Postpubertal Gilts Research Articles

Impact of heat stress on prolactin-mediated ovarian JAK-STAT signaling in postpubertal gilts.

Heat stress (HS) compromises almost every aspect of animal agriculture including reproduction. In pigs, this infecundity is referred to as seasonal infertility (SI), a phenotype including ovarian dysfunction. In multiple species, HS-induced hyperprolactinemia has been described; hence, our study objectives were to characterize and compare HS effects on circulating prolactin (PRL) and ovarian Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling during the follicular (FOL) or luteal (LUT) phases of the estrous cycle in postpubertal gilts. Gilts were estrus synchronized using altrenogest and environmental treatments began immediately after altrenogest withdrawal. For the FOL study: postpubertal gilts were allocated to constant thermoneutral (TN; n = 6; 20 ± 1.2 °C) or cyclical HS (n = 6; 25 to 32 ± 1.2 °C) conditions for 5 d. In the LUT study: postpubertal gilts were assigned to either TN (n = 7; 20 ± 2.6 °C) or cyclical HS (n = 7; 32 to 35 ± 2.6 °C) conditions from 2 to 12 days postestrus (dpe). Blood was collected by jugular venipuncture for PRL quantification on day 5 in the FOL and on day 0 and day 12 in the LUT gilts. Ovaries and corpora lutea (CL) were obtained from euthanized FOL and LUT gilts on day 5 and day 12, respectively. Western blotting was performed to quantify prolactin receptor (PRLR) and JAK/STAT pathway protein abundance. In the FOL phase, no difference (P = 0.20) in circulating PRL between thermal groups was observed. There was no effect (P ≥ 0.34) of HS on PRLR, signal transducer and activator of transcription 3 (STAT3), signal transducer and activator of transcription 5α (STAT5α), and phosphorylated signal transducer and activator of transcription α/β tyrosine 694/699 (pSTAT5α/βTyr694/699) abundance and Janus kinase 2 (JAK2), phosphorylated janus kinase 2 tyrosine 1007/1008 (pJAK2Tyr1007/1008), STAT1, phosphorylated signal transducer and activator of transcription 1 tyrosine 701 (pSTAT1Tyr701), phosphorylated signal transducer and activator of transcription 1 serine 727 (pSTAT1Ser727), and phosphorylated signal transducer and activator of transcription 3 tyrosine 705 (pSTAT3Tyr705) were undetectable in FOL gilt ovaries. Ovarian pSTAT5α/βTyr694/699 abundance tended to moderately increase (4%; P = 0.07) in FOL gilts by HS. In the LUT phase, circulating PRL increased progressively from 2 to 12 dpe, but no thermal treatment-induced difference (P = 0.37) was noted. There was no effect (P ≥ 0.16) of HS on CL abundance of PRLR, pJAK2Tyr1007/1008, JAK2, STAT1, pSTAT1Tyr701, pSTAT1Ser727, pSTAT3Tyr705, STAT5α, or pSTAT5α/βTyr694/699. In LUT phase, CL STAT3 abundance was increased (11%; P < 0.03) by HS. There was no impact of HS (P ≥ 0.76) on levels of pJAK2Tyr1007/1008 and pSTAT5α/βTyr694/699 in LUT gilts; however, the CL pSTAT3Tyr705:STAT3 ratio tended to be decreased (P = 0.10) due to HS. These results indicate an HS-induced estrous cycle-stage-dependent effect on the ovarian JAK/STAT pathway, establishing a potential role for this signaling pathway as a potential contributor to SI.

Porcine endometrial heat shock proteins are differentially influenced by pregnancy status, heat stress, and altrenogest supplementation during the peri-implantation period.

Heat stress (HS) deleteriously affects multiple components of porcine reproduction and is causal to seasonal infertility. Environment-induced hyperthermia causes a HS response (HSR) typically characterized by increased abundance of intracellular heat shock proteins (HSP). Gilts exposed to HS during the peri-implantation period have compromised embryo survival, however if (or how) HS disrupts the porcine endometrium is not understood. Study objectives were to evaluate the endometrial HSP abundance in response to HS during this period and assess the effect of oral progestin (altrenogest; ALT) supplementation. Postpubertal gilts (n = 42) were artificially inseminated during behavioral estrus (n = 28) or were kept cyclic (n = 14), and randomly assigned to thermal neutral (TN; 21 ± 1 °C) or diurnal HS (35 ± 1 °C for 12 h/31.6 ± 1 °C for 12 h) conditions from day 3 to 12 postestrus (dpe). Seven of the inseminated gilts from each thermal treatment group received ALT (15 mg/d) during this period. Using quantitative PCR, transcript abundance of HSP family A (Hsp70) member 1A (HSPA1A, P = 0.001) and member 6 (HSPA6, P < 0.001), and HSP family B (small) member 8 (HSB8, P = 0.001) were increased while HSP family D (Hsp60) member 1 (HSPD1, P = 0.01) was decreased in the endometrium of pregnant gilts compared to the cyclic gilts. Protein abundance of HSPA1A decreased (P = 0.03) in pregnant gilt endometrium due to HS, while HSP family B (small) member 1 (HSPB1) increased (P = 0.01) due to HS. Oral ALT supplementation during HS reduced the transcript abundance of HSP90α family class B member 1 (HSP90AB1, P = 0.04); but HS increased HSP90AB1 (P = 0.001), HSPA1A (P = 0.02), and HSPA6 (P = 0.04) transcript abundance irrespective of ALT. ALT supplementation decreased HSP90α family class A member 1 (HSP90AA1, P = 0.001) protein abundance, irrespective of thermal environment, whereas ALT only decreased HSPA6 (P = 0.02) protein abundance in TN gilts. These results indicate a notable shift of HSP in the porcine endometrium during the peri-implantation period in response to pregnancy status and heat stress.

