Effects of physical or fenceline boar exposure and exogenous gonadotropins on puberty induction and subsequent fertility in gilts.

The present study was part of a larger experiment that evaluated litter of origin effects on gilt production. The objectives of this study were to determine the effect of physical or fenceline boar exposure and exogenous gonadotropins on puberty induction and subsequent fertility in a commercial farm environment. The experiment was performed in three replicates. Prepubertal gilts were assigned by pen (13/pen) to receive 15 min of daily Fenceline (FBE, n = 153) or Physical (PBE, n = 154) Boar Exposure (BE) for 3 weeks starting at 184 d of age in a purpose-designed Boar Exposure Area (BEAR). At the start of week 3, prepubertal gilts were randomly assigned to receive PG600 or none (Control). From weeks 4 to 6, estrus was checked using only FBE. During weeks 1 to 3, measures of reproductive status were obtained weekly or until expression of estrus. Upon detection of first estrus, gilts were relocated into stalls and inseminated at second estrus. PBE reduced age (P = 0.001) and days to puberty (P = 0.002), increased the proportion of gilts in estrus (P = 0.04) in week 1 (38.3 vs. 27.5%), and tended (P = 0.08) to improve estrus in week 2 (37.6 vs. 26.1%) compared to FBE, respectively. In week 3, more prepubertal gilts receiving PBE-PG600 exhibited estrus (P = 0.04; 81.8%) compared to PBE-Control (40.3%), FBE-PG600 (56.4%), and FBE-Control (47.8%). Overall, expression of estrus through week 6 tended (P = 0.08) to be greater for PBE than FBE (91.5 vs. 85.0%). PBE increased (P ≤ 0.05) or tended to increase (P > 0.05 and ≤0.10) service and farrowing rates in parities 1 through 4, but within parity, there were no effects (P > 0.10) on pig production or wean to service interval. Analyses also indicated that weeks from start of boar exposure to puberty, litter of origin traits, and follicle measures at puberty were related to the subsequent fertility. The results of this study confirm the advantages of using increased intensity of boar exposure, combined with PG600 treatment, for effective induction of pubertal estrus in a commercial setting.

Associations among individual gilt birth weight, litter birth weight phenotype, and the efficiency of replacement gilt production.

Selection for larger litter size has increased the number of low individual birth weight (BWi) pigs and produced sows with a repeatable low average litter birth weight phenotype (BWP). Using an average of 3.6 litters records per sow, BWP was established in 644 nucleus-multiplication sows producing replacement gilts in a large commercial operation and classified as low (L-BWP, <1.18 kg, n = 85), medium (M-BWP, ≥1.18 to ≤1.35 kg, n = 250), or high (H-BWP, >1.35 kg, n = 309) on the basis of a BWi of 1.18 kg below which there was a high risk of early mortality and the average BWi (1.35 kg) for the population. In subsequent litters, potential replacement gilts born to these sows (n = 7,341) received a unique identification tag that allowed the impact of BWi, BWP, and their interactions on the efficiency of replacement gilt production to be evaluated. Negative effects of BWi on mortality until day 4 after birth were confirmed (P < 0.05) and cumulative losses to weaning, to day 70 of age, and to final pre-selection at 165 d of age were affected (P ≤ 0.05) by the interaction between BWP and BWi. Among the 2,035 gilts for which records for selection efficiency and production to fourth parity were available, a lower BWi decreased the probability of gilts reaching pubertal estrus (P < 0.05) after 21 and 28 d of boar stimulation starting at 180 d of age, with no effect of BWP. Overall, neither BWi, BWP, nor their interaction affected age at puberty. After breeding, only the main effect of BWP affected productivity and retention in the sow herd. In parities 1 and 2, percent stillborn was higher in litters born to gilts from H-BWP compared with L-BWP dams (P < 0.05), and in parity 2, total born and born alive were lower in sows derived from H-BWP compared with other BWPs. There were no differences in retention based on BWP classes until parity 2, after which retention tended (P ≤ 0.09) to be lower in sows derived from H-BWP compared with L-BWP dams. These results provide evidence that sow BWP is an important factor in the overall efficiency of replacement gilt management. This study also confirms that effective gilt selection and pre-breeding management protocols support excellent sow lifetime productivity and mitigate the risk of a high BWP in the litter of origin affecting retention in the breeding herd.

