Sexual dimorphism in placental development and function: Comparative physiology with an emphasis on the pig.

Across mammalian species, it has been demonstrated that sex influences birth weight, with males being heavier than females; a characteristic that can be observed from early gestation. Male piglets are more likely to be stillborn and have greater preweaning mortality than their female littermates, despite the additional maternal investment into male fetal growth. Given the conserved nature of the genome between the sexes, it is hypothesized that these developmental differences between males and females are most likely orchestrated by differential placental adaptation. This review summarizes the current understanding of fetal sex-specific differences in placental and endometrial structure and function, with an emphasis on pathways found to be differentially regulated in the pig including angiogenesis, apoptosis, and proliferation. Given the importance of piglet sex in agricultural enterprises, and the potential for skewed litter sex ratios, it is imperative to improve understanding of the relationship between fetal sex and molecular signaling in both the placenta and endometria across gestation.

Associations between maternal vitamin D status and porcine litter characteristics throughout gestation.

Emerging evidence suggests an important role of vitamin D in the establishment and maintenance of pregnancy, and the regulation of foetal growth across mammalian species. However, the temporal changes in maternal vitamin D status throughout gestation in the pig and the relationship between maternal vitamin D status and litter characteristics of interest across gestation remain poorly understood and under-investigated. The abundance of 25(OH)D in maternal plasma was quantified by HPLC-MS/MS at gestational days (GD) 18, 30, 45, 60 and 90 (n = 5-11 gilts/GD). Maternal plasma 25(OH)D concentrations significantly increased between GD18 and GD30 (P < 0.05). The relationship between maternal vitamin D metabolite concentrations and litter characteristics of interest including gilt weight, ovulation rate, mean litter weight, number of live foetuses, percentage prenatal survival, and sex ratio of the litter was assessed. Maternal 25(OH)D (P = 0.059) concentrations tended to be positively associated with percentage prenatal survival on GD60. On GD90, maternal 25(OH)D (P < 0.05) concentrations were inversely associated with gilt weight. Maternal plasma 25(OH)D concentrations were inversely associated with the percentage of male foetuses in the litter on GD90 (P < 0.05). This study has provided novel insights into temporal changes in maternal vitamin D status throughout gestation and the relationship between maternal vitamin D status and the economically important litter characteristics of gilt weight, percentage prenatal survival and percentage of male foetuses in the litter. Improving the understanding of the role of vitamin D across important developmental timepoints in relation to foetal growth is essential to improve reproductive success in livestock species.

Birthweight leads to seminal and testicular morphofunctional commitment in sexually mature boars.

Given the importance of males as semen donors for artificial insemination (AI) and the high incidence of low birthweight piglets at commercial farms, the impact of birthweight on fertility in boars deserves special attention. The aim of this study was to evaluate testicular morphofunctional parameters and semen characteristics in different birthweight boars. Forty littermate males were selected at birth and divided into two experimental groups, according to birthweight: high (HW, birthweight ranging from 1.80 to 2.15 kg, n = 20) and low (LW, birthweight ranging from 0.75 to 1.10 kg, n = 20). At 170 days of age, a sub-set of 24 littermate boars (n = 12 HW and n = 12 LW) were randomly selected for semen collection, which was performed once a week, at a 15-day interval, during five weeks. At 300 days of age, boars were orchiectomized, and the testis processed for histological and molecular analyses. The HW group was heavier and presented larger testes compared to LW animals (P < 0.05). Despite that, birthweight did not significantly affect semen volume or sperm quality parameters (concentration, motility, vigor or morphology), although LW boars produced 38.2% fewer total sperm and 24% lower semen concentration, leading to 36.8% less inseminating doses. The histomorphometrical evaluation showed that seminiferous tubules diameter and germinal epithelium height were similar between experimental groups. However, LW boars presented shorter seminiferous tubules and, consequently, fewer Sertoli cells per testis (P 0.05). Even though plasma testosterone levels were equivalent in both birthweight groups, LW testis presented less androgen receptors (P < 0.05). Additionally, birthweight was positively correlated with total seminiferous tubule length and number of Sertoli cells (P < 0.01), and with body and testis weights (P < 0.01). Taken together, even though adult LW boars showed no evident semen pathologies or spermatogenesis commitment, mature HW males have the potential to produce more spermatozoa, consequently more semen doses per ejaculate, being more valuable to an industry that relies on AI.