Effects of increased ambient temperature and supplemental altrenogest before 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 the benefit of supplemental altrenogest (ALT) as a mitigation strategy, 30 crossbred postpubertal gilts (157 ± 11 kg body weight) 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: HS (35 ± 1 °C for 12 h/31.60 ± 1 °C for 12 h) with (HSALT, n = 7) or without (HSCON, n = 7) 15 mg/d ALT supplementation or thermal neutral (TN; 20 ± 1 °C) 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 hours from 3 to 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 the collection of ovarian tissue, and uterine flushing for conceptus collection. In HS compared with TN gilts, RR and TR were increased (P < 0.01) but unaffected by ALT supplementation. Feed intake was reduced (P < 0.01) by HS but unaltered by the ALT treatment. Corpora lutea (CL) weight was reduced (P < 0.01) in HSCON gilts when compared with TNCON and HSALT gilts despite progesterone 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 with 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 with 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 before pregnancy establishment, and ALT increases conceptus elongation by 12 dpe.

Evidence that pubertal status impacts kisspeptin/neurokinin B/dynorphin neurons in the gilt†.

Puberty onset is a complex physiological process, which enables the capacity for reproduction through increased gonadotropin-releasing hormone and subsequently luteinizing hormone secretion. While cells that coexpress kisspeptin, neurokinin B (NKB), and dynorphin in the hypothalamic arcuate nucleus are believed to govern the timing of puberty, the degree to which kisspeptin/NKB/dynorphin (KNDy) neurons exist and are regulated by pubertal status remains to be determined in the gilt. Hypothalamic tissue from prepubertal and postpubertal, early follicular phase gilts was used to determine the expression of kisspeptin, NKB, and dynorphin within the arcuate nucleus. Fluorescent in situ hybridization revealed that the majority (>74%) of arcuate nucleus neurons that express mRNA for kisspeptin coexpressed mRNA for NKB and dynorphin. There were fewer arcuate nucleus cells that expressed mRNA for dynorphin in postpubertal gilts compared to prepubertal gilts (P < 0.05), but the number of arcuate nucleus cells expressing mRNA for kisspeptin or NKB was not different between groups. Within KNDy neurons, mRNA abundance for kisspeptin, NKB, and dynorphin of postpubertal gilts was the same as, less than, and greater than, respectively, prepubertal gilts. Immunostaining for kisspeptin did not differ between prepubertal and postpubertal gilts, but there were fewer NKB immunoreactive fibers in postpubertal gilts compared to prepubertal gilts (P < 0.05). Together, these data reveal novel information about KNDy neurons in gilts and support the idea that NKB and dynorphin play a role in puberty onset in the female pig.

242 Divergent Selection for Early Puberty Impacts KNDy Neuron Gene Expression in Gilts

Abstract Advancing gilt puberty onset is financially desirable for swine production. Neurons in the hypothalamic arcuate nucleus (ARC) that co-express kisspeptin, neurokinin B (NKB), and dynorphin (i.e. KNDy cells) are believed to control gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion, but their role in gilt pubertal development is unknown. We hypothesized that puberty onset in gilts would coincide with greater expression of mRNA for kisspeptin and NKB, and less expression of dynorphin. Using fluorescent in situ hybridization (RNAscope), we examined expression of kisspeptin, NKB, and dynorphin in pre- and postpubertal gilts from two genetic lines divergently selected for age at puberty. Prepubertal (n = 6/line) and postpubertal (n = 6/line) gilts were used, and postpubertal animals all received Matrix (0.22% altrenogest) orally for 14 days with tissue collection two days after the final dose. Gilts were euthanized and heads were perfused with 8 L of 4% paraformaldehyde (PFA). Hypothalamic brain tissue was removed, placed in 4% PFA for 24 hrs, and then in 20% sucrose until sectioning (50 µm). Sectioned tissue was stored in cryopreservative at -20°C until RNAscope. Data were analyzed using SAS software (Version 9.4, SAS Institute, Cary NC) with significance declared at P &amp;lt; 0.05. We determined mRNA expression for kisspeptin was not different between groups (P &amp;gt; 0.05). In addition, we found that mRNA expression for NKB was higher in prepubertal gilts compared to postpubertal gilts (P &amp;lt; 0.05) but was not different between lines; mRNA expression was lowest in postpubertal late puberty gilts. Furthermore, total number of dynorphin cells were higher in prepubertal gilts compared to postpubertal gilts (P &amp;lt; 0.05), while individual cell mRNA expression for dynorphin was greatest in postpubertal early puberty gilts (P &amp;lt; 0.05). Taken together, we suggest puberty onset in gilts is more dependent on NKB and dynorphin than kisspeptin.

