of HS tolerance phenotypes and environmental conditions that constitute HS in commercially housed lactating sows.

Climate change and the associated increase in global temperatures have a well-described negative impact on swine production. Therefore, improving swine heat stress resilience is of utmost importance to reduce the deleterious effects of heat stress on swine health, performance, and welfare. Genomic selection for heat stress resilience may be a viable strategy to improve swine productivity in a changing climate. However, identifying environmental conditions that constitute heat stress and deriving novel traits that can be easily collected on farm and provide accurate and precise predictions of heat stress tolerance is a necessary step. The present study demonstrated that housing conditions had a limited influence on heat stress tolerance phenotypes, several anatomical and thermoregulatory measures were correlated, and housing conditions impacted heat stress threshold temperatures. Results from this study may be applied to large-scale phenotyping initiatives to develop or refine genomic selection indexes for heat stress resilience in pigs.

Feeding a whole-cell inactivated Pichia guilliermondii yeast to gestating and lactating sows in a commercial production system.

A total of 606 sows (PIC 1050) and their progeny (PIC 1050 × 280) were used to determine if feeding gestating and lactating sows a proprietary strain of Pichia guilliermondii as a whole-cell inactivated yeast product (WCY; CitriStim, ADM Animal Nutrition, Quincy, IL) improves sow and litter performance in a commercial production system. Once confirmed pregnant at d 35 post-breeding pregnancy check, sows were fed a basal gestation control (CON) diet (0.55% SID lysine) or the control diet fortified with 0.15% of the WCY replacing corn in the CON diet. Dietary treatments were also fed in lactation (1.05% SID lysine) once sows were moved into farrowing crates on approximately d 112 of gestation until weaning. Sows supplemented with WCY in gestation and lactation had increased total born piglets by 0.45 pigs (P < 0.04), piglets born alive (14.27 vs. 13.85; P < 0.04), and, therefore, heavier born alive litter weights (P < 0.001) compared to CON fed sows. A greater post cross-foster litter size (P < 0.001) meant that litter size at weaning was increased by 0.54 pigs when sows were fed WCY compared to CON (P < 0.001). However, litter weaning weights and 21-d adjusted litter weaning weights were similar (P > 0.158), although numerically greater, for WCY fed sows. Pigs from CON fed sows were 0.35 kg heavier at weaning compared to pigs from WCY fed sows (P < 0.001). This increase in weaning weight of pigs from CON fed sows is partially explained by their 0.93 d longer lactation (P < 0.001) and may also be due to the smaller litter size throughout lactation. The percent of litters treated for scours decreased from 38.3 to 14.2% when sows were fed WCY (P < 0.001). The distribution of birth and weaning weights was not impacted (P > 0.2461) by treatment. In conclusion, feeding gestating and lactating sows a proprietary strain of Pichia guilliermondii as a whole-cell inactivated yeast product increased the number of pigs born and weaned, and decreased the prevalence of scours during lactation.

The importance of optimal body condition to maximise reproductive health and perinatal outcomes in pigs.

Overnutrition or undernutrition during all or part of the reproductive cycle predisposes sows to metabolic consequences and poor reproductive health which contributes to a decrease in sow longevity and an increase in perinatal mortality. This represents not only an economic problem for the pig industry but also results in poor animal welfare. To maximise profitability and increase sustainability in pig production, it is pivotal to provide researchers and practitioners with synthesised information about the repercussions of maternal obesity or malnutrition on reproductive health and perinatal outcomes, and to pinpoint currently available nutritional managements to keep sows' body condition in an optimal range. Thus, the present review summarises recent work on the consequences of maternal malnutrition and highlights new findings.

A Functional Single Nucleotide Polymorphism in the 3' Untranslated Region of the Porcine JARID2 Gene Is Associated with Aggressive Behavior of Weaned Pigs after Mixing.