Gilt Management for Fertility and Longevity.

Substantial evidence supports successful management of gilts as an absolutely necessary component of breeding herd management and the pivotal starting point for the future fertility and longevity of the breeding herd. Therefore, gilt management practices from birth have the potential to influence the future reproductive performance of the sow herd. A good gilt management program will address several key components such as birth traits that determine the efficiency of replacement gilt production; effective selection of the most fertile gilts for entry to the breeding herd; effective management programs that provide a consistent supply of service eligible gilts; and appropriate management of weight, physiological maturity, and a positive metabolic state at breeding. Good gilt management can largely resolve the existing gap between excellent genetic potential and the more modest sow lifetime productivity typically achieved in the industry. Investment in good gilt development programs from birth represents a foundational opportunity for improving the efficiency of the pork production industry.

Identification of differentially expressed genes in sexed pig embryos during post-hatching development in primiparous sows exposed to differing intermittent suckling and breeding strategies.

The aim of commercial pig breeding programs is to maximize the number of pigs produced per sow per year. Given that sows exhibit an estrus during lactation is a potential means of increasing productivity of a pig breeding herd without reducing in lactation length, conventionally, weaning of piglets at a relatively young age is often related to post-weaning piglet performance which compromises piglet welfare. Therefore, intermittent suckling (IS) is a management technique in which lactating sows are separated from their piglets for a fixed period of the days and allowing sows to continue nursing piglets while exhibiting estrus and being breed during lactation, thereby promoting both piglet well-being and sow reproductive performance [1]. For this study, primiparous sows (PP) were exposed to 28 day (D28) lactation with intermittent suckling (IS) during the final week prior to weaning. The sows detected to be in estrus during lactation were either bred at this first estrus (FE) during lactation (IS21FE), or were "skipped" and bred at their second estrus which occurred after final weaning at D28 (IS21SE). Despite the benefits of IS, the effects of the maternal physiology related to breeding during lactation on embryonic transcriptome are largely unknown. Recent advances in the ability to assess embryonic gene expression in both sexes have made these analyses possible. Here, we describe the experimental procedures of two color microarray analyses and annotation of differentially expressed (DE) genes in detail corresponding to data deposited at NCBI in the Gene Expression Omnibus under accession number GSE53576 and GSE73020 for day 9 embryos (D9E) and day 30 embryos (D30E) respectively. Although only a few DE genes were discovered between IS21FE and IS21SE in both sexes from D9E or D30E, the raw data are still valuable for future use to understand the gene expression profiling from two different developmental stages.

Transcriptomic and Epigenetic Profiling of the Lung of Influenza-Infected Pigs: A Comparison of Different Birth Weight and Susceptibility Groups.

Influenza viruses are a common cause of respiratory disease in swine. Infections range in severity from asymptomatic to causing significant morbidity. The main objective of this study was to compare lung transcriptomic and epigenetic responses to influenza infection in pigs from high or low birth weight litters. The latter is a potential indicator of intrauterine growth restriction, a significant risk factor for prenatal programming effects. Individual pigs from high (HBW) or low birth weight (LBW) litters (n = 17) were inoculated with influenza A virus and euthanized 48 hours later. Lesion severity and viral loads were assessed as previously described. The transcriptional response to infection in LBW and HBW groups (n = 16) was assessed by microarray. A separate analysis of pigs classified as 'Resilient' (RES) or 'Susceptible' (SUS) (n = 6) on the basis of severity of lung pathology was also conducted. Eight genes were confirmed as differentially expressed for the birth weight comparison, including three antiviral genes with lower expression in LBW: ISG15, OAS1, and OAS2 (P<0.05). The promoter region methylation status of these three genes was assessed for each birth weight group, and no differences were found. These expression data are consistent with our previous finding that LBW pigs had less severe lesion scores and a trend towards lower viral titres in lung than the HBW cohort. The SUS v RES comparison identified 91 differentially expressed genes (FDR<0.05) that were enriched with functional annotation terms and pathways associated with inflammation. The cytokine genes IL6, IL8, and CCL2 were all upregulated in SUS pigs, and may have driven disease severity in these animals. In conclusion, this study found no evidence that the transcriptional immune response to influenza was adversely affected by low litter birth weight, but did identify several candidate genes for driving disease pathology.

