A simple analytical and experimental procedure for selection of reference genes for reverse-transcription quantitative PCR normalization data.

Variation in cellular activity in a tissue induces changes in RNA concentration, which affects the validity of gene mRNA abundance analyzed by reverse transcription quantitative PCR (RT-qPCR). A common way of accounting for such variation consists of the use of reference genes for normalization. Programs such as geNorm may be used to select suitable reference genes, although a large set of genes that are not co-regulated must be analyzed to obtain accurate results. The objective of this study was to propose an alternative experimental and analytical protocol to assess the invariance of reference genes in porcine mammary tissue using mammary RNA and DNA concentrations as correction factors. Mammary glands were biopsied from 4 sows on d 110 of gestation (prepartum), on d 5 (early) and 17 (peak) of lactation, and on d 5 after weaning (postweaning). Relative expression of 7 potential reference genes, API5, MRPL39, VAPB, ACTB, GAPDH, RPS23, and MTG1, and one candidate gene, SLC7A1, was quantified by RT-qPCR using a relative standard curve approach. Variation in gene expression levels, measured as cycles to threshold at each stage of mammary physiological activity, was tested using a linear mixed model fitting RNA and DNA concentrations as covariates. Results were compared with those obtained with geNorm analysis, and genes selected by each method were used to normalize SLC7A1. Quantified relative mRNA abundance of GAPDH and MRPL39 remained unchanged across stages of mammary physiological activity after accounting for changes in tissue RNA and DNA concentration. In contrast, geNorm analysis selected MTG1, MRPL39, and VAPB as the best reference genes. However, when target gene SLC7A1 was normalized with genes selected either based on our proposed protocol or by geNorm, fold changes in mRNA abundance did not differ. In conclusion, the proposed analytical protocol assesses expression invariance of potential reference genes by accounting for variation in tissue RNA and DNA concentrations and thus represents an alternative method to select suitable reference genes for RT-qPCR analysis.

Transcript abundance of amino acid transporters, ß-casein, and α-lactalbumin in mammary tissue of periparturient, lactating, and postweaned sows.

The objective of these experiments was to test the hypothesis that transcript abundance of cationic AA transporter- and milk protein-encoding genes increase in the porcine mammary gland in response to higher lactation demand. Genes of interest included those encoding for the milk proteins α-lactalbumin (α-LA) and ß-casein (ß-CN; LALBA and CSN2, respectively), and AA transporter b(0,+)AT, y(+)LAT1, y(+)LAT2, ATB(0,+), CAT-1, and CAT-2b (SLC7A9, SLC7A7, SLC7A6, SLC6A14, SLC7A1, and SLC7A2, respectively). Mammary tissue was biopsied from 4 sows on d 110 of gestation (prepartum), on d 2 (early postpartum), on d 5 (early), and d 17 (peak) of lactation, and on d 5 after weaning (postweaning), and mRNA of target genes quantified by reverse transcription quantitative PCR. Compared with prepartum, CAT-1, ATB(0,+), y(+)LAT2, ß-CN, and α-LA mRNA abundance was higher at early lactation, whereas compared with early lactation, only CAT-1 and α-LA mRNA abundance was higher at peak lactation. The CAT-2b, y(+)LAT1, and b(0,+)AT mRNA abundance did not differ when comparing either prepartum or peak lactation to early lactation. Compared with peak lactation, postweaning mRNA abundance of CAT-1, ATB(0,+), α-LA, and ß-CN decreased, y(+)LAT2, CAT-2b, and b(0,+)AT remained unchanged, and y(+)LAT1 increased. The mRNA abundance of y(+)LAT2 increased from early postpartum to early lactation, and remained unchanged for CAT-1, ATB(0,+), α-LA, and ß-CN. From prepartum to peak lactation, the mRNA abundance of CAT-1, y(+)LAT2, and ATB(0,+) was positively correlated with that of ß-CN and α-LA. In conclusion, the expression of genes encoding for y(+)LAT1, CAT-2b, and b(0,+)AT remained unchanged in porcine mammary glands over prepartum to peak lactation period, whereas expression of genes encoding for CAT-1, ATB(0,+), and y(+)LAT2 was upregulated and positively correlated to expression of genes encoding for the mammary synthesized milk proteins ß-CN and α-LA.