Evaluation of the molecular response of corpora lutea to manganese-amino acid complex supplementation in gilts.

Porcine pregnancy establishment and maintenance are dependent on the formation of functional corpora lutea (CL). Manganese (Mn) is critical for CL function as it is a cofactor for Mn superoxide dismutase and enzymes involved in cholesterol synthesis. Previously, we have shown that luteal Mn content increased and luteal progesterone (P4) concentration decreased in the CL of gilts fed diets supplemented with an Mn–amino acid complex (Availa-Mn; Zinpro Corporation) compared with controls fed Mn sulfate. Importantly, serum P4 increased from 0 (estrus onset) to 12 d post estrus (dpe), as expected, but P4 abundance in circulation was not affected by dietary Mn source (P = 0.15). We hypothesized that a more bioavailable Mn source (which results in increased luteal Mn content) would alter the luteal proteome and abundance of mRNA associated with steroid biogenesis during the mid-luteal phase of the estrous cycle. Postpubertal gilts (n = 32) were assigned to one of the four gestation diets. The control diet (CON) contained 20 ppm of supplemental Mn in the form of Mn sulfate. Three additional diets included 20 (TRT1), 40 (TRT2), or 60 (TRT3) ppm of supplemental Mn in the form of a Mn–amino acid complex instead of Mn sulfate. Dietary treatment began at estrus synchronization (approximately 20 d before estrus) and continued through 12 dpe when gilts were euthanized and tissues were collected. Protein and total RNA extracts from the CL were used for proteomic analysis via label-free liquid chromatography with tandem mass spectrometry to assess global protein abundance and quantitative real-time polymerase chain reaction (qRT-PCR) to assess specific mRNA abundance, respectively. Compared with CON, 188, 382, and 401 proteins were differentially abundant (P < 0.10) in TRT1, TRT2, and TRT3, respectively. Gene Ontology enrichment software revealed that proteins involved in P4 signaling and cholesterol synthesis were downregulated in CL of gilts fed Mn–amino acid complex compared with controls. Quantitative RT-PCR showed that relative transcript abundance of genes encoding steroidogenic enzymes (CYP11A1 and StAR) in CL tissue was decreased in gilts from TRT2 compared with CON (P = 0.02), but TRT1 and TRT3 were not affected (P ≥ 0.30). Collectively, these data support our hypothesis that a more bioavailable dietary Mn source may influence luteal function by altering the abundance of protein and mRNA involved in steroidogenesis.

Impact of manganese amino acid complex on tissue-specific trace mineral distribution and corpus luteum function in gilts.

Functional corpora lutea (CL) are required for pregnancy establishment and gestational maintenance in swine, and CL function is susceptible to environmental influences. Manganese (Mn) could be critical in regulating CL function since it is a component of the antioxidant enzyme Mn superoxide dismutase (MnSOD) as well as enzymes involved in cholesterol and steroid hormone synthesis. We hypothesized that a more bioavailable dietary Mn source would increase Mn content in the CL thereby influencing luteal function during the mid-luteal phase of the estrous cycle. Postpubertal gilts (n = 32) were assigned to one of four gestation diets. The control diet (CON) met or exceeded National Research Council (2012) requirements and was formulated to contain 20 parts per million (ppm) of added Mn in the form of Mn sulfate. Three additional diets included 20 (treatment [TRT]1), 40 (TRT2), or 60 (TRT3) ppm of added Mn from a Mn-amino acid complex (Availa-Mn; Zinpro Corporation) instead of Mn sulfate. Dietary treatment began at estrus synchronization onset and continued through 12 days post estrus (dpe) of the ensuing estrous cycle. Blood samples were collected at estrus onset, which was assigned as 0 dpe, as well as 4, 8, and 12 dpe. Gilts were euthanized and tissues were collected at 12 dpe. Serum progesterone (P4) increased (P < 0.01) from 0 to 12 dpe but was unaffected by dietary treatment (P = 0.15) and there was no effect of the interaction between day and treatment (P = 0.85). Luteal Mn content increased (P ≤ 0.05) by 19%, 21%, and 24% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON. Luteal P4 concentrations decreased (P = 0.03) 25%, 26%, and 32% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON. Relative to CON gilts, CL calcium content decreased (P = 0.02) by 36%, 24%, and 34% for TRT1, TRT2, and TRT3 gilts, respectively. Collectively, these data support the hypothesis that feeding a more bioavailable Mn source increases Mn accumulation in CL tissue. If and how this influences CL function may be related to altered luteal P4 concentrations.

