Gestational heat stress alters skeletal muscle gene expression profiles and vascularity in fetal pigs in a sexually dimorphic manner.

BACKGROUND: There is evidence that sow heat stress (HS) during gestation affects fetal development with implications for impaired muscle growth. We have previously demonstrated that maternal HS during early to mid-gestation compromised muscle fibre hyperplasia in developing fetal pigs. Thus, we hypothesised these phenotypic changes are associated with a change in expression of genes regulating fetal skeletal muscle development and metabolism. To test this, at d 60 of gestation, RNA sequencing and immunohistochemistry were performed on fetal longissimus dorsi (LD) muscle biopsies collected from pregnant gilts that had experienced either thermoneutral control (CON, 20 °C, n = 7 gilts, 18 LD samples) or controlled HS (cyclic 28 to 33 °C, n = 8 gilts, 23 LD samples) conditions for 3 weeks. RESULTS: A total of 282 genes were differentially expressed between the HS and CON groups in female LD muscles (false discovery rate (FDR) ≤ 0.05), whereas no differentially expressed genes were detected in male LD muscles between the two groups (FDR > 0.05). Gestational HS increased the expression of genes associated with transcription corepressor activity, adipogenesis cascades, negative regulation of angiogenesis and pro-inflammatory signalling in female LD muscles. Immunohistochemical analyses revealed a decreased muscle vascularity density in fetuses from HS group for both sexes compared to those from the CON group (P = 0.004). CONCLUSIONS: These results reveal gilt HS during early to mid-gestation altered gene expression profiles in fetal LD muscles in a sexually dimorphic manner. The molecular responses, including transcription and angiogenesis repressions and enhanced adipogenesis cascades, were exclusively observed in females. However, the associated reductions in muscle vascularity were observed independently of sexes. Collectively this may indicate female fetal pigs are more adaptive to gestational HS in terms of gene expression changes, and/or there may be sexually dimorphic differences with respect to the timing of muscle molecular responses to gestational HS.

Compensatory feeding during early gestation for sows with a high weight loss after a summer lactation increased piglet birth weight but reduced litter size.

Sows mated in summer produce a greater proportion of born-light piglets (<1.1 kg) which contributes to increased carcass fatness in the progeny population. The reasons for the low birth weight of these piglets remain unclear, and there have been few successful mitigation strategies identified. We hypothesized that: 1) the low birth weight of progeny born to sows mated in summer may be associated with weight loss during the previous summer lactation; and 2) increasing early gestation feed allowance for the sows with high lactational weight loss in summer can help weight recovery and improve progeny birth weight. Sows were classified as having either low (av. 1%) or high (av. 7%) lactational weight loss in their summer lactation. All the sows with low lactational weight loss (LLStd) and half of the sows with high lactational weight loss received a standard gestation feeding regime (HLStd) (2.6 kg/d; day 0-30 gestation), whereas the rest of the sows with high lactational weight loss received a compensatory feed allowance (HLComp) (3.5 kg/d; day 0-30 gestation). A comparison of LLStd (n = 75) versus HLStd sows (n = 78) showed that this magnitude of weight loss over summer lactation did not affect the average piglet or litter birth weight, but such results may be influenced by the higher litter size (P = 0.030) observed in LLStd sows. A comparison of HLStd versus HLComp (n = 81) sows showed that the compensatory feeding increased (P = 0.021) weight gain of gestating sows by 6 kg, increased (P = 0.009) average piglet birth weight by 0.12 kg, tended to reduce (P = 0.054) the percentage of born-light piglets from 23.5% to 17.1% but reduced the litter size by 1.4 (P = 0.014). A subgroup of progeny stratified as born-light (0.8-1.1 kg) or -normal (1.3-1.7 kg) from each sow treatment were monitored for growth performance from weaning until 100 kg weight. The growth performance and carcass backfat of progeny were not affected by sow treatments. Born-light progeny had lower feed intake, lower growth rate, higher G:F, and higher carcass backfat than born-normal progeny (all P < 0.05). In summary, compensatory feeding from day 0 to 30 gestation in the sows with high weight loss during summer lactation reduced the percentage of born-light progeny at the cost of a lower litter size, which should improve growth rate and carcass leanness in the progeny population born to sows with high lactational weight loss.

Maternal Heat Stress Alters Expression of Genes Associated with Nutrient Transport Activity and Metabolism in Female Placentae from Mid-Gestating Pigs.

