Effects of increased ambient temperature and supplemental altrenogest prior to pregnancy establishment in gilts

Heat stress (HS) mitigation strategies are critically needed to combat the substantial economic effects on animal agriculture. The manifestations of seasonal infertility include delayed puberty onset, reduced conception rates, decreased litter size, and increased wean to estrus interval. To assess the effects of HS during early gestation and evaluate a benefit of supplemental altrenogest (ALT) as a mitigation strategy, thirty crossbred post-pubertal gilts (157 ± 11 kg) were subjected to estrous synchronization via 14 d oral administration of ALT. Artificial insemination during estrus was performed and gilts were then placed into one of four treatment groups; heat stress (HS; 35 ± 1 οC for 12h/31.60 ± 1 οC for 12h) with (HSALT, n = 7) or without (HSCON, n = 7) 15 mg/d ALT supplementation or thermal neutral (TN; 20 ± 1 οC) conditions with (TNALT, n = 8) or without (TNCON, n = 8) 15 mg/d ALT supplementation until 12 d post-estrus (dpe). Administrating ALT occurred at 0600 h from 3-12 dpe and rectal temperatures (TR) and respiration rates (RR) were recorded. Blood was collected via jugular venipuncture on 0, 4, 8 and 12 dpe. Gilts were euthanized humanely at 12 dpe followed by collection of ovarian tissue, and uterine flushing for conceptus collection. In HS compared to TN gilts, RR and TR were increased (P < 0.01) but unaffected by ALT supplementation. Feed intake (FI) was reduced (P < 0.01) by HS but unaltered by ALT treatment. Corpora lutea (CL) weight was reduced (P < 0.01) in HSCON gilts when compared to TNCON and HSALT gilts despite progesterone (P4) concentrations in serum and luteal tissue not being affected by treatment (P ≥ 0.10). CL diameter was reduced (P ≤ 0.05) in HSALT gilts compared to other treatments. Interleukin-1β (IL1B) uterine flush concentration was not affected (P > 0.20) by environment or ALT supplementation, although moderate (P = 0.06) interaction between environment and ALT existed, as IL1B concentration in TNALT was increased (P = 0.03) compared to TNCON gilts. While environment did not affect conceptus development (P = 0.90), ALT supplementation advanced conceptus elongation (P < 0.01). Collectively, these data demonstrate that HS may affect luteal development prior to pregnancy establishment, and ALT increases conceptus elongation by12 dpe.

Conceptus interferon gamma is essential for pregnancy maintenance in the pig

Establishment and maintenance of pregnancy in the pig is a complex process that relies on adequate communication between the conceptus and maternal uterine endometrium. During the peri-implantation period, the conceptuses produce and secrete estrogens, interleukin 1 beta 2, prostaglandins and other biological factors into the uterine lumen that change the uterine epithelium to become receptive to the attaching conceptuses as well as promote proper conceptus development. Following elongation, beginning on day 12 of pregnancy, the conceptus is known to secrete two different types of interferons. The pig conceptus secretes both type I (interferon delta, IFND) and type II (interferon gamma, IFNG) interferons during this time. CRISPR/Cas9 gene editing and somatic cell nuclear transfer (SCNT) technologies were used to create an IFNG loss-of-function study in pigs. Blastocyst stage embryos that were IFNG[superscript +/+] or IFNG[superscript -/-] were transferred into recipient gilts and their reproductive tracts were collected on days 15 and 17 of pregnancy. Elongated conceptuses were flushed from recipient gilts on day 15 IFNG[superscript +/+] (4/4) and IFNG[superscript -/-] (4/4) recipient gilts. On day 17 of pregnancy, all IFNG[superscript +/+] recipient gilts (4/4) contained elongated viable conceptuses; however, conceptuses were only recovered from 2 of 8 IFNG[superscript -/-] embryo recipient gilts. In all IFNG[superscript -/-] pregnancies, the conceptuses were thin and fragmented compared to IFNG+/+ conceptuses. Additionally, the reproductive tracts that received IFNG[superscript -/-] conceptuses which were not pregnant on day 17 appeared hyperemic, inflamed and edematous. IFNG was localized to the trophectoderm of IFNG[superscript +/+] conceptuses on both day 15 and 17 of pregnancy. However, IFNG expression was not detected in IFNG[superscript -/-] conceptuses on either day 15 or day 17 of pregnancy. Conceptus IFNG mRNA expression was significantly affected by genotype (P [equals] 0.0006) and day (P [less than] 0.0001). Total IFNG was significantly lower (P [equals] 0.0018) in ULF of IFNG[superscript -/-] embryo recipient gilts compared to IFNG[superscript +/+] embryo recipient gilts. IFNG[superscript +/+] conceptuses induced endometrial folding and recruited large numbers of immune cells to the endometrial stroma beneath the site of conceptus attachment compared to less endometrial folding and presence of immune cells in IFNG[superscript -/-] pregnancies on day 15. An additional group of recipient gilts received either IFNG[superscript +/+] embryos (n [equals] 6) or both IFNG[superscript -/-] and IFNG[superscript +/+] embryos (n [equals] 5) to determine if the presence of IFNG producing conceptuses could rescue the IFNG-/- embryos beyond day 17 of pregnancy. On day 30 of pregnancy, 3/6 of IFNG[superscript +/+] embryo recipient gilts contained 3-4 viable embryos, however, only 1 of 5 IFNG[superscript -/-] and IFNG[superscript +/+] cotransferred recipient gilts maintained pregnancy to day 30. Genotyping indicated that all five (1 healthy, 4 degenerating) embryos were IFNG[superscript +/+]. These results indicate conceptus IFNG production is not essential for early conceptus development, rapid elongation or establishment of pregnancy. However, conceptus IFNG production does appear to be necessary for survival during the period of placental attachment beyond day 15.