29 Influence of dietary manganese source on the corpus luteum proteome in pigs

Abstract Functional corpora lutea (CL) are required for pregnancy establishment and gestational maintenance in swine. Manganese (Mn) could be critical in regulating CL function since it is a cofactor for the enzyme Mn superoxide dismutase (Mn-SOD) as well as enzymes involved in progesterone (P4) synthesis. We hypothesized a more bioavailable dietary Mn source would increase CL Mn content thereby influencing luteal function during the mid-luteal phase of the estrous cycle. Post-pubertal gilts (n = 32) were assigned to one of four gestation diets. The control diet (CON) met or exceeded NRC requirements and was formulated to contain 20 ppm added Mn in the form of Mn sulfate. Three additional diets included 20 (TRT1), 40 (TRT2) or 60 (TRT3) ppm Mn from a Mn-amino acid complex (Availa-Mn; Zinpro Corporation) in place of Mn sulfate. Dietary treatment began at estrus synchronization onset and continued through D12 of the ensuing estrous cycle when gilts were euthanized. Mn content increased (P ≤ 0.06) 19, 21 and 24% in CLs of gilts fed TRT1, TRT2, and TRT3, respectively, and luteal P4 concentration decreased (P ≤ 0.03) 25, 26, and 32% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON. Total CL protein was extracted and liquid chromatography with tandem mass spectrometry (LC-MS/MS) was performed to assess global protein abundance. Compared to CON, 29, 105, and 118 proteins were differentially abundant (P &amp;lt; 0.01) in TRT1, TRT2, and TRT3, respectively. KEGG pathway analysis revealed proteins involved in P4 signaling (membrane-associated P4 receptor component 2) and cholesterol synthesis and transport (mevalonate kinase, diphosphomevalonate decarboxylase, low density lipoprotein receptor) were downregulated in response to Availa-Mn. Collectively, these data support the posit that dietary Mn source affects Mn accumulation and P4 concentration in CL tissue and influences protein abundance which may affect CL function.

Heat stress during the luteal phase decreases luteal size but does not affect circulating progesterone in gilts1.

Heat stress (HS) occurs when heat dissipation mechanisms are insufficient to maintain euthermia, and it is associated with seasonal infertility (SI), which manifests as smaller litters, longer wean-to-estrus interval, increased abortions, and reduced conception rates. To understand HS-induced mechanisms underlying SI, crossbred post-pubertal gilts (167 ± 10 kg; n = 14) experienced either thermal neutral (TN, 20 ± 1 °C, n = 7) or cyclical HS (35 ± 1 °C for 12 h and 31.6 °C for 12 h, n = 7) conditions from 2 to 12 d post-estrus (dpe). Estrous cycles were synchronized via altrenogest administration for 14 d, phenotypic manifestation of estrus was observed and gilts were assigned to experimental treatment. Gilts were limit fed 2.7 kg daily with ad libitum water access. Blood was collected at 0, 4, 8, and 12 dpe via jugular venipuncture and animals were humanely euthanized at 12 dpe. The corpora lutea (CL) width were measured via digital calipers on both ovaries, and CL from one ovary were excised, weighed, and protein and steroid abundance analyzed via western blotting and ELISA, respectively. Relative to TN, HS increased (P < 0.01) rectal temperature and respiration rates and reduced (P < 0.01) feed intake. The CL from HS ovaries were reduced in diameter (P < 0.05) and weight (P < 0.01) relative to those from TN animals. No difference (P = 0.38) in CL or serum progesterone concentrations between groups was observed at any time point, though at 12 dpe the serum progesterone:CL weight was increased (P < 0.10) by HS. No treatment differences (P = 0.84) in circulating insulin were observed. Luteal protein abundance of steroid acute regulatory protein, 3 beta-hydroxysteroid, or prostaglandin F2α receptor were not different between treatments (P = 0.73). Taken together, these data demonstrate that the CL mass is HS sensitive, but this phenotype does not appear to be explained by the metrics evaluated herein. Regardless, HS-induced decreased CL size may have important implications to pig SI and warrants additional attention.

Genome-Wide DNA Methylation Analysis of Hypothalamus During the Onset of Puberty in Gilts

