National Research Council Requirements Research Articles

PSIII-1 The effects of maternal diet on ram lamb reproductive characteristics

Abstract Maternal nutrition during gestation and resulting impacts on ram reproduction and epigenetics are poorly understood. We previously reported altered mRNA and epigenetic markers in F1 ram sperm due to poor maternal diet (restricted- and over-feeding) during gestation, yet the multigenerational effects on ram reproduction are unexplored. We hypothesized that F2 offspring born from restricted- and over-fed granddams would exhibit altered reproductive characteristics, and F1 and F2 offspring would display altered circulating concentrations of reproductive hormones. The objectives of this study were to determine the effects of poor maternal diet on 1) the reproductive function of F2 ram offspring, and 2) key reproductive hormones in F1 and F2 offspring. To evaluate these objectives, multiparous Dorset ewes (F0, n = 46) were fed 100% (CON), 60% (RES), or 140% (OVER) of National Research Council requirements from d 30 of gestation through parturition and a control diet during lactation. Offspring are identified as CON-F1 (n = 10 ewes; 12 rams), RES-F1 (n = 13 ewes; 21 rams), and OVER-F1 (n = 16 ewes; 15 rams). The F1 ewes (n = 37) were maintained on a control diet throughout and were bred between 16 and 19 mo of age to establish the F2 generation. Resulting male offspring are referred to as CON-F2 (n = 6 rams), RES-F2 (n = 13 rams), and OVER-F2 (n = 9 rams) in correspondence to the diets of the granddam. Semen samples of F2 rams were collected via electroejaculation at d 248.4 ± 0.36 (collection 1) and d 255.4 ± 0.36 (collection 2). Semen quality (volume, pH, sperm motility, and sperm concentration) was evaluated at both collections using mCASA; iSperm, Aidmics Biotechnology Co., LTD. Scrotal circumference was measured at collection 2. Sperm morphology was determined by contrast microscopy. Testosterone (T) and follicle stimulating hormone (FSH) were measured in F1 and F2 serum samples collected at d 256 ± 0.13 using commercially available kits. Data were analyzed using R Studio with P ≤ 0.05 considered significant and 0.05 < P ≤ 0.10 considered a tendency. An effect of granddam diet was not observed for F2 ram semen volume, pH, sperm motility, sperm concentration, scrotal circumference, nor histological sperm morphology (P ≥ 0.25). An effect of F0 diet was not observed for F1 or F2 offspring serum concentrations of T and FSH (P ≥ 0.28). In our model, F0 diet during gestation did not affect F2 male offspring sperm quality or circulating T and FSH in F1 and F2 generations. However, based on our previous reports that F0 diet altered F1 sperm epigenome, there may be an impact of maternal diet on epigenetic inheritance which warrants further exploration.

Dietary Supplementation with Rumen-Protected Arginine or N-Carbamylglutamate Enhances Fetal Liver Development in Nutrient-Restricted Pregnant Hu Ewes.

This study was conducted in nutrient-restricted pregnant Hu ewes to determine whether rumen-protected arginine (RP-Arg) or N-carbamylglutamate (NCG) supplementation affects fetal liver growth and development. From 35 d to 110 d of gestation, 32 Hu ewes were randomly divided into four groups: a control group (100% of the National Research Council (NRC) requirements), a nutrient-restricted group (50% of the NRC requirements), and two treatment groups (ARG and NCG, 50% of the NRC requirements, supplemented with 20 g/day RP-Arg or 5 g/day NCG, respectively). Fetal body weights, fetal liver growth performance, the capability of antioxidation, and the expression of the mRNA and proteins of apoptosis-related genes in the fetal liver were determined and analyzed at 110 d of gestation. The dry matter, water, fat, protein, and ash components of the fetal livers in the RG group were found to be lower than in the CG group, and these components were significantly higher in the NCG group than in the RG group (p < 0.05). A decrease in DNA, RNA, and protein concentrations and contents, as well as in protein/DNA ratios, was observed in the RG group in comparison to the CG group (p < 0.05). Compared with the RG group, the NCG group had higher concentrations of DNA, RNA, and protein, as well as higher protein/DNA ratios (p < 0.05). The RG group had lower concentrations of cholinesterase, nitric oxide, nitric oxide synthase, superoxide dismutase, alanine aminotransferase, and total protein than the CG group (p < 0.05). The RG group had higher levels of glutathione peroxidase, maleic dialdehyde, and aspartate aminotransferase than the CG group (p < 0.05). In the RG group, the mRNA and protein expression of p53 and Bax was significantly increased (p < 0.05) compared with the CG group, and the gene expression of FasL and Bcl-2, the ratio of Bcl-2 to Bax, and the protein expression of Bcl-2 in the RG group were lower (p < 0.05) than in the CG group. It appears that RP-Arg and NCG supplementation during pregnancy could influence fetal liver growth and development. A nutrition-based therapeutic intervention to alleviate reduced fetal growth can be developed based on this study, which has demonstrated that maternal undernutrition during pregnancy induces the maldevelopment of the fetal liver.

Effects of poor maternal diet during gestation are detected in F2 offspring.