Birth weight, intrauterine growth retardation and fetal susceptibility to porcine reproductive and respiratory syndrome virus.

The severity of porcine reproductive and respiratory syndrome was compared in pregnant gilts originating from high and low birth weight litters. One-hundred and eleven pregnant gilts experimentally infected with porcine reproductive and respiratory syndrome virus on gestation day 85 (±1) were necropsied along with their fetuses 21 days later. Ovulation rates and litter size did not differ between groups, but fetuses from low birth weight gilts were shorter, lighter and demonstrated evidence of asymmetric growth with large brain:organ weight ratios (i.e. brain sparing). The number of intrauterine growth retarded fetuses, defined by brain:organ weight ratios greater than 1 standard deviation from the mean, was significantly greater in low, compared to high, birth weight gilts. Although γδ T cells significantly decreased over time in high compared to low birth weight gilts, viral load in serum and tissues, gilt serum cytokine levels, and litter outcome, including the percent dead fetuses per litter, did not differ by birth weight group. Thus, this study provided no substantive evidence that the severity of porcine reproductive and respiratory syndrome is affected by dam birth weight. However, intrauterine growth retarded fetuses had lower viral loads in both fetal thymus and in endometrium adjacent to the umbilical stump. Crown rump length did not significantly differ between fetuses that survived and those that died at least one week prior to termination. Taken together, this study clearly demonstrates that birth weight is a transgenerational trait in pigs, and provides evidence that larger fetuses are more susceptible to transplacental PRRSv infection.

Characterization of the altered gene expression profile in early porcine embryos generated from parthenogenesis and somatic cell chromatin transfer.

The in vitro production of early porcine embryos is of particular scientific and economic interest. In general, embryos produced from in vitro Assisted Reproductive Technologies (ART) manipulations, such as somatic cell chromatin transfer (CT) and parthenogenetic activation (PA), are less developmentally competent than in vivo-derived embryos. The mechanisms underlying the deficiencies of embryos generated from PA and CT have not been completely understood. To characterize the altered genes and gene networks in embryos generated from CT and PA, comparative transcriptomic analyses of in vivo (IVV) expanded blastocysts (XB), IVV hatched blastocyst (HB), PA XB, PA HB, and CT HB were performed using a custom microarray platform enriched for genes expressed during early embryonic development. Differential expressions of 1492 and 103 genes were identified in PA and CT HB, respectively, in comparison with IVV HB. The "eIF2 signalling", "mitochondrial dysfunction", "regulation of eIF4 and p70S6K signalling", "protein ubiquitination", and "mTOR signalling" pathways were down-regulated in PA HB. Dysregulation of notch signalling-associated genes were observed in both PA and CT HB. TP53 was predicted to be activated in both PA and CT HB, as 136 and 23 regulation targets of TP53 showed significant differential expression in PA and CT HB, respectively, in comparison with IVV HB. In addition, dysregulations of several critical pluripotency, trophoblast development, and implantation-associated genes (NANOG, GATA2, KRT8, LGMN, and DPP4) were observed in PA HB during the blastocyst hatching process. The critical genes that were observed to be dysregulated in CT and PA embryos could be indicative of underlying developmental deficiencies of embryos produced from these technologies.

Development of a porcine (Sus scofa) embryo-specific microarray: array annotation and validation.