Although selection of the early age at puberty in gilts will make for a favorable effect on the reproductivity of sow, a large proportion of phenotypic variation in age at puberty of gilts cannot be explained by genetics. Previous studies have implicated hypothalamic DNA methylation in the onset of puberty in mammals. However, the underlying molecular mechanism regarding the regulation of the onset of puberty has remained largely unexplored in gilts. Herein, the genome-scale DNA methylation of hypothalamus was acquired, using the reduced representation bisulfite sequencing, to compare and describe the changes of DNA methylation across Pre-, In- and Post-pubertal gilts. In this study, the average methylation levels of CpGs and CpHs (where H = C, T, or A) in CpG islands- and gene-related regions were gradually decreased in hypothalamic methylomes during the pubertal transition. Comparisons of Pre- vs. In-, In- vs. Post-, and Pre- vs. Post-pubertal stage revealed that there were 85726, 92914, and 100421 differentially methylated CpGs and 5940, 14804, and 16893 differentially methylated CpHs (where H = C, T, or A) in the hypothalamic methylomes. The methylation changes of CpHs were more dynamic than that of CpGs, and methylation changes of CpGs and CpHs were likely to be, respectively, involved in the developmental processes of reproduction and the molecular processes of cellular communications in the hypothalamus. Moreover, methylation changes of CpHs were observed to overrepresent in the quantitative trait loci of age at puberty, and the biological function of these CpH methylation changes was enriched in the pancreas development in gilts. Furthermore, the mRNA levels of several differentially CpG or CpH methylated genes related to the transcription of RNA II polymerase, GnRH signaling pathway, Estrogen signaling pathway, PI3K-AKt signaling pathway, and Insulin signaling pathway, including MAX, MMP2, FGF11, IGF1R, FGF21, and GSK3B, were significantly changed across these pubertal stages in the hypothalamus. These results will help our understanding of how DNA methylation contributes to phenotypic variation of age at puberty.

Impact of repeated lipopolysaccharide administration on ovarian signaling during the follicular phase of the estrous cycle in post-pubertal pigs.

Increased circulating lipopolysaccharide (LPS) results from heat stress (HS) and bacterial infection, both of which are associated with reduced female fertility. Specific effects of low-level, repeated LPS exposure on the ovary are unclear, as many studies utilize a bolus model and/or high dosage paradigm. To better understand the effects of chronic LPS exposure on ovarian signaling and function, post-pubertal gilts (n = 20) were orally administered altrenogest for 14 d to synchronize the beginning of the follicular phase of the ovarian cycle. For 5 d after synchronization, gilts (163 ± 3 kg) received IV administration of LPS (0.1 µg/kg BW, n = 10) or saline (CT, n = 10) 4× daily. Blood samples were obtained on days 1, 3, and 5 of LPS treatment. Follicular fluid was aspirated from dominant follicles on day 5, and whole ovarian homogenate was used for transcript and protein abundance analysis via quantitative real-time PCR and western blotting, respectively. There were no treatment differences detected in rectal temperature on any day (P ≥ 0.5). Administering LPS increased plasma insulin (P < 0.01), LPS-binding protein (LBP; P < 0.01), and glucose (P = 0.08) on day 1, but no treatment differences thereafter were observed (P = 0.66). There were no treatment differences in follicular fluid concentration of LBP or 17β-estradiol (P = 0.42). Gilts treated with LPS had increased abundance of ovarian TLR4 protein (P = 0.01), but protein kinase B (AKT) and phosphorylated AKT (pAKT) were unchanged and no effect of LPS on components of the phosphatidylinositol 3 kinase (PI3K) pathway were observed. There was no impact of LPS on ovarian abundance of STAR or CYP19A1, nor ESR1, LDLR, CYP19A1, CYP17A1, or 3BHSD. In conclusion, repeated, low-level LPS administration alters inflammatory but not steroidogenic or PI3K signaling in follicular phase gilt ovaries.

Impact of heat stress during the follicular phase on porcine ovarian steroidogenic and phosphatidylinositol-3 signaling.

Environmental conditions that impede heat dissipation and increase body temperature cause heat stress (HS). The study objective was to evaluate impacts of HS on the follicular phase of the estrous cycle. Postpubertal gilts (126.0 ± 21.6 kg) were orally administered altrenogest to synchronize estrus, and subjected to either 5 d of thermal-neutral (TN; 20.3 ± 0.5 °C; n = 6) or cyclical HS (25.4 - 31.9 °C; n = 6) conditions during the follicular phase preceding behavioral estrus. On d 5, blood samples were obtained, gilts were euthanized, and ovaries collected. Fluid from dominant follicles was aspirated and ovarian protein homogenates prepared for protein abundance analysis. HS decreased feed intake (22%; P = 0.03) and while plasma insulin levels did not differ, the insulin:feed intake ratio was increased 3-fold by HS (P = 0.02). Insulin receptor protein abundance was increased (29%; P < 0.01), but insulin receptor substrate 1, total and phosphorylated protein kinase B, superoxide dismutase 1, and acyloxyacyl hydrolase protein abundance were unaffected by HS (P > 0.05). Plasma and follicular fluid 17β-estradiol, progesterone, and lipopolysaccharide-binding protein concentrations as well as abundance of steroid acute regulatory protein, cytochrome P450 19A1, and multidrug resistance-associated protein 1 were not affected by HS (P > 0.05). HS increased estrogen sulfotransferase protein abundance (44%; P = 0.02), toll-like receptor 4 (36%; P = 0.05), and phosphorylated REL-associated protein (31%; P = 0.02). Regardless of treatment, toll-like receptor 4 protein was localized to mural granulosa cells in the porcine ovary. In conclusion, HS altered ovarian signaling in postpubertal gilts during their follicular phase in ways that likely contributes to seasonal infertility.