Associations between testicular development and fetal size in the pig.

BACKGROUND: Impaired reproductive performance is the largest contributing factor for the removal of boars from commercial systems. Intrauterine growth restricted piglets represent 25% of the total number of piglets born and have impaired reproductive performance. This study aimed to improve the understanding of temporal changes in testicular gene expression during testes development in fetuses of different size. The lightest and closest to mean litter weight (CTMLW) male Large White × Landrace littermates were collected at gestational days (GD) 45, 60 and 90 (n = 5-6 litters/GD). RESULTS: Testes weight and testes weight as a percentage of fetal weight were not associated with fetal size at GD60 or 90. Fetal plasma testosterone was not associated with fetal size at GD90. There was no association between fetal size and seminiferous tubule area and number, number of germ or Sertoli cells per tubule. The lightest fetuses tended to have wider seminiferous tubules compared to the CTMLW fetuses at GD90 (P = 0.077). The testicular expression of KI67 (P ≤ 0.01) and BAX:BCL2 ratio (P = 0.058) mRNAs decreased as gestation progressed. Greater SPP1 mRNA expression was observed at GD60 when compared with GD45 and 90 (P ≤ 0.05). Lower expression of DMRT1 and SPP1 (P < 0.01) mRNAs was observed in testes associated with the lightest fetuses compared to the CTMLW fetuses at GD90. CONCLUSIONS: These findings provide novel insights into the expression profiles of genes associated with testicular development and function. Further, these data suggest that programming of reproductive potential in IUGR boars occurs late in gestation, providing a platform for further mechanistic investigation.

KLB dysregulation mediates disrupted muscle development in intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is a leading cause of neonatal morbidity and mortality in humans and domestic animals. Developmental adaptations of skeletal muscle in IUGR lead to increased risk of premature muscle loss and metabolic disease in later life. Here, we identified ß-Klotho (KLB), a fibroblast growth factor 21 (FGF21) co-receptor, as a novel regulator of muscle development in IUGR. Using the pig as a naturally-occurring disease model, we performed transcriptome-wide profiling of fetal muscle (day 90 of pregnancy) from IUGR and normal-weight (NW) littermates. We found that, alongside large-scale transcriptional changes comprising multiple developmental, tissue injury and metabolic gene pathways, KLB was increased in IUGR muscle. Moreover, FGF21 concentrations were increased in plasma in IUGR fetuses. Using cultures of fetal muscle progenitor cells (MPCs), we showed reduced myogenic capacity of IUGR compared to NW muscle in vitro, as evidenced by differences in fusion indices and myogenic transcript levels, as well as mechanistic target of rapamycin (mTOR) activity. Moreover, transfection of MPCs with KLB small interfering RNA promoted myogenesis and mTOR activation, whereas treatment with FGF21 had opposite and dose-dependent effects in porcine and also in human fetal MPCs. In conclusion, our results identify KLB as a novel and potentially critical mediator of impaired muscle development in IUGR, through conserved mechanisms in pigs and humans. Our data shed new light onto the pathogenesis of IUGR, a significant cause of lifelong ill-health in humans and animals. KEY POINTS: Intrauterine growth restriction (IUGR) is associated with large-scale transcriptional changes in developmental, tissue injury and metabolic gene pathways in fetal skeletal muscle. Levels of the fibroblast growth factor 21 (FGF21) co-receptor, ß-Klotho (KLB) are increased in IUGR fetal muscle, and FGF21 concentrations are increased in IUGR fetal plasma. KLB mediates a reduction in muscle development through inhibition of mechanistic target of rapamycin signalling. These effects of KLB on muscle cells are conserved in pig and human, suggesting a vital role of this protein in the regulation of muscle development and function in mammals.

Profiling of open chromatin in developing pig (Sus scrofa) muscle to identify regulatory regions.