BACKGROUND: The domestic pig is an important livestock species and there is strong interest in the factors that affect the development of viable embryos and offspring in this species. A limited understanding of the molecular mechanisms involved in early embryonic development has inhibited our ability to fully elucidate these factors. Next generation deep sequencing and microarray technologies are powerful tools for delineation of molecular pathways involved in the developing embryo. RESULTS: Here we present the development of a porcine-embryo-specific microarray platform created from a large expressed sequence tag (EST) analysis generated by Roche/454 next-generation sequencing of cDNAs constructed from critical stages of in vivo or in vitro porcine preimplantation embryos. Two cDNA libraries constructed from in vitro and in vivo produced preimplantation porcine embryos were normalized and sequenced using 454 Titanium pyrosequencing technology. Over one million high-quality EST sequences were obtained and used to develop the EMbryogene Porcine Version 1 (EMPV1) microarray composed of 43,795 probes. Based on an initial probe sequence annotation, the EMPV1 features 17,409 protein-coding, 473 pseudogenes, 46 retrotransposed, 2,359 non-coding RNA, 4,121 splice variants in 2,862 genes and a total of 12,324 Novel Transcript Regions (NTR). After re-annotation, the total unique genes increased from 11,961 to 16,281 and 1.9% of them belonged to a large olfactory receptor (OR) gene family. Quality control on the EMPV1 was performed and revealed an even distribution of ten clusters of spiked-in control spots and array to array (dye-swap) correlation was 0.97. CONCLUSIONS: Using next-generation deep sequencing we have produced a large EST dataset to allow for the selection of probe sequences for the development of the EMPV1 microarray platform. The quality of this embryo-specific array was confirmed with a high-level of reproducibility using current Agilent microarray technology. With more than an estimated 20,000 unique genes represented on the EMPV1, this platform will provide the foundation for future research into the in vivo and in vitro factors that affect the viability of porcine embryos, as well as the effects of these factors on the live offspring that result from these embryos.

Pig preovulatory oocytes modulate cumulus cell protein and gene expression in vitro.

This study investigated the changes in protein and gene expression in oocytectomized cumulus cells (OOX) of medium-sized follicles from gilts, cultured with or without denuded oocytes isolated from large oestrogenic sow follicles. Proteomic analysis identified 14 proteins that were differentially expressed in OOX, of which the protein 14-3-3 eta, a signal transduction pathway modulator, was down-regulated in the presence of oocytes. Oocyte co-culture also down-regulated FSHR mRNA expression in OOX, as measured by real-time PCR, and FSHR and 14-3-3 eta mRNA abundance were positively correlated. The oocyte also up-regulated HSD3B mRNA, suggesting an effect on cumulus cell progesterone synthesis. Together with data on gene expression in granulosa cells during the follicular phase of the sow oestrous cycle, this study suggests that modulation of the expression of steroidogenesis related proteins and genes in cumulus cells by the porcine preovulatory oocyte reflects the specific physiological requirements of the preovulatory follicle.

Seminal plasma proteins as potential markers of relative fertility in boars.

This study investigated whether specific proteins from distinct seminal plasma fractions of boars could be related to in vivo fertility. Nine boars with acceptable sperm motility and morphology for use in artificial insemination demonstrated major differences in total number born and pregnancy rate when low sperm doses (1.5 billion sperm) were used to breed a minimum of 50 gilts per boar. The 2 lowest-fertility and 2 highest-fertility boars were chosen for evaluation of specific seminal plasma proteins. On 4 occasions, semen was collected and separated into 3 fractions based on sperm concentration (Sperm-Peak, Sperm-Rich, and Sperm-Free), and the fractions were analyzed for total protein concentration and abundance of major seminal plasma glycoprotein (PSP-I), AWN-1, and osteopontin protein using Western blotting techniques. The concentrations of these seminal plasma proteins were lower in the Sperm-Peak fractions compared with the Sperm-Free fractions (P < .05). Seminal plasma from the pooled Sperm-Rich fraction used for artificial insemination was also subjected to two-dimensional gel electrophoresis to investigate novel protein markers related to in vivo fertility. Total piglets born (r = -0.76, P = .01) and sperm motility at day 7 (r = -0.74, P = .037) were again negatively correlated with a 22-kDa protein identified by mass spectrometry as PSP-I. However, fertility index and farrowing rate tended to be positively correlated (P < .10) with a 25-kDa protein, identified as glutathione peroxidase (GPX5), an antioxidant enzyme that may protect sperm membranes from oxidative damage. These candidate proteins merit further investigation as markers of fertility in boars.