1090 Lipopolysaccharide exposure in swine alters ovarian toll-like receptor 4 expression

Heat stress (HS) is associated with decreased fertility and endotoxemia as evidenced by increased systemic lipopolysaccharide (LPS) arising from decreased intestinal integrity. Across multiple species, LPS is associated with reduced female fecundity; phenotypic responses similar to HS-induced infertility, including spontaneous abortion and increased wean-to-estrus interval length. LPS binds to toll-like receptor 4 (TLR4), a membrane bound receptor, to initiate a signaling cascade culminating in the phosphorylation of nuclear factor kappa-B (pNFκB) and pro-inflammatory cytokine production. Acyloxyacyl hydrolase (AOAH) participates in LPS detoxification by cleaving the lipid A moiety, rendering the deacylated LPS unable to effectively bind TLR4. We hypothesized that endotoxemia could impact ovarian function in swine. Post-pubertal gilts were synchronized to the follicular phase of their estrous cycle using Matrix® administered orally for 14 d. Immediately following Matrix® removal, gilts were treated with vehicle control (CT; 3 mL sterile saline; n = 6) or LPS (0.1 μg/kg BW; E. coli 055:B5; n = 6) via jugular catheter four times daily (0000, 0600, 1200, 1800 h) for 5 d during the follicular phase preceding estrus. Six hours after the final LPS infusion, animals were euthanized and ovaries collected. Whole ovarian protein homogenates were prepared and western blotting performed to quantify abundance of TLR4 and AOAH protein. Relative to CT, ovaries from LPS treated gilts had reduced (P < 0.05) abundance of TLR4 protein. No effect (P > 0.05) of LPS infusion on AOAH abundance was observed. These data demonstrate that the ovary to be responsive to chronic, low-level LPS exposure, and that endotoxemia potentially contributes to seasonal infertility in swine. Funded by the Global Food Security Consortium.

0691 Translational genomics for improving sow reproductive longevity

Approximately 50% of sows are culled annually with more than one-third due to poor fertility. Age at puberty, the earliest prebreeding indicator of reproductive longevity, can be measured early in life and has a moderate heritability. Selection for age at puberty is challenging due to labor-intensive phenotyping. Genomic selection for this trait would be a more viable option because it could increase accuracy and selection response. This study aims to identify DNA markers that will predict, at weaning, gilts with early age at puberty and superior reproductive longevity. Our hypothesis is that genetic sources that affect age at puberty also explain variation in sow reproductive longevity. To test the hypothesis, data and tissues from a UNL resource population (n > 1,700 gilts) were integrated with genomewide association analyses, genome/RNA sequencing, and polymorphism discovery to uncover DNA variants that could predict age at puberty and reproductive longevity. A BeadArray panel of 56,424 SNP explained 25.2% of the phenotypic variation in age at puberty in a training set (n = 820). In an evaluation data set consisting of subsequent batches of similar genetics (n = 412), we compared a model based on all SNP from major 1-Mb windows with one based on SNP with the largest estimated effect. The model based on all SNP from the major windows explained more of the phenotypic variance compared with the model based on large effect SNP (12.3 to 36.8% vs. 6.5 to 23.7%). One major pleiotropic region included AVPR1A, for which the favorable genotype was associated with higher probability of the gilts to produce the first parity compared with the other genotypes (P < 0.05). Genome sequencing of 20 sires using Proton technology provided sources of genetic variation outside the limited capability of the BeadArray. Sequencing reads averaged 165 bp with a depth that varied from 16.2x to 29.7x. A substantial proportion (38%) of the total SNP discovered (140,000) were located in known genes. Transcriptome profile was evaluated by RNA sequencing of the microdissected arcuate nucleus (ARC) in pre-/postpubertal gilts (n = 12) subjected to different dietary treatments. Using a combination of Tophat and local Bowtie, the majority of the reads were aligned to the reference genome/transcriptome (>93%). This integrated knowledge accompanied by economic modeling will be evaluated in commercial populations to understand and improve expression of puberty and sow reproductive potential through genomic selection. This project is supported by Agriculture and Food Research Initiative Competitive Grant number 2013-68004-20370 from the USDA–National Institute of Food and Agriculture. The USDA is an equal opportunity provider and employer.

318 Impact of cyclical heat stress during follicular development on ovarian TLR4, PI3K and steroidogenic proteins in synchronized post-pubertal gilts

318 Impact of cyclical heat stress during follicular development on ovarian TLR4, PI3K and steroidogenic proteins in synchronized post-pubertal gilts

Nutrient restriction induces failure of reproductive function and molecular changes in hypothalamus–pituitary–gonadal axis in postpubertal gilts