Placental insufficiency is a known consequence of maternal heat stress during gestation in farm animals. The molecular regulation of placentae during the stress response is little known in pigs. This study aims to identify differential gene expression in pig placentae caused by maternal heat exposure during early to mid-gestation. RNA sequencing (RNA-seq) was performed on female placental samples from pregnant pigs exposed to thermoneutral control (CON; constant 20 °C; n = 5) or cyclic heat stress (HS; cyclic 28 to 33 °C; n = 5) conditions between d40 and d60 of gestation. On d60 of gestation, placental efficiency (fetal/placental weight) was decreased (p = 0.023) by maternal HS. A total of 169 genes were differentially expressed (FDR ≤ 0.1) between CON and HS placentae of female fetuses, of which 35 genes were upregulated and 134 genes were downregulated by maternal HS. The current data revealed transport activity (FDR = 0.027), glycoprotein biosynthetic process (FDR = 0.044), and carbohydrate metabolic process (FDR = 0.049) among the terms enriched by the downregulated genes (HS vs. CON). In addition, solute carrier (SLC)-mediated transmembrane transport (FDR = 0.008) and glycosaminoglycan biosynthesis (FDR = 0.027), which modulates placental stroma synthesis, were identified among the pathways enriched by the downregulated genes. These findings provide evidence that heat-stress induced placental inefficiency may be underpinned by altered expression of genes associated with placental nutrient transport capacity and metabolism. A further understanding of the molecular mechanism contributes to the identification of placental gene signatures of summer infertility in pigs.

The Greater Proportion of Born-Light Progeny from Sows Mated in Summer Contributes to Increased Carcass Fatness Observed in Spring.

The backfat of pig carcasses is greater in spring than summer in Australia. The unexplained seasonal variation in carcass backfat creates complications for pig producers in supplying consistent lean carcasses. As a novel explanation, we hypothesised that the increased carcass fatness in spring was due to a greater percentage of born-light progeny from sows that were mated in summer and experienced hot conditions during early gestation. The first part of our experiment compared the birth weight of piglets born to the sows mated in summer (February, the Southern Hemisphere) with those born to sows mated in autumn (May; the Southern Hemisphere), and the second part of the experiment compared the growth performance and carcass fatness of the progeny that were stratified as born-light (0.7-1.1 kg) and born-normal (1.3-1.7 kg) from the sows mated in these two seasons. The results showed that the sows mated in summer experienced hotter conditions during early gestation as evidenced by an increased respiration rate and rectal temperature, compared with those mated in autumn. The sows mated in summer had a greater proportion of piglets that were born ≤1.1 kg (24.2% vs. 15.8%, p < 0.001), lower average piglet birth weight (1.39 kg vs. 1.52 kg, p < 0.001), lower total litter weights (18.9 kg vs. 19.5 kg, p = 0.044) and lower average placental weight (0.26 vs. 0.31 kg, p = 0.011) than those mated in autumn, although litter sizes were similar. Feed intake and growth rate of progeny from 14 weeks of age to slaughter (101 kg live weight) were greater for the born-normal than born-light pigs within the progeny from sows mated in autumn, but there was no difference between the born-light and normal progeny from sows mated in summer, as evidenced by the interaction between piglet birth weight and sow mating season (Both p < 0.05). Only the born-light piglets from the sows mated in summer had a greater backfat thickness and loin fat% than the progeny from the sows mated in autumn, as evidenced by a trend of interaction between piglet birth weight and sow mating season (Both p < 0.10). In conclusion, the increased proportion of born-light piglets (0.7-1.1 kg range) from the sows mated in summer contributed to the increased carcass fatness observed in spring.

Controlled elevated temperatures during early-mid gestation cause placental insufficiency and implications for fetal growth in pregnant pigs.