In pig production, pigs often show more aggressive behavior after mixing, which adversely affects animal welfare and growth performance. The Jumonji and structural domain-rich AT interaction domain 2 (JARID2) gene plays an important role in neurodevelopment in mice and various psychiatric disorders in humans. The JARID2 gene may impact the aggressive behavior of pigs. By observing the behavior of 500 weaned pigs during the first 72 h after mixing, the ear tissue samples of the 12 most aggressive and 12 least aggressive pigs were selected for DNA resequencing based on the intensity of their aggressive behavior. Large group correlation analysis indicated that the rs3262221458 site located in the 3'-UTR region of the porcine JARID2 gene has a strong relationship with the aggressive behavior of weaned pigs. Pigs with the mutant TT genotype of rs3262221458 have more aggressive behavior than those pigs with the GG and GT genotypes. The dual luciferase assay indicated that the luciferase activity of the plasmids containing the G allele of rs326221458 was significantly less than that of plasmids containing the T allele of rs326221458 and control groups. The binding ability of miR-9828-3p to sequences containing the T allele was less than that of sequences containing the G allele. The overexpression of miR-9828-3p in porcine neuroglial cells (PNGCs) and PK15 cells significantly decreased the mRNA and protein levels of the JARID2 gene. In addition, miR-9828-3p inhibited the proliferation of PNGCs. After inhibiting miR-9828-3p, the mRNA and protein expression levels of JARID2 increased, and the proliferation of PNGCs showed an opposite trend to the cells that forced the expression of miR-9828-3p. In addition, interference with the JARID2 gene by siRNA can effectively inhibit the proliferation of PNGCs. In summary, we found that the rs326221458 locus regulates the expression of the JARID2 gene by affecting the binding of miR-9828-3p and the JARID2 gene, thereby affecting the aggressive behavior of weaned pigs after mixing.

A behavior and physiology-based decision support tool to predict thermal comfort and stress in non-pregnant, mid-gestation, and late-gestation sows.

BACKGROUND: Although thermal indices have been proposed for swine, none to our knowledge differentiate by reproductive stage or predict thermal comfort using behavioral and physiological data. The study objective was to develop a behavior and physiology-based decision support tool to predict thermal comfort and stress in multiparous (3.28 ± 0.81) non-pregnant (n = 11), mid-gestation (n = 13), and late-gestation (n = 12) sows. RESULTS: Regression analyses were performed using PROC MIXED in SAS 9.4 to determine the optimal environmental indicator [dry bulb temperature (TDB) and dew point] of heat stress (HS) in non-pregnant, mid-gestation, and late-gestation sows with respiration rate (RR) and body temperature (TB) successively used as the dependent variable in a cubic function. A linear relationship was observed for skin temperature (TS) indicating that TDB rather than the sow HS response impacted TS and so TS was excluded from further analyses. Reproductive stage was significant for all analyses (P < 0.05). Heat stress thresholds for each reproductive stage were calculated using the inflections points of RR for mild HS and TB for moderate and severe HS. Mild HS inflection points differed for non-pregnant, mid-gestation, and late gestation sows and occurred at 25.5, 25.1, and 24.0 °C, respectively. Moderate HS inflection points differed for non-pregnant, mid-gestation, and late gestation sows and occurred at 28.1, 27.8, and 25.5 °C, respectively. Severe HS inflection points were similar for non-pregnant and mid-gestation sows (32.9 °C) but differed for late-gestation sows (30.8 °C). These data were integrated with previously collected behavioral thermal preference data to estimate the TDB that non-pregnant, mid-gestation, and late-gestation sows found to be cool (TDB < TDB preference range), comfortable (TDB = TDB preference range), and warm (TDB preference range < TDB < mild HS). CONCLUSIONS: The results of this study provide valuable information about thermal comfort and thermal stress thresholds in sows at three reproductive stages. The development of a behavior and physiology-based decision support tool to predict thermal comfort and stress in non-pregnant, mid-gestation, and late-gestation sows is expected to provide swine producers with a more accurate means of managing sow environments.

Genome-Wide Detection of Copy Number Variations and Evaluation of Candidate Copy Number Polymorphism Genes Associated With Complex Traits of Pigs.