People on a diet to lose weight may be at risk of reproductive failure. To investigate the effects of nutrient restriction on reproductive function and the underlying mechanism, changes of reproductive traits, hormone secretions and gene expressions in hypothalamus-pituitary-gonadal axis were examined in postpubertal gilts at anestrus induced by nutrient restriction. Gilts having experienced two estrus cycles were fed a normal (CON, 2.86 kg/d) or nutrient restricted (NR, 1 kg/d) food regimens to expect anestrus. NR gilts experienced another three estrus cycles, but did not express estrus symptoms at the anticipated fourth estrus. Blood samples were collected at 5 days' interval for consecutive three times for measurement of hormone concentrations at the 23th day of the fourth estrus cycle. Individual progesterone concentrations of NR gilts from three consecutive blood samples were below 1.0 ng/mL versus 2.0 ng/mL in CON gilts, which was considered anestrus. NR gilts had impaired development of reproductive tract characterized by absence of large follicles (diameter ≥ 6 mm), decreased number of corepus lutea and atrophy of uterus and ovary tissues. Circulating concentrations of IGF-I, kisspeptin, estradiol, progesterone and leptin were significantly lower in NR gilts than that in CON gilts. Nutrient restriction down-regulated gene expressions of kiss-1, G-protein coupled protein 54, gonadotropin-releasing hormone, estrogen receptor α, progesterone receptor, leptin receptor, follicle-stimulating hormone and luteinizing hormone and insulin-like growth factor I in hypothalamus-pituitary-gonadal axis of gilts. Collectively, nutrient restriction resulted in impairment of reproductive function and changes of hormone secretions and gene expressions in hypothalamus-pituitary-gonadal axis, which shed light on the underlying mechanism by which nutrient restriction influenced reproductive function.

Estrogenic effects of genistein on reproductive tissues of ovariectomized gilts1

The soybean phytoestrogen genistein has a range of estrogenic actions demonstrated in various species; however, only limited research has been done to investigate its effects in swine. The objective of this study was to characterize the effects of a graded dose of genistein on estrogen-sensitive uterine and cervical tissues in ovariectomized gilts. Thirty-four postpubertal gilts were ovariectomized and assigned randomly to 1 of 6 treatment groups 15 d postovariectomy. Treatment groups received vehicle, estradiol benzoate (2 mg/d), or genistein (50, 100, 200, or 400 mg/d) via intramuscular injection at 12-h intervals for 10 d. Following the treatment period, gilts were euthanized, and uterine and cervical tissues were collected and processed for chemical or histological analysis. Uterine and cervical tissue mass, as indicated by wet, dry, and protein weights and total DNA content (expressed per 100 kg of BW), increased as the dosage of genistein increased (P < 0.001 for each regression). Uterine and cervical wet weights were increased by a dosage of 200 mg of genistein/d (P < 0.001 and P < 0.01, respectively) but not by 100 mg of genistein/d (P = 0.38 and P = 0.14, respectively) compared with those of control gilts. Height of epithelial cells lining the uterine glands and the lumen of uterus and cervix increased when gilts were treated with estradiol benzoate or 400 mg of genistein/d (P < 0.01). When the gilts were treated with estradiol benzoate or 400 mg of genistein/d, immunohistochemical staining demonstrated an increase in the percentage of cells that stained positive for progesterone receptor in the uterine glands and in the cells lining the vaginal cervix (P < 0.05). In gilts treated with 400 mg of genistein/d, the percentage of cells stained positive for proliferating cell nuclear antigen increased in the epithelium of the uterine glands, uterine lumen, and vaginal cervix (P < 0.05). Tissue growth was stimulated by genistein in a dosage-dependent manner, although no dosage of genistein induced a response as great as that of estradiol benzoate. Estrogen-sensitive tissues of the ovariectomized gilt, such as the cervix and uterus, are affected by injection of large dosages of the phytoestrogen genistein. The sensitivity of the uterus of the gilt to estrogenic substances makes it a potential model to examine the impact of environmental endocrine modulators on reproductive tissues.

Evaluation of the uterine environment and embryos of prepubertal gilts.

A series of three experiments was conducted to test the functional status of the uterus and embryos in prepubertal gilts. In Exp. 1, gilts were induced to ovulate by treating with gonadotropins followed by hCG 72 or 96 h later, and were artificially inseminated 24 h after hCG. Five of the 10 gilts treated at 120 d of age, but none of the gilts treated at 100 of age, maintained pregnancies. We next tested the function of the uterine environment by transferring embryos from postpubertal females into gilts of various ages that had been induced to ovulate but not inseminated (Exp. 2). Pregnancy rate at d 50 of gestation was 44% (4/9) for 100-d-old recipients, 67% (2/3) for 140-d-old recipients, and 60% (3/5) for postpubertal recipients (P > 0.20). Therefore, uteri of 100-d-old gilts are able to maintain pregnancies with conceptuses from postpubertal gilts. In Exp. 3, embryos from 100-d-old and postpubertal gilts were transferred into postpubertal recipients. Uterine horns of recipients were surgically separated before transfer, and embryos from 100-d-old and post-pubertal females were transferred to opposite horns of some recipients (experimental). Other recipients received embryos from postpubertal females in both uterine horns (control). When examined on d 50 to 60 of gestation, three of five control gilts were pregnant and three of seven experimental gilts were pregnant (P > 0.50). In experimental recipients, the survival of embryos from 100-d-old gilts was 38% (8/21) compared to 57% (15/26) for embryos from postpubertal gilts (P > 0.30). Because all uterine horns of pregnant recipients contained fetuses, these results support the hypothesis that embryos from 100-d-old gilts are able to initiate and maintain pregnancies in the uteri of postpubertal gilts. Therefore, the uterine environment of 100-d-old gilts provides an environment that supports development of embryos produced by postpubertal gilts, and the embryos produced by 100-d-old gilts can survive and develop in the uteri of postpubertal gilts. It was only the combination of embryos and uteri of 100-d-old gilts that did not permit pregnancy to be maintained.