There is very little information about how the genome is regulated in domestic pigs (Sus scrofa). This lack of knowledge hinders efforts to define and predict the effects of genetic variants in pig breeding programs. To address this knowledge gap, we need to identify regulatory sequences in the pig genome starting with regions of open chromatin. We used the "Improved Protocol for the Assay for Transposase-Accessible Chromatin (Omni-ATAC-Seq)" to identify putative regulatory regions in flash-frozen semitendinosus muscle from 24 male piglets. We collected samples from the smallest-, average-, and largest-sized male piglets from each litter through five developmental time points. Of the 4661 ATAC-Seq peaks identified that represent regions of open chromatin, >50% were within 1 kb of known transcription start sites. Differential read count analysis revealed 377 ATAC-Seq defined genomic regions where chromatin accessibility differed significantly across developmental time points. We found regions of open chromatin associated with downregulation of genes involved in muscle development that were present in small-sized fetal piglets but absent in large-sized fetal piglets at day 90 of gestation. The dataset that we have generated provides a resource for studies of genome regulation in pigs and contributes valuable functional annotation information to filter genetic variants for use in genomic selection in pig breeding programs.

Associations between foetal size and ovarian development in the pig.

It is estimated that intra-uterine growth restricted piglets represent 25 % of the total number of piglets born. Growth restricted female pigs have impaired reproductive performance postnatally. HHowever, when during gestation this phenotype arises is not known. With this study, the aim was to improve the understanding of foetal ovarian development in normal and small foetuses throughout gestation. Female Large White X Landrace foetuses were obtained at gestational day (GD) 45, 60 and 90 (n = 5-6 litters/GD). Histological analysis of GATA4 stained foetal ovaries at GD60 and 90 indicated there were fewer primary follicles (P ≤ 0.05) in the foetuses weighing the least compared to those with a weight similar to the mean for the litter (CTMLW) at GD90. Plasma oestradiol concentrations were less in the foetuses with lesser weights compared with greater weight foetuses at GD90 (P ≤ 0.05). The RNA was extracted from ovaries of the lesser weight and CTMLW foetuses at GD45, 60 and 90 and qPCR was performed to quantify relative abundance of 12 candidate mRNAs for which encoded proteins that modulate ovarian function and development. Gestational changes in relative abundances of CD31, PTGFR, SPP1 and VEGFA mRNA transcripts were observed. Relative abundance of KI67 (P = 0.066) and P53 (P ≤ 0.05) was less in ovaries of the lesser weight compared to CTMLW foetuses at GD60. There was a lesser relative abundance of PTGFR mRNA transcript in ovaries from the foetuses with lesser weight compared to CTMLW foetuses at GD45 and 60 (P ≤ 0.05). These findings indicate that postnatal differences in the reproductive potential of growth restricted females are programmed early in gestation. It is hoped that further investigation will improve the understanding of the relationship between prenatal reproductive development and postnatal reproductive performance.

Identification of appropriate reference genes for qPCR analyses of porcine placentae and endometria, supplying foetuses of different size and sex, at multiple gestational days.

Recent studies suggest associations exist between foetal size and sex, and gene expression at the porcine feto-maternal interface. It is essential to identify reference genes which have stable expression throughout gestation in feto-placental units associated with foetuses of different size and sex. qPCR was performed for 11 genes within porcine placentae and endometria at gestational days (GD) 30, 60 and 90. Several reference genes were found to have stable expression in these samples. The combination of B2m1 and Tbp1, and Hprt1 and Tbp1 had the most stable expression in endometria and placentae, respectively. Reference genes identified as having stable expression were utilized in a larger experiment with placentae and endometria associated with foetuses of different size and sex at four GD. The average expression of B2m1 and Tbp1 mRNAs was suitable for the normalization of temporal changes in endometria, and comparison between endometria supplying foetuses of different size throughout gestation. The average expression of Hprt1 and Tbp1 mRNAs was suitable for the normalization of placental mRNA expression for comparison of temporal changes and sex differences between placentae supplying foetuses of different sex throughout gestation. This combination was suitable for the normalization of mRNA expression in placentas supplying GD30, GD60 and GD90 foetuses of different size. This study has identified reference genes with stable expression in placentae and endometria across multiple gestational days, in tissues associated with foetuses of different size and sex. The results of these experiments highlight the importance of selecting appropriate reference genes for the biological comparison under investigation.

Novel relationships between porcine fetal size, sex, and endometrial angiogenesis†.