Poor maternal nutrition of F0 ewes impairs F1 offspring growth, with minimal differences in glucose tolerance or select metabolic circulating factors, and independent of differences in residual feed intake (RFI). To determine if poor maternal nutrition in F0 ewes alters F2 offspring growth, circulating leptin, feed efficiency, or glucose tolerance, F0 ewes (n = 46) pregnant with twins were fed 100% (control), 60% (restricted), or 140% (over) of National Research Council requirements from days 30 ± 0.02 of gestation until parturition. At 16 to 19 mo of age, female F1 (n = 36) offspring were bred to generate F2 offspring [CON-F2 (n = 12 ewes; 6 rams), RES-F2 (n = 7 ewes; 13 rams), or OVER-F2 (n = 13 ewes; 9 rams) corresponding to diets of the granddam (F0)]. Lamb body weights (BW) and blood samples were collected weekly from days 0 to 28 and every 14 d until day 252 of age. Circulating leptin was measured in serum at days 0, 7, 14, 56, 210, and 252. An intravenous glucose tolerance test was performed at days 133 ± 0.28. At days 167 ± 0.33, individual daily intake was recorded over a 77-d feeding period to determine RFI. Rams were euthanized at days 285 ± 0.93, and body morphometrics, loin eye area (LEA), back fat thickness, and organ weights were collected and bone mineral density (BMD) and length were determined in the right hind leg. During gestation, OVER-F1 ewes tended to be 8.6% smaller than CON-F1 ewes (P ≤ 0.06). F2 offspring were of similar BW from birth to day 70 (P ≥ 0.20). However, from days 84 to 252, RES-F2 offspring tended to be 7.3% smaller than CON-F2 (P ≤ 0.10). Granddam diet did not influence F2 ram body morphometrics, organ or muscle weights, LEA, adipose deposition, or leg BMD (P ≥ 0.84). RES-F2 (-0.20) and CON-F2 (-0.45) rams tended to be more feed efficient than CON-F2 ewes (0.31; P ≤ 0.08). No effects of granddam diet were observed on glucose or insulin average or baseline concentrations, area under the curve, first-phase response, or ratio (P ≥ 0.52). However, CON-F2 rams (297mg/dL ± 16.5) had a greater glucose peak compared with RES-F2 rams (239mg/dL ± 11.2; P = 0.05). Peak insulin concentrations were not influenced by granddam diet (P = 0.75). At d 56, RES-F2 and OVER-F2 offspring had 53.5% and 61.8% less leptin compared with CON-F2 offspring, respectively (P ≤ 0.02). These data indicate that poor maternal nutrition impacts offspring growth into the second generation with minimal impacts on offspring RFI, glucose tolerance, and circulating leptin.

PSIV-1 Effects of Poor Maternal Nutrition During Gestation on F0 and F1 Ewe Colostrum and Milk Composition and Colostrum Igg

Abstract High-quality colostrum and milk are critical for proper neonatal growth and immune system development. Immunoglobulin G (IgG) is the predominant antibody class in colostrum and is vital for passive immune transfer in sheep. There is limited understanding of how poor maternal nutrition during gestation (restricted- and over-feeding) might influence colostrum and milk composition and colostrum IgG concentrations and how F1 offspring colostrum and milk are impacted as a result of F0 maternal diet. The objective of this experiment was to determine if poor maternal nutrition during gestation impacts F0 and F1 colostrum and milk composition, including IgG concentrations. We hypothesized that maternal restricted-feeding would decrease, and maternal over-feeding would increase total solids, fat, and protein during early and transitional lactation and that restricted-feeding would increase IgG concentrations and over-feeding would decrease IgG concentrations in F0 and F1 ewe colostrum. Multiparous Dorset ewes pregnant (n = 46) with twins were fed 100% (control; n = 13), 60% (restricted; n = 17) or 140% (over; n = 16) of National Research Council (NRC) requirements. After birth and during lactation, dams were fed 100% NRC for lactating ewes, so the nutritional insult only occurred during gestation. Resulting ewe offspring (n = 32) were raised on control diets and were bred between 16 and 19 mo of age. Colostrum samples were collected within 24 h of parturition and milk samples were collected at d 3 and d 21 postpartum. Colostrum and milk composition were evaluated using Brix Refractometry (total solids, g/dL) and spectrophotometry (crude fat, %; crude protein, %). Additionally, a radial immunodiffusion assay was used to determine IgG concentrations (mg/dL) in colostrum. A treatment by time interaction was observed for F0 colostrum crude fat where restricted-fed ewes had less fat than control-fed ewes (P ≤ 0.024). No interaction was detected for all other F0 and F1 composition variables (P ≥ 0.16). We did not detect a main effect of diet on F0 or F1 total solids, crude fat, and crude protein of colostrum nor milk (P ≥ 0.24). A main effect of time was observed for all variables where colostrum had greater total solids, crude fat, and crude protein compared with milk at d 3 and d 21 (P ≤ 0.0001). No effect of maternal diet on IgG concentrations were detected in F0 or F1 ewe colostrum (P ≥ 0.46). These data indicate that poor maternal diet has minimal effects on F0 and F1 ewe colostrum composition and IgG concentrations, and d 3 and d 21 milk composition. In addition, the impact of F0 diet on colostrum fat composition did not persist into the F1 generation.