Attenuation of estrogenic effects by dihydrotestosterone in the pig uterus is associated with downregulation of the estrogen receptors.

Androgens are known to attenuate some effects of estradiol-17beta (E) in the uterus. The objectives of the present experiment were to determine effects of 5alpha-dihydrotestosterone (DHT) on estrogenic actions in the pig uterus and its associations with changes in expression of the estrogen receptor (ER) alpha and ERbeta. Postpubertal gilts (120-130 kg of body weight; n = 16) were ovariectomized, and 3-4 weeks later received once-a-day injections (i.m.) of one of the following treatments during four consecutive days: 1) vehicle (corn oil), 2) E (250 microg), 3) E (250 microg) plus 1 mg DHT, or 4) E (250 microg) plus 10 mg DHT. Uterine tissues were collected 24 h after the last treatment. Gilts receiving E or E plus 1 mg DHT had greater uterine wet weight, uterine horn diameter, luminal epithelium thickness, and endometrial gland diameter compared with gilts treated with vehicle or E plus 10 mg DHT. Gilts receiving E or E plus 1 mg DHT were not different in these characteristics. Relative amounts of mRNAs in the endometrium for the cell proliferation marker histone H2a and the E-inducible protein complement component C3 increased in gilts treated with E compared with gilts treated with vehicle. E-induced increases in histone H2a and C3 mRNAs were not altered by cotreatment with E plus 1 mg DHT but were inhibited by E plus 10 mg DHT. Androgen receptor (AR) mRNA in the endometrium increased by treatment with E. Cotreatment of gilts with E and DHT did not alter the E-induced AR mRNA increase. Gilts treated with E plus 10 mg DHT had lesser amounts of immunoreactive ERalpha in cell nuclei of the myometrium and endometrial stroma and a tendency for a decrease in luminal epithelium compared with gilts treated with E. Amounts of immunoreactive ERalpha in glandular epithelium were not influenced by the treatments. Relative amounts of ERalpha and ERbeta mRNAs decreased in the endometrium of gilts treated with E plus 10 mg DHT compared with gilts treated with E. Downregulation of the ERs, particularly ERalpha in the myometrium and endometrial stroma, might be a relevant mechanism in the antagonism of estrogenic effects by DHT in the pig uterus.

Development of stereotypic behaviour in sows fed a starch diet or a non-starch polysaccharide diet during gestation and lactation over two parities

The effect of feeding sows a starch diet or a diet with a high level of fermentable non-starch polysaccharides (NSP) during gestation, lactation or both gestation and lactation over the first two parities on the development of stereotypic behaviour was studied in sows housed in groups during gestation and individually during lactation. A total of 119 postpubertal gilts were allotted to a 2×2 factorial experiment. Treatments were diet composition during gestation (G-Starch: 274g/kg starch and 123g/kg fermentable NSP or G-NSP: 86g/kg starch and 300g/kg fermentable NSP) and diet composition during lactation (L-Starch: 293g/kg starch and 113g/kg fermentable NSP or L-NSP: 189g/kg starch and 216g/kg fermentable NSP). Sows on both gestation diets were fed iso-energetic. During lactation, sows were given free access to the lactation diets. Behavioural measurements were carried out in weeks 3, 12 and 15 after start of the experiment or after weaning in parity 1 and 2 sows, respectively, 3 days before the expected date of parturition and 1 week before weaning in the first 3h after the morning meal using a scan-sampling technique. Feeding group-housed sows a diet high in fermentable NSP during gestation reduced the frequency of total non-feeding oral activities (=sham chewing + other non-feeding oral activities) in gestation compared with a starch diet (P<0.001 and <0.05, in parities 1 and 2, respectively). In both parity 1 and 2 sows, the greatest reduction was realised in week 15 and the smallest reduction in week 3. During lactation, the composition of the gestation diet did not affect the frequency of total non-feeding oral activities. Feeding sows a diet high in fermentable NSP during lactation reduced the frequency of total non-feeding oral activities in lactation in parity 2 sows (P<0.05) but not in parity 1 sows compared with a starch diet. Moreover, it tended to reduce, although not statistically significant (P=0.15), the frequency of total non-feeding oral activities during subsequent gestation. This study shows that diets high in fermentable NSP during gestation reduce the level of stereotypic behaviour in gestation. It seems that diets high in fermentable NSP during lactation may have an additional reducing effect on the development of stereotypic behaviour in subsequent gestation.