It is hypothesized that growth restriction occurs due to inadequate vascularization of the feto-maternal interface. Evidence exists for sexual dimorphism in placental function although associations between fetal sex and the endometrium remain poorly investigated. This study investigated the relationship between porcine fetal size, sex and endometrial angiogenesis at multiple gestational days (GD). Endometrial samples supplying the lightest and closest to mean litter weight (CTMLW), male and female Large White X Landrace conceptuses or fetuses were obtained at GD18, 30, 45, 60, and 90 (n = 5-9 litters/GD). Immunohistochemistry for CD31 revealed a greater number of blood vessels in endometrium supplying females compared to those supplying males at GD45. Endometrial samples supplying the lightest fetuses had fewer blood vessels (GD60) and uterine glands (GD90) compared to those supplying the CTMLW fetuses. Quantitative PCR revealed decreased CD31 (GD60), HPSE and VEGFA (GD90) expression, alongside increased HIF1A (GD45) expression in endometrial samples supplying the lightest compared to the CTMLW fetuses. At GD30, PTGFR, CD31, and VEGFA mRNA expression was increased in samples supplying female fetuses compared to those supplying male fetuses. Intriguingly, decreased expression of ACP5, CD31, HIF1A, and VEGFA mRNAs was observed at GD60 in endometrial samples supplying female fetuses compared to those supplying their male littermates. Endothelial cell branching assays demonstrated impaired endothelial cell branching in response to conditioned media from endometrial samples supplying the lightest and female fetuses compared with the CTMLW and male fetuses, respectively. This study has highlighted that endometrial tissues supplying the lightest and female fetuses have impaired angiogenesis when compared with the CTMLW and female fetuses respectively. Importantly, the relationship between fetal size, sex and endometrial vascularity is dynamic and dependent upon the GD investigated.

Association of foetal size and sex with porcine foeto-maternal interface integrin expression.

Integrins regulate adhesion at the foeto-maternal interface by interacting with secreted phosphoprotein 1 (SPP1) and fibronectin (FN). It is hypothesised that impaired foetal growth of 'runt' piglets is linked to altered integrin signalling at the foeto-maternal interface. Placental and endometrial samples associated with the lightest and closest to mean litter weight (CTMLW) (gestational day (GD18, 30, 45, 60 and 90), of both sex (GD30, 45, 60 and 90) (n = 5-8 litters/GD), Large White × Landrace conceptuses or foetuses were obtained. The mRNA expression of the integrin subunits (ITG) ITGA2, ITGAV, ITGB1, ITGB3, ITGB5, ITGB6, ITGB8, SPP1 and FN was quantified by qPCR. Temporal changes in mRNA expression were observed, with different profiles in the two tissues. Endometrial ITGB1 (P ≤ 0.05, GD45) and SPP1 (P ≤ 0.05, all GD combined and GD60) expression was decreased in samples supplying the lightest compared to the CTMLW foetuses. Placentas supplying female foetuses had decreased expression of ITGB6 (GD45, P ≤ 0.05) and FN (GD90, P ≤ 0.05) compared to those supplying male foetuses. Endometrial samples supplying females had increased ITGB3 (P ≤ 0.05, GD60) and FN (P ≤ 0.05, GD30) expression and decreased SPP1 (P ≤ 0.05, GD60) expression compared to male foetuses. Correlations between mean within-gilt mRNA expression and percentage prenatal survival, number of live foetuses or conceptuses and percentage male foetuses were observed. This study has highlighted novel and dynamic associations between foetal size, sex and integrin subunit mRNA expression at the porcine foeto-maternal interface. Further studies should be performed to improve the understanding of the mechanisms behind these novel findings.

Associations between fetal size, sex and placental angiogenesis in the pig.

Inadequate fetal growth cannot be remedied postnatally, leading to severe consequences for neonatal and adult development. It is hypothesized that growth restriction occurs due to inadequate placental vascularization. This study investigated the relationship between porcine fetal size, sex, and placental angiogenesis at multiple gestational days (GD). Placental samples supplying the lightest and closest to mean litter weight (CTMLW), male and female Large White X Landrace fetuses were obtained at GD30, 45, 60, and 90. Immunohistochemistry revealed increased chorioallantoic membrane CD31 staining in placentas supplying the lightest compared to those supplying the CTMLW fetuses at GD60. At GD90, placentas supplying the lightest fetuses had decreased CD31 staining in the chorioallantoic membrane compared to those supplying the CTMLW fetuses. The mRNA expression of six candidate genes with central roles at the feto-maternal interface increased with advancing gestation. At GD60, ACP5 expression was increased in placentas supplying the lightest compared to the CTMLW fetuses. At GD45, CD31 expression was decreased in placentas supplying the lightest compared to the CTMLW fetuses. In contrast, CD31 expression was increased in placentas supplying the lightest compared the CTMLW fetuses at GD60. In vitro endothelial cell branching assays demonstrated that placentas supplying the lightest and male fetuses impaired endothelial cell branching compared to placentas from the CTMLW (GD45 and 60) and female fetuses (GD60), respectively. This study has highlighted that placentas supplying the lightest and male fetuses have impaired angiogenesis. Importantly, the relationship between fetal size, sex, and placental vascularity is dynamic and dependent upon the GD investigated.