Copy number variation (CNV) has been considered to be an important source of genetic variation for important phenotypic traits of livestock. In this study, we performed whole-genome CNV detection on Suhuai (SH) (n = 23), Chinese Min Zhu (MZ) (n = 11), and Large White (LW) (n = 12) pigs based on next-generation sequencing data. The copy number variation regions (CNVRs) were annotated and analyzed, and 10,885, 10,836, and 10,917 CNVRs were detected in LW, MZ, and SH pigs, respectively. Some CNVRs have been randomly selected for verification of the variation type by real-time PCR. We found that SH and LW pigs are closely related, while MZ pigs are distantly related to the SH and LW pigs by CNVR-based genetic structure, PCA, VST, and QTL analyses. A total of 14 known genes annotated in CNVRs were unique for LW pigs. Among them, the cyclin T2 (CCNT2) is involved in cell proliferation and the cell cycle. The FA Complementation Group M (FANCM) is involved in defective DNA repair and reproductive cell development. Ten known genes annotated in 47 CNVRs were unique for MZ pigs. The genes included glycerol-3-phosphate acyltransferase 3 (GPAT3) is involved in fat synthesis and is essential to forming the glycerol triphosphate. Glutathione S-transferase mu 4 (GSTM4) gene plays an important role in detoxification. Eleven known genes annotated in 23 CNVRs were unique for SH pigs. Neuroligin 4 X-linked (NLGN4X) and Neuroligin 4 Y-linked (NLGN4Y) are involved with nerve disorders and nerve signal transmission. IgLON family member 5 (IGLON5) is related to autoimmunity and neural activities. The unique characteristics of LW, MZ, and SH pigs are related to these genes with CNV polymorphisms. These findings provide important information for the identification of candidate genes in the molecular breeding of pigs.

Comprehensive Transcriptome Analysis of Follicles from Two Stages of the Estrus Cycle of Two Breeds Reveals the Roles of Long Intergenic Non-Coding RNAs in Gilts.

Visible and long-lasting estrus expression of gilts and sows effectively sends a mating signal. To reveal the roles of Long Intergenic Non-coding RNAs (lincRNAs) in estrus expression, RNA-seq was used to investigate the lincRNAs expression of follicular tissues from Large White gilts at diestrus (LD) and estrus (LE), and Chinese Mi gilts at diestrus (MD) and estrus (ME). Seventy-three differentially expressed lincRNAs (DELs) were found in all comparisons (LE vs. ME, LD vs. LE, and MD vs. ME comparisons). Eleven lincRNAs were differentially expressed in both LD vs. LE and MD vs. ME comparisons. Fifteen DELs were mapped onto the pig corpus luteum number Quantitative Trait Loci (QTL) fragments. A protein-protein interaction (PPI) network that involved estrus expression using 20 DEGs was then constructed. Interestingly, three predicted target DEGs (PTGs) (CYP19A1 of MSTRG.10910, CDK1 of MSTRG.10910 and MSTRG.23984, SCARB1 of MSTRG.1559) were observed in the PPI network. A competitive endogenous RNA (ceRNA) network including three lincRNAs, five miRNAs, and five genes was constructed. Our study provides new insight into the lincRNAs associated with estrus expression and follicular development in gilts.

A Single-Nucleotide Polymorphism in the Promoter of Porcine ARHGAP24 Gene Regulates Aggressive Behavior of Weaned Pigs After Mixing by Affecting the Binding of Transcription Factor p53.

Pigs are important biomedical model animals for the study of human neurological diseases. Similar to human aggressive behavior in children and adolescents, weaned pigs also show more aggressive behavior after mixing, which has negative effects on animal welfare and growth performance. The identification of functional single-nucleotide polymorphisms (SNPs) related to the aggressive behavior of pigs would provide valuable molecular markers of the aggressive behavioral trait for genetic improvement program. The Rho GTPase-activating protein 24 (ARHGAP24) gene plays an important role in regulating the process of axon guidance, which may impact the aggressive behavior of pigs. By resequencing the entire coding region, partially adjacent introns and the 5' and 3' flanking regions, six and four SNPs were identified in the 5' flanking region and 5' untranslated region (UTR) of the porcine ARHGAP24 gene, respectively. Association analyses revealed that nine SNPs were significantly associated with aggressive behavioral traits (p = < 1.00 × 10-4-4.51 × 10-2), and their haplotypes were significantly associated with aggressive behavior (p = < 1.00 × 10-4-2.99 × 10-2). The core promoter region of the ARHGAP24 gene has been identified between -670 and -1,113 bp. Furthermore, the luciferase activity of allele A of rs335052970 was significantly less than that of allele G, suggesting that the transcriptional activity of the ARHGAP24 gene was inhibited by allele A of rs335052970. It was identified that the transcription factor p53 bound to the transcription factor binding sites (TFBSs) containing allele A of rs335052970. In porcine primary neural cells, p53 binds to the target promoter region of the ARHGAP24 gene, reduces its promoter transcriptional activity, and then reduces its messenger RNA (mRNA) and protein expression. The results demonstrated that the ARHGAP24 gene had significant genetic effects on aggressive behavioral traits of pigs. Therefore, rs335052970 in the ARHGAP24 gene can be used as a molecular marker to select the less aggressive pigs.