446 The Effects of Maternal Diet During Gestation on Oxidative Status of Offspring

Abstract Poor maternal nutrition during gestation is common in livestock production and includes both nutrient restriction and over-nutrition. Poor maternal nutrition affects offspring pre- and post-natal growth including decreases in muscularity and increases in adiposity which results in poor quantity and quality of animal products. Oxidative stress, when free radicals exceed the capacity of antioxidants, damages lipid membranes, proteins, and DNA. Oxidative stress may contribute to the poor growth outcomes observed in offspring from poorly nourished dams. Therefore, the objective of this research was to determine the effects of poor maternal nutrition during gestation on offspring circulating oxidative status in sheep. We hypothesized that offspring of restricted- and over-fed ewes would have an altered oxidative status in circulation. To test this hypothesis, multiparous Dorset ewes (n = 45) pregnant with twins were fed 100% (CON), 60% (RES), or 140% (OVER) of National Research Council requirements from d 30 of gestation until parturition. At parturition, ewes were fed a control diet such that the nutritional insult only occurred during gestation. Blood samples were collected from offspring by jugular venipuncture at d 112 and d 224 of age to evaluate oxidative status in circulation. Data were analyzed in SAS 9.4 using PROC MIXED with fixed effects of offspring sex and maternal diet with sampling timepoint as repeated measure. Data were considered significant at P &amp;lt; 0.05. In offspring plasma, superoxide dismutase, an endogenous antioxidant, activity was 17% greater in CON offspring compared with RES and OVER (P ≤ 0.04). Malondialdehyde (MDA), a marker of lipid peroxidation, concentration was not different at d 112 but RES animals had 10% lesser concentration of MDA than CON at d 224 (P ≤ 0.02). Protein carbonyl (PC), a marker of protein oxidation, concentration was 9% greater in CON animals than RES and OVER (P = 0.01). Additionally, PC concentrations were 19% greater at d 224 compared with d 112 (P ≤ 0.001). These results indicate that at the chosen timepoints, RES and OVER offspring had less antioxidant activity but also less indication of oxidative damage. Overall, the alterations in antioxidant activity and oxidative damage suggest dysregulation in circulating oxidative status that may contribute to the negative consequences of poor maternal nutrition during gestation. Future work should focus on the impacts of poor maternal nutrition on oxidative stress in muscle.

PSVI-10 Effects of Poor Maternal Nutrition During Gestation on Offspring Basal Immune Status

Abstract Poor maternal nutrition during gestation affects offspring pre- and post-natal growth, decreasing muscularity and increasing adiposity. Chronic inflammation may be a potential mechanism for the alterations reported. The objective of this research was to determine the effects of poor maternal nutrition during gestation on offspring inflammatory status. We hypothesized that offspring of restricted- and over-fed ewes would have an altered inflammatory status which would be dependent on offspring sex. To test this hypothesis, multiparous Dorset ewes (n = 45) pregnant with twins were fed 100% (CON), 60% (RES), or 140% (OVER) of National Research Council requirements from d 30 of gestation until parturition. At parturition, all ewes were fed a control diet, such that the nutritional insult only occurred during gestation. At d 120, whole blood was collected from offspring and RNA was isolated to evaluate 86 genes related to the innate and adaptive immune responses. Data were analyzed in SAS 9.4 using PROC MIXED with fixed effects of offspring sex, maternal diet, and the interaction. Maternal diet affected 7 genes. Bovine major histocompatibility complex, a gene in the innate immune response, was downregulated by 27% and 37% in OVER compared with CON and RES, respectively (P = 0.001). Cluster of differentiation 86, a marker of T cell activation, was upregulated by 17% in OVER compared with CON (P = 0.04). Lymphocyte antigen 96, critical in the response to pathogens, expression was 48% greater in RES compared with CON (P = 0.04). A transcription factor in cytokine signaling, Signal transducer and activator of transcription 3 was upregulated in OVER and RES offspring by 19 and 21%, respectively, compared with CON (P = 0.02). Pattern recognition receptors Toll-like receptor-1 (TLR1), TLR4, and TLR8 were upregulated in RES and OVER compared with CON (P ≤ 0.04). The interaction of offspring sex and maternal diet significantly altered gene expression in 2 genes. A regulator of T cell differentiation, GATA Binding Protein 3 was downregulated in OVER females by 21 to 43% compared with all other offspring and was upregulated in OVER males by 26 to 76% compared with all female offspring (P = 0.04). A gene related to cytokine signaling, Janus Kinase 2 was upregulated in RES and OVER males by 29 to 64% compared with all other offspring (P = 0.05). Sex altered gene expression of 54 genes (P ≤ 0.05). Overall, maternal diet altered gene expression of the innate and adaptive immune responses in a sex-specific manner. Alterations in inflammatory status may contribute to altered body composition and may predispose offspring to impaired response to immune challenges. Future work should focus on the impacts of poor maternal nutrition during gestation on the response to an immune challenge.

Maternal restricted- and over- feeding during gestation perturb offspring sperm epigenome in sheep.

Inadequate maternal nutrition during gestation can have immediate and lifelong effects on offspring. This study shows that maternal restricted - and over- nutrition during gestation do not affect semen characteristics in F1 male offspring but alters offspring sperm sncRNA profiles and DNA methylome in sheep. There is a growing body of evidence that inadequate maternal nutrition during gestation can have immediate and lifelong effects on offspring. However, little is known about the effects of maternal nutrition during gestation on male offspring reproduction. Here, using a sheep model of maternal restricted - and over - nutrition (60 or 140% of the National Research Council requirements) during gestation, we found that maternal restricted - and over - nutrition do not affect semen characteristics (i.e. volume, sperm concentration, pH, sperm motility, sperm morphology) or scrotal circumference in male F1 offspring. However, using small RNA sequencing analysis, we demonstrated that both restricted - and over - nutrition during gestation induced marked changes in composition and expression of sperm small noncoding RNAs (sncRNAs) subpopulations including in male F1 offspring. Whole-genome bisulfite sequencing analysis further identified specific genomic loci where poor maternal nutrition resulted in alterations in DNA methylation. These findings indicate that maternal restricted - and over - nutrition during gestation induce epigenetic modifications in sperm of F1 offspring sperm in sheep, which may contribute to environmentally influenced phenotypes in ruminants.