Associations between fetal size, sex and both proliferation and apoptosis at the porcine feto-maternal interface.

INTRODUCTION: Inadequate fetal growth has severe consequences for both neonatal and adult development. It is hypothesised that the feto-maternal interface associated with the lightest and male fetuses will undergo more apoptosis and less proliferation than those supplying the closest to mean litter weight (CTMLW) and female fetuses respectively. METHODS: Placental and endometrial samples associated with the lightest and CTMLW (gestational day (GD) 18 and 30), male and female (GD45, 60 and 90) Large White X Landrace conceptuses or fetuses were obtained. The mRNA expression of candidate genes involved in apoptosis or proliferation (BAX, BCL2, P53 and KI67) was quantified by qPCR. TUNEL staining was performed on placental samples supplying the lightest and CTMLW fetuses (GD45 and 60), of both sex (GD60). RESULTS: Placentas associated with the lightest fetuses had decreased P53 and KI67 expression compared to the CTMLW fetuses at GD45. At GD60, P53 expression was increased in placentas supplying the lightest compared to CTMLW fetuses. P53 expression was increased in endometrial samples associated with the lightest compared to the CTMLW fetuses at GD45. At GD30 and GD60 respectively, BAX expression was increased and BCL2, P53 and KI67 expression were decreased in endometrial samples associated with females compared to their male littermates. TUNEL staining revealed no association between fetal size or sex, and apoptotic cell number. DISCUSSION: This study has highlighted dynamic associations between fetal size, sex, and apoptosis and proliferation at the porcine feto-maternal interface. Further studies should be performed to improve the understanding of the mechanisms behind these findings.

The role of dietary fibre in pig production, with a particular emphasis on reproduction.

Fibres from a variety of sources are a common constituent of pig feeds. They provide a means to utilise locally-produced plant materials which are often a by-product of the food or drink industry. The value of a high fibre diet in terms of producing satiety has long been recognised. However the addition of fibre can reduce feed intake, which is clearly detrimental during stages of the production cycle when nutrient needs are high, for example in growing piglets and during lactation. More recently, fibre has been found to promote novel benefits to pig production systems, particularly given the reduction in antimicrobial use world-wide, concern for the welfare of animals fed a restricted diet and the need to ensure that such systems are more environmentally friendly. For example, inclusion of dietary fibre can alter the gut microbiota in ways that could reduce the need for antibiotics, while controlled addition of certain fibre types may reduce nitrogen losses into the environment and so reduce the environmental cost of pig production. Of particular potential value is the opportunity to use crude fibre concentrates as 'functional' feed additives to improve young pig growth and welfare. Perhaps the greatest opportunity for the use of high fibre diets is to improve the reproductive efficiency of pigs. Increased dietary fibre before mating improves oocyte maturation, prenatal survival and litter size; providing a consumer-acceptable means of increasing the amount of saleable meat produced per sow. The mechanisms responsible for these beneficial effects remain to be elucidated. However, changes in plasma and follicular fluid concentrations of key hormones and metabolites, as well as effects of the hypothalamic satiety centre on gonadotrophin secretion and epigenetic effects are strong candidates.

Periparturient Behavior and Physiology: Further Insight Into the Farrowing Process for Primiparous and Multiparous Sows.