Extracellular vesicles, lipids, and lipoproteins in early pregnant sheep

In sheep, pregnancy establishment encompasses conceptus elongation, implantation, and placentation. These events are regulated by factors present within the uterine luminal fluid (ULF) from the endometrial epithelium and the conceptus itself that affect proliferation, migration, attachment, and adhesion of the conceptus trophectoderm. As the peri-implantation period is especially susceptible to pregnancy loss, it is essential to understand the various components and functional roles of substances within the ULF. The central hypothesis of this dissertation is that lipids and lipid associated macromolecules are components of the ULF and mediate endometrial-embryonic crosstalk and regulate conceptus development. This work sought to identify, characterize, and/or determine the roles of: (1) extracellular vesicles (EVs); (2) lipids and metabolites; (3) prostaglandins (PGs); and (4) apolipoproteins present within the ULF of ewes during early gestation. Collectively, the present studies established that: (1) EVs increase within the ULF during the estrous cycle but are depleted in the uterine lumen of pregnant ewes due to uptake by the elongating conceptus; (2) the lipid and protein cargo of uterine EVs is diverse and altered by pregnancy; (3) uterine EVs regulate cellular processes in the conceptus trophectoderm and endometrium including cell proliferation and secretions; (4) various lipids (specifically phospholipids, ceramides, and triglycerides) and metabolites are elevated in the ULF of pregnant ewes; (5) the conceptus lipidome and metabolome is distinct from the ULF and endometrium suggesting selective uptake of ULF substances; (6) the production of PGs by PTGS2 in the conceptus is not required for conceptus elongation; (7) the secretion of APOA1 by the conceptus does not mobilize endometrial lipids into the ULF and is not required for early pregnancy development or survival. Collectively, these studies highlight the complex and dynamic composition of the ULF and support the overall hypothesis that lipids and lipid-associated macromolecules are critical components of the ULF that mediate conceptus-endometrial crosstalk and regulate important developmental processes in the conceptus. Future investigation and expansion of these findings will fill crucial gaps in our knowledge of early pregnancy events and may provide biomarkers or help develop therapies to improve pregnancy outcomes and reproductive efficiency in agricultural species.

mRNA Expressions of Candidate Genes in Gestational Day 16 Conceptus and Corresponding Endometrium in Repeat Breeder Dairy Cows with Suboptimal Uterine Environment Following Transfer of Different Quality Day 7 Embryos

Effect of the gestational day (GD) 7 embryo quality grade (QG) and subclinical endometritis (SCE) on mRNA and protein expressions of candidate genes [Interferon-τ (IFNT), IFN stimulated genes (ISG15, CTSL1, RSAD2, SLC2A1, CXCL10, and SLC27A6), Peroxisome proliferator activated receptors (PPARA, D, and G), Retinoid X receptors (RXRA, B, and G), and Mucin-1 (MUC1)] in GD16 conceptus and corresponding endometrium were evaluated. After screening of performance records (n = 2389) and selection of repeat breeders (n = 681), cows with SCE (≥6% polymorphonuclear neutrophils—PMN; n = 180) and no-SCE (<6%PMN; n = 180) received GD7 embryos of different QGs. Based on GD16 conceptus recovery, cows with SCE (n = 30) and No- SCE (n = 30) that received GD7 embryos QG1 (good, n = 20), 2 (fair, n = 20), and 3 (poor, n = 20) were included for gene analysis. mRNA and protein expressions (IFNT, ISG15, CXCL10, PPARG, RXRG, SLC2A1, and SLC27A6) differed between SCE and embryo QG groups. All genes but MUC1 and all proteins but MUC1 expression was greater in filamentous conceptus and corresponding endometrium vs. tubular conceptus and matching endometrium in SCE and embryo QG groups. In conclusion, disrupted embryo-uterine communication by altered expression of candidate genes in SCE cows, and in cows following the transfer of poor embryo negatively programs the conceptus development and plausibly affects conceptus survival.