Temporal regulation of BMP2, BMP6, BMP15, GDF9, BMPR1A, BMPR1B, BMPR2 and TGFBR1 mRNA expression in the oocyte, granulosa and theca cells of developing preovulatory follicles in the pig.

This study aimed to describe the abundance and localization of BMP2, BMP6, BMP15, GDF9, BMPR1A, BMPR1B, BMPR2 and TGFBR1 mRNA during pig preovulatory follicular development and to evaluate their implication in improving follicular maturity in the preovulatory period preceding the second versus first post-weaning oestrus. Oocytes, granulosa (GC) and theca cells (TC) were recovered from antral follicles of primiparous sows at day 1, 2 and 4 after weaning and at day 14, 16 and 20 of their subsequent oestrous cycle. Real-time PCR analysis revealed that with the exception of BMP6 mRNA, which was absent in GC, all genes were expressed in every cell type. Although BMP6, BMP15 and GDF9 mRNA were most abundant in the oocyte, their expression remained relatively constant during follicular development. By contrast, receptor BMPR1B and TGFBR1 expressions in the GC and TC were temporally regulated. BMPR1B mRNA abundance was positively correlated with plasma oestradiol (E2) suggesting that its regulation by oestrogen may be implicated in normal folliculogenesis. Interestingly, the increase in BMPR1B mRNA and protein abundance during the periovulatory period in GC and TC suggests a role for bone morphogenetic protein (BMP) 15 in the ovulatory process. Finally, expression of these ligands and receptors was not associated with potential differences in follicle maturity observed during the second versus first post-weaning preovulatory follicular wave. In conclusion, our results clearly demonstrate the presence of a complex signalling system within the pig follicle involving the transforming growth factor-beta superfamily and their receptors, and provide evidence to support a role for BMP15 and BMPR1B during ovulation.

Satellite cell ablation attenuates short-term fast-to-slow fibre type transformations in rat fast-twitch skeletal muscle.

The purpose of this time-course study was to determine whether satellite cell ablation within rat tibialis anterior (TA) muscles exposed to short-term chronic low-frequency stimulation (CLFS) would limit fast-to-slow fibre type transformations. Satellite cells of the left TA were ablated by exposure to gamma-irradiation before 1, 2, 5 or 10 days of CLFS and 1 week later where required. Control groups received only CLFS or a sham operation. Continuous infusion of 5-bromo-2'-deoxyuridine revealed that CLFS first induced an increase in satellite cell proliferation at 1 day, up to a maximum at 10 days over control (mean +/- SEM, 5.7 +/- 0.7 and 20.4 +/- 1.0 versus 1.5 +/- 0.2 mm(-2), respectively, P < 0.007) that was abolished by gamma-irradiation. Myosin heavy chain mRNA, immunohistochemical and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses revealed CLFS-induced fast-to-slow fibre type transformation began at 5 days and continued at 10 days; in those muscles that were also exposed to gamma-irradiation, attenuation occurred within the fast fibre population, and the final fast-twitch to slow-twitch adaptation did not occur. These findings indicate satellite cells play active and obligatory roles early on in the time course during skeletal muscle fibre type adaptations to CLFS.

Effect of satellite cell ablation on low-frequency-stimulated fast-to-slow fibre-type transitions in rat skeletal muscle.