Identification of functional single nucleotide polymorphisms in the porcine SLC6A4 gene associated with aggressive behavior in weaned pigs after mixing.

Variation in genes of the serotonergic system influences aggressive behavior by affecting serotonin levels in the central and cortical nervous system. SLC6A4 (serotonin transporter) is a master regulator of 5-HT signaling and involved in the regulation of aggressive behavior in humans and rodents. To identify potential functional single nucleotide polymorphisms (SNPs) for the porcine SLC6A4 gene associated with aggressive behavior, a total of 500 pigs (268 barrows and 232 gilts) were selected and mixed in 51 pens. Their behavior was recorded and observed for 72 h after mixing. Based on a composite aggressive score (CAS), the most aggressive and the least aggressive pigs within each pen were selected separately (a total of 204 pigs). Ear tissue was sampled to extract genomic DNA. Eight SNPs in the 5'-flanking region, coding region, and 3'-untranslated region (3'-UTR) of SLC6A4 were genotyped, of which 6 SNPs had significant differences (P < 0.05) in allele frequency between the most aggressive and least aggressive pigs. Luciferase activity was greater in plasmids of genotype GG than plasmids of genotype CC of rs345058216 (P < 0.01). Computational analysis nominated MAZ as putative transcription factor (TF) with higher probability to bind the SLC6A4 promoter at the SNP (rs345058216) site. Also, we demonstrated that MAZ overexpression modulates SLC6A4 promoter activity in allele-specific manner with an in vitro assay. In addition, we demonstrated that SLC6A4 was a direct target of miR-671-5p. The dual luciferase reporter gene assay and cell transfection were performed to examine the role of miR-671-5p in regulating SLC6A4 expression. The luciferase assays revealed that the SNP rs332335871 affects regulation of miR-671-5p in SLC6A4 expression. After overexpression of miR-671-5p in porcine primary neural cells, the SLC6A4 mRNA levels can be significantly reduced. In conclusion, we here found that miR-671-5p and MAZ mediated porcine SLC6A4 expression level, which provides the possible molecular mechanism of aggressive behavior.

The current study identified the functional single nucleotide polymorphisms for the porcine SLC6A4 (serotonin transporter) gene associated with aggressive behavior of pigs after mixing. We investigated the underlying molecular regulation mechanism of those functional variations. SNP rs345058216 (c.-1694C > G) affects binding of the transcription factor MAZ (MYC associated zinc finger protein) to the core promoter region of the SLC6A4 gene, whereas SNP rs332335871 (c.*1586G > A) affects the binding of miR-671-5p to the 3ʹ-UTR of SLC6A4 gene. These results suggest that rs345058216 and rs332335871 could be used as candidate molecular markers for aggressive behavior of pigs after mixing.

Functionally Antagonistic Transcription Factors IRF1 and IRF2 Regulate the Transcription of the Dopamine Receptor D2 Gene Associated with Aggressive Behavior of Weaned Pigs.

Aggressive behavior has negative effects on animal welfare and growth performance in pigs. The dopamine receptor D2 (DRD2) has a critical neuromodulator role in the dopamine signal pathway within the brain to control behavior. A functional single-nucleotide polymorphism (SNP), rs1110730503, in the promoter region of the porcine DRD2 gene was identified, which affects aggressive behavior in pigs. A chromatin immunoprecipitation (ChIP) assay was used to identify the interactions between interferon regulatory factor 1 (IRF1) and IRF2 with the DRD2 gene. The overexpression or knockdown of these two transcription factors in porcine kidney-15 (PK15) and porcine neuronal cells (PNCs) indicate that the binding of IRF1 to DRD2 promotes the transcription of the DRD2 gene, but the binding of IRF2 to the DRD2 gene inhibits its transcription. Furthermore, IRF1 and IRF2 are functionally antagonistic to each other. The downregulation of DRD2 or upregulation of IRF2 increased the apoptosis rate of porcine neuroglial cells. Taken together, we found that transcriptional factors IRF1 and IRF2 have vital roles in regulating the transcription of the DRD2 gene, and rs1110730503 (-915A/T) is a functional SNP that influences IRF2 binding to the promoter of the DRD2 gene. These findings will provide further insight towards controlling aggressive behavior in pigs.