Restricted- and over-feeding during gestation decreases growth of offspring throughout maturity.

To determine the effects of poor maternal nutrition on the growth and metabolism of offspring into maturity, multiparous Dorset ewes pregnant with twins (n = 46) were fed to either 100% (control; n = 13), 60% (restricted; n = 17), or 140% (over; n = 16) of National Research Council requirements from day 30 ± 0.02 of gestation until parturition. Offspring of these ewes are referred to as CON (n = 10 ewes; 12 rams), RES (n = 13 ewes; 21 rams), or OVER (n = 16 ewes; 13 rams), respectively. Lamb body weights (BW) and blood samples were collected weekly from birth (day 0) to day 28 and then every 14 d until day 252. Intravenous glucose tolerance test (infusion of 0.25 g dextrose/kg BW) was performed at day 133 ± 0.25. At day 167 ± 1.42, individual daily intake was recorded over a 77 d feeding period to determine residual feed intake (RFI). Rams were euthanized at day 282 ± 1.82 and body morphometrics, loin eye area (LEA), back fat thickness, and organ weights were collected. The right leg was collected from rams at necropsy and dual-energy x-ray absorptiometry was used to determine bone mineral density (BMD) and length. Averaged from day 0 until day 252, RES and OVER offspring weighed 10.8% and 6.8% less than CON offspring, respectively (P ≤ 0.02). When adjusted for BW, liver and testes weights tended to be increased and decreased, respectively, in RES rams compared with CON rams (P ≤ 0.08). Additionally, RES BMD and bone length were less than CON rams (P ≤ 0.06). Treatment did not influence muscle mass, LEA, or adipose deposition (P ≥ 0.41). Rams (-0.17) were more feed efficient than ewes (0.23; P < 0.01); however, no effect of maternal diet was observed (P ≥ 0.57). At 2 min post glucose infusion, glucose concentrations in OVER offspring were greater than CON and RES offspring (P = 0.04). Concentrations of insulin in CON rams tended to be greater than OVER and RES ewes at 5 min (P ≤ 0.07). No differences were detected in insulin:glucose or area under the curve (AUC) for glucose or insulin (P ≤ 0.29). Maternal diet did not impact offspring triglycerides or cholesterol (P ≤ 0.35). Pre-weaning leptin tended to be 70% greater in OVER offspring than CON (P ≤ 0.07). These data indicate that poor maternal nutrition impairs offspring growth throughout maturity but does not affect RFI. Changes in metabolic factors and glucose tolerance are minimal, highlighting the need to investigate other mechanisms that may contribute to negative impacts of poor maternal diet.

PSII-9 Preliminary Investigation of Nutrient Challenge During Pregnancy on Wether Lamb Development, Growth, and Performance from Birth to Harvest

Abstract Inadequate nutrition during gestation has been shown to impair growth and metabolism of affected offspring and can predispose them to lifelong inefficiency and poor performance. Development throughout preweaning has been studied and compared to carcass performance; however, the impact of nutrient challenge during gestation on post weaning growth, efficiency, and nutrition utilization is not well known. Thus, our objective was to investigate the impacts of maternal nutrient restriction in late gestation on wether lambs' lifelong performance, from birth through harvest. We hypothesized that lambs from nutrient restricted dams would have reduced growth efficiency, taking longer to reach market weight and yielding altered body and carcass composition. Twenty-two bred yearling ewes were fed a diet meeting 100% of National Research Council (NRC) requirements until the 110th day of gestion (dGA). From 110 dGA until lambing occurred, ewes were either maintained on a diet meeting 100% of NRC requirements (n = 10) or reduced to a diet of 65% of NRC requirements (n = 12). Beginning at birth, body weight and morphometrics were collected weekly for control (CONT; n = 4) and treatment (NR; n = 5) wether lambs. At approximately 71 days of age, wethers were weaned and placed into individual pens to simulate a controlled feedlot setting. Wether lambs were fed a commercial lamb finishing diet ad libitum and orts were collected to evaluate feed efficiency and growth. At 115 days of age, wethers were placed in metabolism crates for 5 days to allow for total collection or urine and feces in order to evaluate nutrient balance. Birthweight, weaning weight, and morphometrics did not differ between NR and CONT lambs from birth until weaning. However, there was a tendency (P &amp;lt; 0.10) for a slower rate of gain from birth until weaning in NR lambs compared to their CONT counterparts. Current research in feedlot performance and digestibility is ongoing; however, thus far there are no differences in feed intake or average daily gain. These data suggest that a moderate nutrient challenge in the last month of gestation is not substantial enough to induce adaptive changes that result in differences in postnatal growth and performance.