Effect of dietary near ideal amino acid profile on heat production of lactating sows exposed to thermal neutral and heat stress conditions.

BACKGROUND: Reduced protein diet manifested potential to mitigate heat production based on the concept of ideal amino acid profile. The hypothesis of this study was that lactating sows fed a low crude protein (LCP) diet with supplemental amino acid produce less heat compared to those fed a high crude protein (HCP) diet under both thermal neutral (TN) and heat stress (HS) conditions. METHODS: Thirty-two lactating sows were allotted to HCP (193 g CP/kg) and LCP (140 g CP/kg) diets under thermal neutral (TN, 21 ± 1.5 °C) or cycling heat stress (HS, 32 ± 1.5 °C daytime and 24 ± 1.5 °C nighttime) conditions. Diets contained 0.90% SID lysine and 10.8 MJ/kg net energy. Positive pressure indirect calorimeters were used to measure gas exchange in individual sows with litters, and individual piglets on days 4, 8, 14 and 18. Sow and litter weights were recorded on days 1, 10 and 21. RESULTS: Sow total heat production (THP) was calculated by subtracting litter THP from sow + litter THP based on BW0.75. Sow BW and body protein (BP) loss was greater for LCP diet compared to HCP diet in peak lactation (P < 0.05 and P < 0.01, respectively) and throughout the entire lactation period (P < 0.05 and P = 0.056, respectively) under HS conditions. Heat-stressed sows fed HCP diet had higher (P < 0.05) rectal temperature at 13:00 (P < 0.05) and 19:00 (P < 0.01), and higher respiration rate at 07:00 (P <  0.05), 13:00 (P < 0.05) and 19:00 (P < 0.05) compared to TN sows fed HCP diet. In sows fed LCP diet, those under HS tended to have higher (P = 0.098) rectal temperature at 13:00 and had higher (P <  0.05) respiration rate at 07:00, 13:00 and 19:00 compared to TN sows. The relationship between daily THP and days in lactation of sows fed LCP diet was quadratic (P < 0.05), with an ascending trend until day 14 and a descending trend from days 14 to 18. Sows fed LCP diet had lower daily THP at day 18 (P < 0.001) compared to those fed the HCP diet under HS conditions. CONCLUSION: Reduction in THP in sows fed LCP diet was largely associated with THP on day 18 of lactation under HS conditions. Feeding LCP diets alleviated the increased body temperature in sows under HS conditions throughout lactation, which was accompanied by a reduction in respiration rate. Total heat production is associated with days in lactation, in particular under HS conditions with THP appearing to peak between days 14 and 18.

Dynamics of nitrogen retention at two feeding levels in gestating parity 2 and 3 sows.