Characterizing the effect of incrementally increasing dry bulb temperature on linear and nonlinear measures of heart rate variability in nonpregnant, mid-gestation, and late-gestation sows.

Characterizing the sow physiological response to an increased heat load is essential for effective heat stress mitigation. The study objective was to characterize the effects of a 400-min heating episode on sow heart rate variability (HRV) at different reproductive stages. HRV is a commonly used noninvasive proxy measure of autonomic function. Twenty-seven sows were enrolled in the study according to their gestation stage at time of selection: 1) nonpregnant (NP; n = 7), 2) mid-gestation (MID; 57.3 ± 11.8 d gestation; n = 11), and 3) late-gestation (LATE; 98.8 ± 4.9 d gestation; n = 8). The HRV data utilized in the study were collected from each pig as the dry bulb temperature in the room increased incrementally from 19.84 ± 2.15 °C to 35.54 ± 0.43 °C (range: 17.1-37.5 °C) over a 400-min period. After data collection, one 5-min set of continuous heart rate data were identified per pig for each of nine temperature intervals (19-20.99, 21-22.99, 23-24.99, 25-26.99, 27-28.99, 29-30.99, 31-32.99, 33-34.99, and 35-36.99 °C). Mean inter-beat interval length (RR), standard deviation of r-r intervals (SDNN), root mean square of successive differences (RMSSD), high frequency spectral power (HF), sample entropy (SampEn), short-term detrended fluctuation analysis (DFAα1), and three measures (%REC, DET, LMEAN) derived from recurrence quantification analysis were calculated for each data set. All data were analyzed using the PROC GLIMMIX procedure in SAS 9.4. Overall, LATE sows exhibited lower RR than NP sows (P < 0.01). The standard deviation of r-r intervals and RMSSD differed between each group (P < 0.01), with LATE sows exhibiting the lowest SDNN and RMSSD and NP sows exhibiting the greatest SDNN and RMSSD. Late-gestation sows exhibited lower HF than both MID and NP sows (P < 0.0001), greater DFA values than NP sows (P = 0.05), and greater DET compared to MID sows (P = 0.001). Late-gestation also sows exhibited greater %REC and LMEAN compared to MID (P < 0.01) and NP sows (all P < 0.01). In conclusion, LATE sows exhibited indicators of greater autonomic stress throughout the heating period compared to MID and NP sows. However, temperature by treatment interactions were not detected as dry bulb increased. Future studies are needed to fully elucidate the effect of gestational stage and increasing dry bulb temperature on sow HRV.

Pregnant pigs may be at a higher risk of poor physiological outcomes due to heat exposure compared to mature female pigs that are not pregnant. The purpose of this study was to evaluate the stress response of pregnant pigs to increasing environmental temperatures using heart rate variability, a noninvasive measure commonly used to evaluate the physiological stress response. Our findings show that pregnant pigs, particularly those who are closer to giving birth, exhibit greater evidence of physiological stress compared to pigs who are not pregnant. However, we did not find evidence that increasing environmental temperature throughout the experimental period was a primary reason for the increased stress exhibited by pregnant pigs. It is possible that the physiological changes that normally occur during pregnancy may have masked the physiological stress response typically associated with increased heat exposure.

Electronically controlled cooling pads can improve litter growth performance and indirect measures of milk production in heat-stressed lactating sows.