204 Awardee Talk: Poor Maternal Nutrition Impacts Metabolic Factors and Glucose Tolerance in Sheep Offspring

Abstract To evaluate the hypothesis that offspring born to restricted- and over-fed ewes would exhibit decreased glucose tolerance, total cholesterol (TC), and triglyceride (TG), and increased leptin, multiparous Dorset ewes (n = 46) pregnant with twins were fed 100%, 60% or 140% of National Research Council (NRC) requirements from d30±0.02 of gestation until parturition. Offspring are referred to as CON (n=10 ewes; 12 rams), RES (n=13 ewes; 21 rams), and OVER (n =16 ewes; 15 rams), respectively. Blood samples were collected from offspring at d0, 7, 14, 58, 210, and 252 of age and plasma TC, plasma TG, and serum leptin were analyzed with commercial kits. Concentrations of TG were greater in rams than ewes (P=0.029). Leptin was greater (P&amp;lt; 0.0001) in ewes than rams at d30 and 36, and greater in CON than RES and OVER at d252 (P=0.058). At d133±0.25 an intravenous glucose tolerance test was performed, and blood samples collected at -30, -15, 0, 2, 5, 10, 15, 30, 60, and 120 min relative to glucose infusion (0.25 g/kg body weight of a 50% dextrose solution). Glucose and insulin concentrations were determined using commercial kits. Glucose concentrations were greater at 5 and 15 min in ewes than rams (P≤0.04) and at 2 min, OVER were greater than CON and RES (P=0.03). Insulin concentrations were greater in rams than ewes between 5 and 15 min (P=0.0251). Insulin in RES rams tended to be greater than OVER and RES ewes between 5 and 10 min (P=0.06) and continued to be greater than RES ewes at 15 min (P=0.05). First-phase response of insulin tended to be greater in RES compared with CON and OVER (P=0.092). There were no differences detected in insulin:glucose (P=0.312). In conclusion, poor maternal nutrition negatively impacts key metabolic hormones in offspring thereby contributing to altered growth and metabolism.

210 Restricted Maternal Nutrition: Impacts on Fetal Development and Metabolism in Sheep

Abstract Adequate nutrition is important for proper growth and metabolism. This is even more important during gestation when insults to the fetus can negatively impact fetal growth and development with persistent effects into adulthood. Using a sheep model, we have demonstrated that maternal restricted feeding (60% of National Research Council requirements) during gestation reduced growth in offspring and muscle fiber cross-sectional area. These changes in growth are associated with reduced stem cell (satellite cells and mesenchymal stem cells) population and function. Offspring of restricted-fed mothers also displayed altered metabolism indicated by increased insulin resistance, leptin dysregulation, and reduced circulating metabolic and growth factors. Based on these persistent negative effects of maternal diet on offspring postnatal growth and metabolism, we evaluated the impacts during fetal growth on whole body and tissue-specific metabolism and the impact of maternal realimentation during gestation. Nutrient restriction caused changes in metabolic pathways in a tissue specific manner in muscle, liver, and blood demonstrating the ability of maternal diet to program offspring metabolism in highly metabolic tissues. Several pathways involved in amino acid, lipid, carbohydrate, and transsulfuration/methionine metabolism were altered in all 3 tissues, demonstrating the whole-body effects of maternal diet on offspring metabolism. Interestingly, realimentation alleviated some of the effects of nutrient restriction on offspring metabolic pathways. In summary, maternal nutrient restriction during gestation negatively affects offspring fetal growth with persistent effects into adulthood which may be caused by changes in stem cell function and key metabolic pathways during fetal development.

Digestible indispensable amino acid scores of animal and plant ingredients potentially used in dog diet formulation: how this protein quality metric is affected by ingredient characteristics and reference amino acid profile.

The ability of a diet or an ingredient to satisfy the indispensable amino acid (IAA) requirements of an individual is a reflection of protein quality (PQ). The concept of PQ is gaining recognition in the pet food industry as a way to identify candidate ingredients for diet formulation. The objective of this report was to use IAA digestibility data from swine and cecectomized rooster assays to generate digestible IAA scores (defined herein as DIAAS-like values) to predict the PQ of ingredients used in dog diets. However, as PQ equation development relies on a reference IAA profile, which is intended to be based on the physiological requirements of a specific population, we sought to generate DIAAS-like values using IAA requirements established by the National Research Council (NRC) as well as practical IAA recommendations presented by the Association of American Feed Control Officials (AAFCO) and European Pet Food Industry Federation (FEDIAF), to assess how these profiles may affect PQ. In total, 30 animal (75 unique inputs) and 27 plant ingredients (94 unique inputs) satisfied all inclusion criteria to be used in the final data set. Ingredients were initially categorized as animal or plant, and further categorized based on AAFCO Official Common and Usual Names and Definitions of Feed Ingredients to allow for additional, more distinct comparisons to be made. Data were analyzed using PROC GLIMMIX in SAS, with ingredient reference as a random effect, and ingredient category, regulatory body, and life stage as fixed effects. As expected, differences were observed in DIAAS-like values for nearly all ingredients and ingredient categories when determined using NRC, AAFCO, or FEDIAF IAA requirements or recommendations as the reference pattern. Moreover, applying reference patterns based on NRC adult maintenance IAA requirements consistently produced the lowest DIAAS-like values. Ultimately, while future studies assessing PQ should utilize NRC minimal requirements, individual ingredient and ingredient category differences in DIAAS-like values when using AAFCO and FEDIAF recommendations underpin the different regulatory approaches to establishing dietary nutrient recommendations that exist globally and support the need for harmonization of dietary recommendations.