Parity-2 ( = 39) and -3 ( = 28; same sows from parity-2 sows) Yorkshire sows were used to measure whole body protein deposition (Pd) during gestation (maternal and pregnancy-associated) at 2 feeding levels to test the hypothesis that the pattern of whole body and maternal Pd during gestation does not differ when feeding either 15% above or 15% below estimated ME requirements, both exceeding AA requirements. Initial BW and backfat (BF) at d 26 ± 0.3 of gestation were 195.4 ± 5.2 kg and 15.1 ± 0.5 mm, and 223.1 ± 5.3 kg and 16.0 ± 0.6 mm for parity-2 and -3, respectively. Sows were assigned to 1 of 2 feeding levels (high and low; 15% above and 15% below estimated ME requirements, respectively) of the same diet (3.30 Mcal ME/kg, 17.8% CP, 0.82% SID Lys) from d 31 to 110 of each gestation cycle. Five N balances were conducted throughout each gestation starting at d 36, 51, 65, 85, and 106 ± 0.5 during 4-d periods. Pregnancy-associated Pd was model-derived for each sow and N balance period using the gestating sow model, based on actual litter size (including stillborn) and mean piglet birth weight. Maternal Pd was calculated as the difference between whole body and pregnancy-associated Pd. Whole body Pd and maternal Pd were greater ( 0.002) for sows on the high feeding level. Whole body Pd increased ( 0.001) with day of gestation and maternal Pd did not differ with day of gestation. Whole body and maternal Pd were greater ( ≤ 0.004) for parity-3 sows from d 51 to 54 and 85 to 88 of gestation only. Estimated efficiency of Lys retention for whole body Pd was not different between the 2 feeding levels, greater ( 0.016) for parity-3 sows, and increased quadratically ( 0.027) with day of gestation. During lactation (21.3 ± 0.3 d), there was no effect on sow ADFI and minimal effect on litter performance due to gestation feeding level or parity. Sows on the high feeding level had greater ( 0.009) BW and BF loss during lactation and these parameters were not affected by parity. Feeding 15% above estimated ME requirements during gestation resulted in consistently greater whole body and maternal. Whole body and maternal Pd were particularly greater for parity-3 sows from d 51 to 54 and 85 to 88 of gestation. The pattern of maternal Pd, regardless of feeding level, was not affected by day of gestation and is in contrast to , where maternal Pd is expected to be greater in early gestation.

Impact of improving dietary amino acid balance for lactating sows on efficiency of dietary amino acid utilization and transcript abundance of genes encoding lysine transporters in mammary tissue.

Lactating multiparous Yorkshire sows ( = 64) were used in 2 experiments to test the hypothesis that reducing dietary CP intake and improving AA balance through crystalline AA (CAA) supplementation improves apparent dietary AA utilization efficiency for milk production and increases transcript abundance of genes encoding Lys transporter proteins in mammary tissue. In Exp. 1, 40 sows were assigned to 1 of 4 diets: 1) high CP (HCP; 16.0% CP, as-fed basis; analyzed concentration), 2) medium-high CP (MHCP; 15.7% CP), 3) medium-low CP (MLCP; 14.3% CP), and 4) low CP (LCP; 13.2% CP). The HCP diet was formulated using soybean meal and corn as the only Lys sources. The reduced-CP diets contained CAA to meet estimated requirements for essential AA that became progressively limiting with reduction in CP concentration, that is, Lys, Ile, Met + Cys, Thr, Trp, and Val. Dietary standardized ileal digestible (SID) Lys concentration was 80% of the estimated requirement. In Exp. 2, 24 sows were assigned to the HCP or LCP diets. In Exp. 1, blood samples were postprandially collected 15 h on d 3, 7, 14, and 18 of lactation and utilization efficiency of dietary AA for milk production was calculated during early (d 3 to 7) and peak (d 14 to 18) lactation. Efficiency values were estimated from daily SID AA intakes and milk AA yield, with corrections for maternal AA requirement for maintenance and AA contribution from body protein losses. In Exp. 2, mammary tissue was biopsied on d 4 and 14 of lactation to determine the mRNA abundance of genes encoding Lys transporter proteins. In peak lactation, Lys, Thr, Trp, and Val utilization efficiency increased with decreasing dietary CP (linear for Trp and Val, < 0.05; in sows fed the MHCP diet vs. sows fed the HCP diet for Lys and Thr, < 0.05). Total essential and nonessential 15-h postprandial serum AA concentrations increased with decreasing dietary CP (linear, = 0.09 and < 0.05, respectively), suggesting increased maternal body protein mobilization. Transcript abundance of several genes involved in Lys transport in mammary tissue did not differ between sows fed the LCP and HCP diets. Feeding lactating sows low-CP diets supplemented with CAA increases the efficiency of utilizing dietary Lys, Thr, Trp, and Val for milk protein production but is unrelated to abundance in mRNA of genes encoding Lys transport proteins in the mammary gland. Dietary Lys utilization for milk protein production in lactating sows appears to be optimized when crystalline Lys is included at a minimum of 0.10% in a diet containing 15.70% CP.