It is known that pig offspring born from pregnant pigs exposed to elevated ambient temperatures during gestation have altered phenotypes, possibly due to placental insufficiency and impaired fetal growth. Therefore, the objective of this study was to quantify the effect of maternal heat exposure during early-mid gestation, when pig placentae grow heavily, on placental and fetal development. Fifteen pregnant pigs were allocated to thermoneutral (TN; 20 °C; n = 7) or cyclic elevated temperature conditions (ET; 28 to 33 °C; n = 8) from d40 to d60 of gestation. Following euthanasia of the pigs on d60, placental and fetal morphometry and biochemistry were measured. Compared to TN fetuses, ET fetuses had increased (P = 0.041) placental weights and a lower (P = 0.013) placental efficiency (fetal/placental weight), although fetal weights were not significantly different. Fetuses from ET pigs had reduced (P = 0.032) M. longissimus fibre number density and a thicker (P = 0.017) placental epithelial layer compared to their TN counterparts. Elevated temperatures decreased (P = 0.026) placental mRNA expression of a glucose transporter (GLUT-3) and increased (P = 0.037) placental IGF-2 mRNA expression. In conclusion, controlled elevated temperatures between d40 to d60 of gestation reduced pig placental efficiency, resulting in compensatory growth of the placentae to maintain fetal development. Placental insufficiency during early-mid gestation may have implications for fetal development, possibly causing a long-term phenotypic change of the progeny.

Developmental Programming and Growth of Livestock Tissues for Meat Production.

Maternal regulation of fetal development has consequences for growth and development of carcass tissues. Severely restricted fetal growth can reduce postnatal growth capacity, resulting in smaller-for-age animals that take longer to reach market weights but has little effect on feedlot efficiency or carcass and meat quality. Specific nutritional supplementation, particularly during later pregnancy, may limit fetal growth retardation and enhance postnatal growth capacity and carcass characteristics, and may improve development of intramuscular fat. Continued improvements in understanding developmental processes and their regulation will increase future capacity to improve growth, efficiency, carcasses, and meat quality through developmental programming.

Plasma leptin is regulated predominantly by nutrition in preruminant lambs.

In juvenile and mature animals, the plasma concentration of leptin is regulated by adiposity and nutrition. However, the timing of these influences on plasma leptin, and their relative importance in early postnatal life, are unknown. We investigated these plasma leptin influences in sheep, a species characterized during fetal life by leanness and insensitivity of leptin to variation in maternal nutrition. Small and large neonatal lambs were randomly assigned to either a diet sustaining an average daily weight gain (ADG) of 148 g/d (Low plane) or ate ad libitum a diet sustaining an ADG of 337 g/d (High plane). A subset of animals were slaughtered at 7.5, 10, 15 and 20 kg of body weight. Birth size had no effect on plasma leptin concentrations and adiposity at birth or at later times. Plasma leptin concentrations increased within 6 d of birth in the High plane lambs (P < 0.01) and continued to rise over time. In contrast, plasma leptin concentrations never changed in the Low plane lambs despite increasing adiposity. The positive association between plasma leptin concentration and adiposity was greater in the High plane than in the Low plane lambs, suggesting an independent effect of nutrition. Consistent with this finding, lipid accretion rates, a variable that is mostly independent of adiposity, was a strong predictor of plasma leptin concentrations only in the High plane lambs (R(2) = 0.77, P < 0.01). A positive association between plasma insulin and leptin developed over time in the High plane lambs (R(2) = 0.75, P < 0.01 on d 40), but was not seen in the Low plane lambs. These data indicate that both nutrition and adiposity regulate plasma leptin synthesis in early postnatal life, but in contrast to adulthood, the effects of nutrition appear to be predominant.

Effect of insulin and growth hormone on plasma leptin in periparturient dairy cows.

After parturition, dairy cows suffer from an intense energy deficit caused by the onset of copious milk secretion and an inadequate increase in voluntary food intake. We previously showed that this energy deficit contributes to a decline in plasma leptin. This decline mirrors that of plasma insulin but is reciprocal to the profile of plasma growth hormone (GH), suggesting that both hormones may regulate plasma leptin in periparturient dairy cows. To study the role of insulin, hyperinsulinemic-euglycemic clamps were performed on six dairy cows in late pregnancy (LP, 31 days prepartum) and early lactation (EL, 7 days postpartum). Infusion of insulin (1 microg.kg body wt-1.h-1) caused a progressive rise in the plasma concentration of leptin that reached maximum levels at 24 h during both physiological states. At steady states, the absolute increase in plasma leptin was greater in LP than in EL cows (2.4 vs. 0.4 ng/ml). Insulin infusion increased leptin mRNA in adipose tissue during LP but not during EL. During lactation, mammary epithelial cells expressed leptin mRNA but insulin did not increase milk leptin output. In contrast, a 3-day period of GH administration had no effect on plasma leptin during LP or EL. Therefore, insulin increases plasma leptin in LP by stimulating adipose tissue synthesis but has only marginal effects in EL, when cows are in negative energy balance. Other factors, such as increased response of adipose tissue to beta-adrenergic signals, probably contribute to the reduction of plasma leptin in early lactating dairy cows.