Poor maternal diet during gestation alters offspring muscle proteome in sheep.

Poor maternal nutrition during gestation can result in reduced offspring muscle growth and altered muscle metabolism. We hypothesized that over- or restricted-nutrition during gestation would alter the longissimus dorsi muscle (LM) proteome of offspring. Pregnant ewes were fed 60% (restricted), 100% (control), or 140% (over) of National Research Council requirements for total digestible nutrients from day 30 of gestation until parturition. Fetal (RES, CON, OVER) LM were collected at days 90 and 135 of gestation, or from offspring within 24 h of birth. Sarcoplasmic proteins were isolated, trypsin digested, and subjected to multiplexed, label-based quantitative mass spectrometry analysis integrating tandem mass tag technology. Differential expression of proteins was identified by ANOVA followed by Tukey's HSD post hoc tests, and regularized regression via the elastic net. Significance was set at P < 0.05. Over-represented pathways containing differentially expressed proteins were identified by Reactome and included metabolism of proteins, immune system, cellular response to stress/external stimuli, developmental biology, and infectious disease. As a result of maternal diet, a total of 312 proteins were differentially expressed (day 90 = 89 proteins; day 135 = 115 proteins; birth = 131 proteins). Expression of eukaryotic initiation factor (EIF) 2S3, EIF3L, and EIF4G2 was lower in OVER fetuses at day 90 of gestation (P < 0.05). Calcineurin A and mitogen-activated protein kinase 1 were greater in RES fetuses at day 90 (P < 0.04). At day 135 of gestation, pyruvate kinase and lactate dehydrogenase A expression were greater in OVER fetuses than CON (P < 0.04). Thioredoxin expression was greater in RES fetuses relative to CON at day 135 (P = 0.05). At birth, proteins of the COP9 signalosome complex were greater in RES offspring relative to OVER (P < 0.05). Together, these data indicate that protein degradation and synthesis, metabolism, and oxidative stress are altered in a time and diet-specific manner, which may contribute to the phenotypic and metabolic changes observed during fetal development and postnatal growth.

Trace mineral source influences digestion, ruminal fermentation, and ruminal copper, zinc, and manganese distribution in steers fed a diet suitable for lactating dairy cows

High solubility of certain trace minerals (TM) in the rumen can alter nutrient digestibility and fermentation. The objectives of the present studies were to determine the effects of TM source on 1) nutrient digestibility and ruminal fermentation, 2) concentrations of soluble Cu, Zn, and Mn in the rumen following a pulse dose of TM, and 3) Cu, Zn, and Mn binding strength on ruminal digesta using dialysis against a chelating agent in steers fed a diet formulated to meet the requirements of a high producing dairy cow. Twelve Angus steers fitted with ruminal cannulae were adapted to a diet balanced with nutrient concentrations similar to a diet for a high producing lactating dairy cow for 21 d. Steers were then randomly assigned to dietary treatments consisting of 10 mg Cu, 40 mg Mn, and 60 mg Zn/kg DM from either sulfate (STM), hydroxychloride (HTM) or complexed trace minerals (CTM). The experimental design did not include a negative control (no supplemental Cu, Mn, or Zn) because the basal diet did not meet the National Research Council requirement for Cu and Zn. Copper, Mn, and Zn are also generally supplemented to lactating dairy cow diets at concentrations approximating those supplied in the present study. Following a 14-d adaptation period, total fecal output was collected for 5-d. Following the fecal collection period, rumen fluid was collected for Volatile fatty acid (VFA) parameters. On the following day, the same diet was provided for 14 d, without supplemental Cu, Zn, and Mn. This period served as a wash-out period. A pulse dose of 100, 400, and 600 mg of Cu, Zn, Mn, respectively, from either STM, HTM, or CTM, was administered via ruminal cannulae to the steers on day 15. Over a 24-h period ruminal samples were obtained every 2-h. Following centrifugation, the supernatant was analyzed for Cu, Mn, and Zn. Ruminal solid digesta samples from times 0, 12, and 24 h after bolus dosing were exposed to dialysis against Tris-EDTA. Digestibility of NDF and ADF were lesser in STM vs. HTM and vs. CTM supplemented steers. Steers receiving HTM and CTM had greater total VFA concentrations than STM, and molar proportions of individual VFA were not affected by treatment. Ruminal soluble Cu and Zn concentrations were greater post dosing in STM and CTM supplemented steers at 2, 4, and 6 h for Cu and 4, 6, 8, 10 and 12 h for Zn when compared to HTM supplemented steers. The release of Cu and Zn from ruminal solid digesta following dialysis against Tris-EDTA at 12 and 24 h postdosing was greater for steers receiving HTM compared to those receiving STM or CTM. Results indicate trace mineral source impacts: 1) how tightly bound Cu and Zn are to ruminal solid digesta; 2) fiber digestion; 3) and ruminal total VFA concentrations.