Heat stress (HS) decreases lactation output in sows due to an attempt to reduce metabolic heat production. However, this negatively affects litter growth performance. Therefore, the study objective was to determine whether electronically controlled cooling pads (ECP) would improve indirect measures of lactation output (e.g., total heat production [THP]) and litter growth performance in HS exposed sows. Over two repetitions, 12 multiparous (2.69 ± 0.85) lactating sows (265.4 ± 26.1 kg body weight [BW]) and litters were assigned to either an ECP (n = 3/repetition) or a non-functional ECP (NECP; n = 3/repetition) and placed into farrowing crates within indirect calorimeters from days 3.7 ± 0.5 to 18.7 ± 0.5 of lactation. Litters were standardized across all sows (11.4 ± 0.7 piglets/litter), and sows were provided ad libitum feed and water. All sows were exposed to cyclical HS (28.27 ± 0.26 °C nighttime to 33.09 ± 0.19°C daytime). On days 4, 8, 14, and 18 of lactation, indirect calorimetry was performed on each individual sow and litter to determine THP and THP/kg BW0.75. Body temperature (TB) was measured hourly using vaginal implants, and respiration rate (RR; breaths per minute [bpm]) was measured daily at 0700, 1100, 1300, 1500, and 1900 h. Sow feed intake (FI) was assessed daily. Litter weights were obtained at birth, on days 4, 8, 14, and 18 of lactation, and at weaning. Data were analyzed using PROC GLIMMIX with sow and/or litter as the experimental unit. An overall decrease (P < 0.01; 25 bpm) in RR and maximum daily TB (P = 0.02; 0.40 °C) was observed in ECP vs. NECP sows. An increase in THP (P < 0.01; 20.4%) and THP/kg BW0.75 (P < 0.01; 23.1%) was observed for ECP when compared with NECP sows and litters. Litter average daily gain and weaning weight were increased (P < 0.05; 25.0 and 19.2%, respectively) for ECP vs. NECP litters. No FI differences were observed (P = 0.40) when comparing ECP (5.66 ± 0.31 kg/d) and NECP (5.28 ± 0.31 kg/d) sows. In summary, ECPs improve litter growth, thermoregulatory measures, and bioenergetic parameters associated with greater milk production in lactating sows exposed to cyclical HS.

Lactating sows are heat stress sensitive due to greater feed intake and metabolic heat production to support milk production. Therefore, heat-stressed lactating sows reduce their feed intake and undergo physiological changes that prioritize survival over productivity which reduces milk production. Reduced milk production has negative downstream effects on the litter and can decrease litter growth performance and health status. The negative effects of heat stress on lactating sows may be ameliorated by using electronically controlled cooling pads. Electronically controlled cooling pads improve the ability of lactating sows to maintain a normal body temperature under heat stress conditions. However, it is unknown whether these pads can improve lactating sow feed intake, milk production, and subsequently litter performance under heat stress conditions. It was determined that electronically controlled cooling pads were effective in increasing piglet body weight gain and weaning weight and increasing indirect measures of sow milk production independent of greater sow feed intake. In addition, the electronically controlled cooling pads allowed sows to maintain a normal body temperature under heat stress conditions. These data suggest that electronically controlled cooling pads can be an effective technology to improve lactating sow and litter performance under heat stress conditions.

MiR-31 targets HSD17B14 and FSHR, and miR-20b targets HSD17B14 to affect apoptosis and steroid hormone metabolism of porcine ovarian granulosa cells.

Porcine 17-hydroxysteroid dehydrogenase type 14 (HSD17B14) and FSH reporter (FSHR) genes play important roles in the metabolism of steroid hormones and the apoptosis of ovarian granulosa cells (GCs). Our bioinformatics analyses and the dual luciferase reporter assays indicated that porcine miR-20b and miR-31 target the 3'-UTR region of HSD17B14 gene, and miR-31 also targets the 3'-UTR region of FSHR gene. Overexpression of porcine HSD17B14 gene promoted the conversion from estradiol (E2) to estrone (E1) and increased the apoptosis of porcine GCs. Overexpression of miR-20b down-regulated the mRNA and protein expression level of HSD17B14 gene, decreased the concentration of progesterone (P4) and E1, increased E2, as well as reduced apoptosis of GCs. Moreover, overexpression of miR-31 also down-regulated the protein expression level of HSD17B14 gene, decreased the concentration of P4 and E1, and increased E2. However, miR-31 promoted apoptosis of GCs by targeting to the 3'-UTR of porcine FSHR gene. Taken together, we found that both porcine miR-20b and miR-31 target HSD17B14 gene, but miR-31 also targets FSHR gene to regulate the metabolism of steroid hormones and the apoptosis of porcine ovarian GCs. These findings expand the epigenetic regulatory mechanism of porcine miR-31 and miR-20b in ovarian GCs.