Giving birth is a critical time for many species and is often the most painful event ever experienced by females. In domestic species, like the pig, pain associated with parturition represents a potential welfare concern, and the consequences of pain can cause economic losses (e.g., by indirectly contributing to piglet mortality as pain could slow post-farrowing recovery, reduce food and water intake, reducing milk let-down). This study investigated pain assessment and its management in primiparous (gilts) and multiparous (sows) breeding pigs, including the provision of a non-steroidal anti-inflammatory drug (NSAID) post-parturition. Individuals were randomly allocated to receive the NSAID ketoprofen (3 mg/kg bodyweight) (n = 11 gilts, 16 sows) or the equivalent volume of saline (n = 13 gilts, 16 sows) by intramuscular injection 1.5 h after the birth of the last piglet. Data collected included putative behavioral indicators of pain (back leg forward, tremble, back arch), salivary cortisol concentrations pre-farrowing and up to 7 days post-injection. In addition, post-partum biomarkers of inflammation, including the acute phase protein C-reactive protein (CRP) and 3 porcine cytokines [interleukin-1 ß (IL1 ß), interleukin-6 (IL6), and tumor necrosis factor α (TNF α)] were measured in plasma collected 6 h following the injection. Behaviors were analyzed using generalized linear mixed models, and physiological variables with linear mixed models. No difference in putative pain behaviors, salivary cortisol, CRP, or cytokines were found between individuals treated with ketoprofen or those administered the saline control. However, there were some differences between gilts and sows, as sows exhibited more putative pain behavior than gilts, had higher salivary cortisol on the day of farrowing and had higher plasma TNF α. Conversely, gilts had higher salivary cortisol than sows on day 3 post-farrowing and had higher CRP. This indicates that, like human females, multiparous sows experience more pain from uterine activity following birth than primiparas. This study provides useful information for developing management practices relating to post-farrowing care for breeding pigs.

Doppler ultrasound can be used to monitor umbilical arterial blood flow in lightly sedated pigs at multiple gestational ages.

Doppler ultrasound was performed under moderate sedation (ketamine and azaperone) for 30min to monitor umbilical arterial (UA) blood flow in one uterine horn of Large White×Landrace gilts (n=23) at Gestational Days (GD) 30, 45, 60 and 90. Gilts were scanned before they were killed to examine relationships between litter size, sex ratio and five UA parameters (peak systolic velocity (PSV), end diastolic velocity (EDV), A/B (PSV to EDV) ratio, fetal heart rate (FHR) and resistance index (RI)). In gilts in which scans were obtained from all fetuses in the scanned horn, relationships between UA parameters, and fetal weight and sex were examined. A subset of gilts were sedated, scanned and recovered (SSR) earlier in gestation (GD30 or GD45) to assess the effects of sedation on later fetal development by comparison with control litters (no previous sedation). Temporal changes were observed in all UA parameters (P≤0.001). At GD60 and GD90, FHR decreased with increasing duration of sedation (P≤0.001). Sex ratio and fetal weight affected UA blood flow, whereas litter size and fetal sex did not. SSR at GD30 and GD45 was associated with decreased fetal weight at GD60 (P≤0.001) and GD90 (P=0.06) respectively, compared with controls. These results suggest maternal sedation during gestation affects fetal development, which should be investigated further. Measuring UA blood flow in growth-restricted porcine fetuses throughout gestation may be feasible.

Sex-specific prenatal stress effects on the rat reproductive axis and adrenal gland structure.

Social stress during pregnancy has profound effects on offspring physiology. This study examined whether an ethologically relevant social stress during late pregnancy in rats alters the reproductive axis and adrenal gland structure in post-pubertal male and female offspring. Prenatally stressed (PNS) pregnant rats (n=9) were exposed to an unfamiliar lactating rat for 10 min/day from day 16 to 20 of pregnancy inclusive, whereas control pregnant rats (n=9) remained in their home cages. Gonads, adrenal glands and blood samples were obtained from one female and one male from each litter at 11 to 12-weeks of age. Anogenital distance was measured. There was no treatment effect on body, adrenal or gonad weight at 11-12 weeks. PNS did not affect the number of primordial, secondary or tertiary ovarian follicles, numbers of corpora lutea or ovarian FSH receptor expression. There was an indication that PNS females had more primary follicles and greater ovarian aromatase expression compared with control females (both P=0.09). PNS males had longer anogenital distances (0.01±0.0 cm/g vs 0.008±0.00 cm/g; P=0.007) and higher plasma FSH concentrations (0.05 ng/mL vs 0.006 ng/mL; s.e.d.=0.023; P=0.043) compared with control males. There were no treatment effects on the number of Sertoli cells or seminiferous tubules, seminiferous tubule area, plasma testosterone concentration or testis expression of aromatase, FSH receptor or androgen receptor. PNS did not affect adrenal size. These data suggest that the developing male reproductive axis is more sensitive to maternal stress and that PNS may enhance aspects of male reproductive development.