Reduced vitamin supplementation with fat-soluble vitamins A, D, and E added at National Research Council requirements may not be adequate for optimal sow and progeny performance

Objective: To evaluate performance and physiological vitamin status of sows and progeny fed 2 vitamin supplementation levels, industry vs reduced (all vitamins reduced with fat-soluble vitamins added at National Research Council recommendations). Materials and methods: Sows (n = 244) were allotted in a randomized complete block design to 1 of 2 vitamin supplementation levels. At weaning, 765 progeny from a subset of sows were allotted to treatments in a 2 × 2 factorial arrangement of two sow and two nursery vitamin supplementation levels with 15 pens/treatment. Performance and vitamin status of sows and progeny were measured from farrowing to nursery exit. Results: Reduced vitamin supplementation reduced sow lactation feed intake (P = .01), hepatic vitamin A (P = .001), and serum vitamin D (P &lt; .001), but did not affect sow body weight or litter performance. Regardless of vitamin levels fed to the sow, progeny fed reduced levels post weaning had decreased circulating (P &lt; .001) and stored (P = .03) vitamin levels and a reduction in average daily gain (P &lt; .001), average daily feed intake (P &lt; .001), gain:feed ratio (P = .002), and body weight (P &lt; .001) at the end of the nursery period compared to progeny fed industry levels. Implications: Reduced vitamin supplementation reduced sow feed intake without affecting sow or litter performance, but decreased circulating and stored vitamin levels in sows could impact long-term reproductive performance. Reduced vitamin inclusion levels in nursery diets reduced performance and serum vitamin concentrations compared to industry vitamin levels.

PSX-A-16 Late-Breaking: Maternal nutrient restriction and re-alimentation influences liver protein expression in fetal sheep

Abstract To determine the effects of maternal nutrient restriction and re-alimentation on the fetal liver proteome, 48 pregnant ewes with singletons were fed a control diet [100% National Research Council (NRC) requirements (CON)] starting at the beginning of gestation. On day 50 of gestation, a subset of ewes were fed either CON or 60% NRC requirements (RES). A subset of ewes were euthanized at day 90 of gestation (n = 7/treatment), and fetal liver samples collected. Remaining ewes were maintained on the current diet (CON-CON, n=6; RES-RES, n = 7) or switched to alternative diet (CON-RES, RES-CON; n=7/treatment). On day 130 of gestation, remaining ewes were euthanized, and fetal samples collected. Fetal liver proteins were extracted, digested by trypsin and subjected to multiplexed, label-based quantitative mass spectrometry analysis integrating Tandem Mass Tags. Proteins were identified and quantified using Proteome Discoverer (v2.5, Thermo Scientific) and differential abundance analysis was performed using ANOVA and post hoc Tukey’s HSD test. Hierarchal clustering analysis showed clustering of treatments by day of gestation. However, differences were also observed between treatments. At day 90 of gestation, 23 proteins were differentially expressed in RES compared with CON among which glycyl tRNA synthetase and pyruvate carboxylase were increased 12% and 10%, respectively (P &amp;lt; 0.03). At day 130 of gestation, 24, 5, and 71 proteins were differentially expressed in CON-RES, RES-CON, and RES-RES, respectively, compared with CON-CON. Carnosine dipeptidase 2 was decreased 7% in CON-RES and rho associated protein kinase and glycogen synthase 2 were increased 20% and 26% in RES-CON and RES-RES, respectively, compared with CON-CON (P &amp;lt; 0.04). These results indicate that nutrient restriction during mid- and late-gestation impacts expression of proteins involved in gluconeogenesis, glycogenesis, and the formation of carnosine, an integral molecule in beta-oxidation, and that re-alimentation alters proteins involved in cell migration pathways. Supported by USDA-AFRI grants 2016-67016-24884 and 2017-67016-26568.

PSV-2 Maternal nutrient restriction and re-alimentation influences liver and muscle tissue development and gene expression

Abstract To determine the effects of maternal nutrient restriction and re-alimentation on fetal liver and muscle development, 48 pregnant ewes with singletons, were fed a control diet [100% National Research Council (NRC) requirements (CON)] starting at the beginning of gestation. On day 50 of gestation, ewes (n = 7) were euthanized and fetal liver and skeletal muscle samples were collected. The remaining animals were fed either CON or 60% NRC requirements (RES), a subset were euthanized at day 90 of gestation (n = 7/treatment), and fetal samples obtained. Remaining ewes were maintained on the current diet (CON-CON, n = 6; RES-RES, n = 7) or switched to alternative diet (CON-RES, RES-CON; n = 7/treatment). On day 130 of gestation, remaining ewes were euthanized, and fetal samples collected. Fetal liver was analyzed for general tissue morphology, and fetal skeletal muscles were analyzed for lipid accumulation. mRNA expression of growth and metabolic factors were quantified in liver and muscle tissues. Hepatocellular vacuolation was increased in RES-CON and RES-RES compared with CON-CON and CON-RES (P &amp;lt; 0.01). In semitendinosus and triceps brachii, intramyocellular lipid content increased 19% and 15%, respectively, in RES-CON and RES-RES compared with CON-CON and CON-RES (P£0.02) and in longissimus dorsi, lipid content was decreased 7% in CON-RES and RES-RES compared with CON-CON and RES-CON (P=0.01). In liver, insulin-like growth factor binding protein-1, glycogen synthase 2, and pyruvate dehydrogenase kinase 1 expression increased 1.92-fold, 1.45-fold, and 1.47-fold, respectively (P£0.03) in CON-RES and RES-RES compared with RES-CON and CON-CON. In LD, IGF1-R expression increased 3.19-fold in CON-RES and RES-RES compared with RES-CON and CON-CON (P = 0.05). These results demonstrate that maternal nutrient restriction followed by re-alimentation restores liver and muscle gene expression of growth and metabolic factors while negatively impacting liver composition and muscle lipid content potentially leading to altered tissue function and metabolism later in life. Supported by USDA-AFRI grants 2016-67016-24884 and 2017-67016-26568.