Gene editing to investigate the role of conceptus factors in the establishment of pregnancy in the pig

Development of viviparity in mammals requires that the placenta evolves as an intermediate interface between the fetus and maternal uterus. In addition to the retention of the fetus and secretion of nutrients to support growth and development to term, it is essential that viviparous species modify or inhibit the maternal immune system from recognizing the semi-allogeneic fetus. Following blastocyst hatching from its zona pellucida, trophoblast differentiation provides the initial communication to the maternal endometrium to regulate maintenance of progesterone production from the corpus luteum and biological pathways in uterine and conceptus development necessary in the establishment and maintenance of pregnancy. Many conceptus factors have been proposed to serve in the establishment and maintenance of pregnancy. CRISPR-Cas9 gene-editing technology provides a specific and efficient method to generate animal models to perform loss-of-function studies to investigate the role of specific conceptus factors. The utilization of CRISPR-Cas9 gene editing has provided a direct approach to investigate the specific role of conceptus factors in the development and establishment of pregnancy in the pig. This technology has helped address a number of questions concerning peri-implantation development and has altered our understanding of maternal recognition and maintenance of pregnancy in the pig.

Pre-implantation exogenous progesterone and pregnancy in sheep: I. polyamines, nutrient transport, and progestamedins

Background Administration of exogenous progesterone (P4) to ewes during the pre-implantation period advances conceptus development and implantation. This study determined effects of exogenous P4 on transport of select nutrients and pathways that enhance conceptus development. Pregnant ewes (n = 38) were treated with either 25 mg P4 in 1 mL corn oil (P4, n = 18) or 1 mL corn oil alone (CO, n = 20) from day 1.5 through day 8 of pregnancy and hysterectomized on either day 9 or day 12 of pregnancy. Endometrial expression of genes encoding enzymes for synthesis of polyamines, transporters of glucose, arginine, and glycine, as well as progestamedins was determined by RT-qPCR. Results On day 12 of pregnancy, conceptuses from P4-treated ewes had elongated while those from CO-treated ewes were spherical. The mRNA expression of AZIN2, an arginine decarboxylase, was lower in endometria of P4-treated than CO-treated ewes on day 9 of pregnancy. Expression of FGF10, a progestamedin, was greater in endometria of CO and P4-treated ewes on day 12 of gestation in addition to P4-treated ewes necropsied on day 9 of gestation. Treatment with P4 down-regulated endometrial expression of amino acid transporter SLC1A4 on day 12 of pregnancy. Conclusions Results indicated that administration of exogenous P4 during the pre-implantation period advanced the expression of FGF10, which may accelerate proliferation of trophectoderm cells, but also was correlated with decreased expression of glycine and serine transporters and polyamine synthesis enzyme AZIN2. Further research with increased sample sizes may determine how differential expression affects endometrial functions and potentially embryonic loss.

Global Transcriptomic Analyses Reveal Genes Involved in Conceptus Development During the Implantation Stages in Pigs

Prenatal mortality remains a significant concern to the pig farming industry around the world. Spontaneous fetal loss ranging from 20 to 45% by term occur after fertilization, with most of the loss happening during the implantation period. Since the factors regulating the high mortality rates of early conceptus during implantation phases are poorly understood, we sought to analyze the overall gene expression changes during this period, and identify the molecular mechanisms involved in conceptus development. This work employed Illumina’s next-generation sequencing (RNA-Seq) and quantitative real-time PCR to analyze differentially expressed genes (DEGs). Soft clustering was subsequently used for the cluster analysis of gene expression. We identified 8236 DEGs in porcine conceptus at day 9, 12, and 15 of pregnancy. Annotation analysis of these genes revealed rRNA processing (GO:0006364), cell adhesion (GO:1904874), and heart development (GO:0007507), as the most significantly enriched biological processes at day 9, 12, and 15 of pregnancy, respectively. In addition, we found various genes, such as T-complex 1, RuvB-like AAA ATPase 2, connective tissue growth factor, integrins, interferon gamma, SLA-1, chemokine ligand 9, PAG-2, transforming growth factor beta receptor 1, and Annexin A2, that play essential roles in conceptus morphological development and implantation in pigs. Furthermore, we investigated the function of PAG-2 in vitro and found that PAG-2 can inhibit trophoblast cell proliferation and migration. Our analysis provides a valuable resource for understanding the mechanisms of conceptus development and implantation in pigs.

Overfeeding Extends the Period of Annual Cyclicity but Increases the Risk of Early Embryonic Death in Shetland Pony Mares

Obesity has been associated with altered reproductive activity in mares, and may negatively affect fertility. To examine the influence of long-term high-energy (HE) feeding on fertility, Shetland pony mares were fed a diet containing 200% of net energy (NE) requirements during a three-year study. The incidence of hemorrhagic anovulatory follicles (HAF) and annual duration of cyclicity were compared to those in control mares receiving a maintenance diet. Day-7 embryos were flushed and transferred between donor and recipient mares from both groups; the resulting conceptuses were collected 21 days after transfer to assess conceptus development. HE mares became obese, and embryos recovered from HE mares were more likely to succumb to early embryonic death. The period of annual cyclicity was extended in HE compared to control mares in all years. The incidence of HAFs did not consistently differ between HE and control mares. No differences in embryo morphometric parameters were apparent. In conclusion, consuming a HE diet extended the duration of cyclicity, and appeared to increase the likelihood of embryos undergoing early embryonic death following embryo transfer.