Heat shock induces interferon-TAU gene expression by in vitro-produced bovine blastocysts.

PROBLEM: The type I interferon (IFN), IFN-tau (τ), is the primary embryonic signal for pregnancy maintenance in ruminants. This study determined the effects of heat shock upon IFN-τ (IFNT) gene expression by bovine blastocysts in vitro. METHOD OF STUDY: In vitro-produced blastocyst-stage embryos were exposed to 42°C for 4 hr, and mRNA for heat-shock protein 70 (HSP70) and IFNT quantified. RESULTS: Heat shock increased both HSP70 and IFNT expression. There was a significant correlation between HSP70 and IFNT transcript levels irrespective of whether a blastocyst had been exposed to heat shock or not. CONCLUSION: The increase in IFNT as a result of heat shock suggests that a proportion of the variation in IFNT expression observed in blastocyst-stage embryos is a response to stress.

Secreted phosphoprotein 1 expression in endometrium and placental tissues of hyperprolific large white and meishan gilts.

Increased litter size and within-litter uniformity in birth weight would improve pig reproductive efficiency. This study compared the location and gene and protein expression of secreted phosphoprotein 1 in placental and uterine tissues supplying a normally sized and the smallest fetus carried by hyperprolific Large White and Meishan gilts on Days 41-42 of pregnancy. Immunohistochemistry and in situ hybridization showed that the protein and gene encoding secreted phosphoprotein 1 were located in the glandular and luminal epithelium of the endometrium and in the placenta. Secreted phosphoprotein 1 protein levels were higher in glandular epithelium, luminal epithelium, and placenta from Meishan gilts compared to corresponding tissues from hyperprolific Large White gilts. Reverse transcription quantitative PCR demonstrated secreted phosphoprotein 1 mRNA levels were higher in endometrium, but not placenta, from Meishan compared to hyperprolific Large White gilts. In hyperprolific Large White gilts, secreted phosphoprotein 1 protein levels were higher in glandular epithelium and placenta surrounding small fetuses than corresponding tissues supplying normal-sized fetuses. Similarly, in Meishan gilts, secreted phosphoprotein 1 protein levels were higher in luminal epithelium surrounding small compared to normal-sized fetuses. Within hyperprolific Large White, but not Meishan, gilts secreted phosphoprotein 1 mRNA was higher in endometrium surrounding the normal-sized fetus than the control fetus. The contradictory relationship between fetal size and secreted phosphoprotein 1 protein and mRNA in the hyperprolific Large White is intriguing and may reflect breed differences in posttranslational modification. The striking breed differences in secreted phospoprotein 1 expression suggest that SPP1 may be associated with placental efficiency.

Genotype and fetal size affect maternal-fetal amino acid status and fetal endocrinology in Large White × Landrace and Meishan pigs.

This study compared maternal plasma amino acid concentrations, placental protein secretion in vitro and fetal body composition and plasma amino acid and hormone concentrations in feto-placental units from the smallest and a normally-sized fetus carried by Large White × Landrace or Meishan gilts on Day 100 of pregnancy. Compared with Large White × Landrace, Meishan placental tissue secreted more protein and Meishan fetuses contained relatively more fat and protein, but less moisture. Fetal plasma concentrations of insulin, triiodothryonine, thyroxine and insulin-like growth factor (IGF)-II were higher in Meishan than Large White × Landrace fetuses. In both breeds, fetal cortisol concentrations were inversely related to fetal size, whereas concentrations of IGF-I were higher in average-sized fetuses. Concentrations of 10 amino acids were higher in Large White × Landrace than Meishan gilts, while glutamine concentrations were higher in Meishan gilts. Concentrations of alanine, aspartic acid, glutamic acid and threonine were higher in Meishan than Large White × Landrace fetuses. Average-sized fetuses had higher concentrations of asparagine, leucine, lysine, phenylalanine, threonine, tyrosine and valine than the smallest fetus. This study revealed novel genotype and fetal size differences in porcine maternal-fetal amino acid status and fetal hormone and metabolite concentrations.