Effects of poor maternal nutrition during gestation on ewe and offspring plasma concentrations of leptin and ghrelin

Poor maternal nutrition during gestation can negatively affect offspring growth, development, and health. Leptin and ghrelin, key hormones in energy homeostasis and appetite control, may mediate these changes. We hypothesized that restricted- and over-feeding during gestation would alter plasma concentrations of leptin and ghrelin in ewes and offspring. Pregnant ewes (n = 37) were fed 1 of 3 diets starting on d 30 ± 0.02 of gestation until necropsy at d 135 of gestation or parturition: restricted- [RES; 60% National Research Council (NRC) requirements for total digestible nutrients, n = 13], control- (CON; 100% NRC, n = 11), or over-fed (OVER; 140% NRC, n = 13). Blood samples were collected from pregnant ewes at days 20, 30, 44, 72, 100, 128, and 142 of gestation. Offspring blood samples were collected within 24 h after birth (n = 21 CON, 25 RES, 23 OVER). Plasma leptin and ghrelin concentrations were determined by RIA. Ewe data were analyzed using the MIXED procedure in SAS with ewe as the repeated subject. Offspring data were analyzed using the MIXED procedure. Correlations between BW and leptin and ghrelin concentrations were identified using PROC CORR. At d 100, RES (5.39 ± 2.58 ng/mL) had decreased leptin concentrations compared with OVER (14.97 ± 2.48 ng/mL; P = 0.008) and at d 128, RES (6.39 ± 2.50 ng/mL) also had decreased leptin concentrations compared with OVER (13.61 ± 2.47 ng/mL; P = 0.04). At d 142, RES (0.26 ± 0.04 ng/mL) had increased ghrelin concentrations compared with CON (0.15 ± 0.04 ng/mL; P = 0.04). Leptin and ghrelin concentrations were also altered between days of gestation within a dietary treatment. In CON ewes, plasma concentrations of leptin were increased at d 30 (19.28 ± 7.43 ng/mL) compared with d 44 (5.20 ± 3.10 ng/mL; P = 0.03), and the plasma concentrations of ghrelin at d 128 (0.20 ± 0.03 ng/mL) were increased compared with d 30 (0.16 ± 0.03 ng/mL; P = 0.01) and d 100 (0.17 ± 0.03 ng/mL; P = 0.04). Maternal diet did not alter plasma ghrelin or leptin concentrations in the offspring (P > 0.50). There were no strong, significant correlations between ewe BW and leptin (r < 0.33; P > 0.06) or ghrelin (r > -0.47; P > 0.001) concentrations or lamb BW and leptin or ghrelin concentrations (r > -0.32, P > 0.06). Maternal alterations in circulating leptin and ghrelin may program changes in energy balance that could result in increased adiposity in adult offspring. Alterations in energy homeostasis may be a mechanism behind the long-lasting changes in growth, body composition, development, and metabolism in the offspring of poorly nourished ewes.

Economic and environmental effects of revised metabolizable protein and amino acid recommendations on Canadian dairy farms

The objective of this research was to evaluate the potential economic and environmental effects of the formulation model used to balance dairy rations for metabolizable protein (MP) or 3 essential AA (EAA: His, Lys, and Met) in 3 regions of Canada with different farming systems. The Maritimes, Central Canada, and the Prairies reference dairy farms averaged 63, 71, 144 mature cows per herd and 135, 95, 255 ha of land, respectively. Using N-CyCLES, a whole-farm linear program model, dairy rations were balanced for (1) MP, based on National Research Council (NRC) requirements (MP_2001); (2) MP plus Lys and Met, based on NRC (AA_2001); (3) MP (MP_Rev); or (4) for His, Lys, and Met (AA_Rev), both based on a revised factorial approach revisiting both supply and requirements of MP and EAA. Energy was balanced to meet requirements based on NRC (2001). Assuming the requirements were met within each approach, it was considered that milk yield and composition were not affected by the type of formulation. Given the assumptions of the study, when compared with MP_2001 formulation, balancing dairy rations using the AA_Rev approach reduced calculated farm N balance by 3.8%, on average from 12.71 to 12.24 g/kg of fat- and protein-corrected milk; it also enhanced farm net income by 4.5%, from 19.00 to 19.70 $CAN/100 kg of fat- and protein-corrected milk, by reducing inclusion of protein concentrate in dairy rations. Calculated animal N efficiency was on average 4.3% higher with AA_Rev than with MP_2001 for mid-lactation cows. This gain in N efficiency would result in a reduction in N2O emission by manure, contributing to a partial decrease of total greenhouse gas emission by 1.7%, through a reduction of N excreted in manure. With the AA_2001 formulation, farm N balance was 1% higher than with MP_2001 formulation while reducing farm net income by 6.4%, due to the need to purchase rumen-protected AA, with no effect on total greenhouse gas emission. Both MP formulations lead to fairly similar outputs. The AA_Rev formulation also indicated that His might be a co-limiting AA with Met in dairy rations balanced with ingredients usually included in Canadian dairy rations. Given the assumptions of the study, balancing dairy rations for 3 EAA (His, Lys, and Met) rather than MP, has some potential positive effects on Canadian dairy farms by increasing net incomes through a reduction of crude protein supply, leading to a decreased environmental effect.

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.