The purpose of this study was to determine whether satellite cell ablation within rat fast-twitch muscles exposed to chronic low-frequency stimulation (CLFS) would limit fast-to-slow fibre-type transitions. Twenty-nine male Wistar rats were randomly assigned to one of three groups. Satellite cells of the left tibialis anterior were ablated by weekly exposure to a 25 Gy dose of gamma-irradiation during 21 days of CLFS (IRR-Stim), whilst a second group received only 21 days of CLFS (Stim). A third group received weekly doses of gamma-irradiation (IRR). Non-irradiated right legs served as internal controls. Continuous infusion of 5-bromo-2'-deoxyuridine (BrdU) revealed that CLFS induced an 8.0-fold increase in satellite cell proliferation over control (mean +/-s.e.m.: 23.9 +/- 1.7 versus 3.0 +/- 0.5 mm(-2), P < 0.0001) that was abolished by gamma-irradiation. M-cadherin and myogenin staining were also elevated 7.7- and 3.8-fold (P < 0.0001), respectively, in Stim compared with control, indicating increases in quiescent and terminally differentiating satellite cells; these increases were abolished by gamma-irradiation. Myonuclear content was elevated 3.3-fold (P < 0.0001) in Stim, but remained unchanged in IRR-Stim. Immunohistochemical analyses revealed attenuation of fast-to-slow fibre-type transitions in IRR-Stim compared with Stim. Comparable changes were observed at the protein level by SDS-PAGE. It is concluded that although considerable adaptive potential exists within myonuclei, satellite cells play a role in facilitating fast-to-slow fibre-type transitions.

Increasing feed intake in late gestation does not affect plasma progesterone concentration in the sow.

Rate of decline in plasma progesterone concentration may influence the success of lactogenesis in the sow. The aim of this experiment was to investigate whether progesterone concentration and rate of decline of progesterone in the periparturient sow could be manipulated by changing her feeding level. Forty-two sows received either 1.15 or 2 times maintenance energy daily from day 100 of gestation up until and including the day of farrowing. Blood samples were taken on days 98 (pre-treatment baseline) and 109 of gestation, during farrowing, 6h after farrowing and at 09:00 h for the 3 days following farrowing. Plasma progesterone concentration was determined and progesterone half-life was calculated for each sow. High intake feeding had no effect on plasma progesterone concentration at any time of sampling. Progesterone half-life averaged 41.2 +/- 3.81 h and did not differ between treatments. There was no relationship between progesterone concentration, or half-life, and litter weight gain, although there was a weak correlation between decline in progesterone in the first 6h after birth and piglet growth rate from birth to 6 days of age (R(2) = 0.109, P < 0.05). It was concluded that increasing feed intake in late gestation cannot be used to increase progesterone clearance rate and hasten the onset of lactogenesis in sows.

Regulation of IGF-I and porcine oviductal secretory protein (pOSP) secretion into the pig oviduct in the peri-ovulatory period, and effects of previous nutrition.

The mechanisms regulating oviduct function were investigated. In Experiment 1, porcine oviductal secretory protein (pOSP) mRNA, and pOSP and insulin-like growth factor (IGF-I) in oviductal flushings, decreased through the peri-ovulatory period. In Experiment 2, higher plasma steroids in oviductal veins, ipsilateral (INT), rather than contralateral (OVX), to the remaining ovary in unilaterally ovariectomized gilts, were associated with higher pOSP in INT oviductal flushings. In Experiment 3, oviduct function was assessed as part of a collaborative study in cyclic gilts. Feed restriction in the late, compared to the early, luteal phase reduced estradiol concentrations in oviductal plasma, pOSP mRNA in oviductal tissue, and IGF-I concentrations and pOSP abundance in oviduct flushings. Previous insulin treatment differentially affected oviduct function. These data provide the first direct evidence for effects of previous feed restriction and insulin treatment on the oviduct environment in the peri-ovulatory period, which may contribute to nutritional effects on embryonic survival.