Prediction of stage of pregnancy in prolific sheep using ultrasound measurement of fetal bones.

The use of ultrasound to estimate stage of pregnancy was assessed in 32 ewes of a prolific genotype carrying 7 singleton fetuses and 9 twin, 10 triplet and 6 quadruplet litters that were scanned on six occasions from 60 to 120 days of gestation. At least one ultrasound measurement per ewe of fetal metacarpal bone length (MCL), biparietal diameter (BPD), or of both bones was made on over 90% of attempts (n = 152). Measurement of MCL was made on 78% of attempts (n = 371), of BPD on 73% of attempts, and of both bones on 62% of attempts. The equation developed from BPD (mean absolute error (MAE) = 3.2 days) was similar to that developed from measurement of MCL (MAE = 3.3 days) in its capacity to predict stage of pregnancy. Accuracy of prediction was improved using equations that included mean values within litters for BPD (MAE = 2.5 days) and MCL (MAE = 2.6 days). Further improvement in predictive capacity was achieved using multiple regression equations developed from measurement of both bones (individual fetuses: MAE = 2.6 days; equations including mean values within litters: MAE = 2.2 days). The results demonstrate that ultrasound can be used to estimate stage of pregnancy in prolific ewes, and that the use of mean values for bone measurements from different fetuses within litters and/or measurement of bones with different growth allometry can increase the reliability of estimates. The utility of the procedure depends on the number of fetuses measured per ewe, the number of bones measured per fetus and, hence, the time required to measure bones and the degree of accuracy required.

Spatial and developmental regulation of leptin in fetal sheep.

To better understand the biology of leptin during prenatal life, the developmental and spatial regulation of leptin was studied in ovine fetuses. Fetal plasma leptin increased steadily between days 40 and 143 postcoitus (PC), but it was unrelated to fetal weight or placental weight at day 135 PC. Leptin gene expression was detected in fetal brain and liver during most of gestation and in fetal adipose tissue after day 100 PC. At day 130 PC, expression in fetal perirenal adipose tissue was approximately 10% of maternal expression. In contrast, leptin gene expression was never detected in the placenta and other uteroplacental tissues. When ewes were fed 55% of requirements between days 122 and 135 PC, fetal plasma leptin remained constant despite acute reduction in maternal concentration. We conclude that fetal plasma leptin originates mostly from nonadipose tissue in early pregnancy and, in addition, from fetal adipose tissue near term. The role of fetal plasma leptin remains uncertain given the lack of nutritional regulation and association with fetal growth.

Regulation of placental nutrient transport and implications for fetal growth.

Fetal macronutrient requirements for oxidative metabolism and growth are met by placental transport of glucose, amino acids, and, to a lesser extent that varies with species, fatty acids. It is becoming possible to relate the maternal-fetal transport kinetics of these molecules in vivo to the expression and distribution of specific transporters among placental cell types and subcellular membrane fractions. This is most true for glucose transport, although apparent inconsistencies among data on the roles and relative importance of the predominant placenta glucose transporters, GLUT-1 and GLUT-3, remain to be resolved. The quantity of macronutrients transferred to the fetus from the maternal bloodstream is greatly influenced by placental metabolism, which results in net consumption of large amounts of glucose and, to a lesser extent, amino acids. The pattern of fetal nutrient supply is also altered considerably by placental conversion of glucose to lactate and, in some species, fructose, and extensive transamination of amino acids. Placental capacity for transport of glucose and amino acids increases with fetal demand as gestation advances through expansion of the exchange surface area and increased expression of specific transport molecules. In late pregnancy, transport capacity is closely related to placental size and can be modified by maternal nutrition. Preliminary evidence suggests that placental expression and function of specific transport proteins are influenced by extracellular concentrations of nutrients and endocrine factors, but, in general, the humoral regulation of placental capacity for nutrient transport is poorly understood. Consequences of normal and abnormal development of placental transport functions for fetal growth, especially during late gestation, and, possibly, for fetal programming of postnatal disorders, are discussed.