Impact of feeding reduced crude protein diets to lactating sows on nitrogen utilization.

Forty lactating multiparous Yorkshire sows were used to test the hypothesis that reducing dietary CP and supplementing with crystalline amino acids (CAA) increases dietary N utilization for milk production during early and peak lactation. Sows were assigned to 1 of 4 diets: 1) 16.0% CP (as-fed; analyzed contents; HCP); 2) 15.7% CP (MHCP); 3) 14.3% CP (MLCP); 4) 13.2% CP (LCP); diet HCP was formulated using soybean meal and corn as the only Lys sources. The reduced CP diets contained CAA to meet requirements of the limiting AA. Sow and piglet BW were measured on d 1, 3, 7, 14, 18, and 21 of lactation. Nitrogen retention was measured on sows between d 3 and 7 (early) and d 14 and 18 (peak) of lactation. Milk true protein output was calculated from estimated milk yield and analyzed true protein concentration. Sow BW change (overall mean: -4.2 ± 3.37 kg over the 21-d lactation period) and average daily DM intake (overall mean: 4.05 ± 0.18 and 6.12 ± 0.20 kg/d, early and peak lactation, respectively) did not differ between diets. Nitrogen intake decreased as dietary CP concentration decreased (114.3, 106.0, 107.4, and 99.0 ± 5.29 g/d and 169.5, 168.3, 161.2, and 145.1 ± 5.29 g/d for HCP, MHCP, MLCP, and LCP in early and peak lactation, respectively; L: < 0.05). Sow loin eye area loss tended to increase as dietary CP concentration decreased (Linear (): = 0.082). Litter growth rate (LGR) over the 21-d lactation period tended to increase with decreasing dietary CP concentration (L: = 0.084). In early lactation, N retention (N intake- fecal and urinary N) and milk true protein and casein output were not affected by dietary treatment. In early lactation, as dietary CP decreased, N retained as percentage of N intake tended to increase (L: = 0.093) and estimated efficiency of using retained N for milk N output was not influenced by dietary CP concentration. In peak lactation, N retention (122.5, 123.8, 121.2, and 109.0 ± 4.88 g/d for HCP, MHCP, MLCP, and LCP, respectively) decreased (L: < 0.05), N retained as percentage of N absorbed (N intake - fecal N) increased (L: < 0.05), milk casein yield increased ( = 0.051), and estimated efficiency of using retained N for milk N output (44.5, 51.0, 54.9, and 62.9 ± 5.9% for HCP, MHCP, MLCP, and LCP, respectively) increased (L: < 0.05). Feeding lactating diets reduced in CP from 16.0% to 14.3% with CAA inclusion as partial replacement for limiting AA improved N retention and N utilization efficiency for milk protein production in peak lactation, while these effects were less pronounced in early lactation.

Dietary and plasma branched-chain amino acids in relation to tryptophan: effect on voluntary feed intake and lactation metabolism in the primiparous sow.

Seventy-six gravid gilts of different genotypes, Yorkshire x Duroc (YD) and Pig Improvement Company (PIC), were used in two trials to test the effect of decreasing the dietary ratio of tryptophan: branched-chain amino acids (BCAA) on feed intake, plasma metabolites, and body weight change during a 4-wk lactation period. The dietary ratio of tryptophan: BCAA was .063 in the Control and .041 in the experimental diet (BC). Gilts were fed 1.8 kg/d from d 107 of gestation until farrowing and given ad libitum access to feed during lactation. Feed consumption was measured daily. Body weights and preprandial blood samples were taken on d 107 of gestation and on d 1, 7, 14, and 21 postpartum. Plasma glucose, NEFA, urea nitrogen, ammonia, N tau-methylhistidine, and alpha-amino nitrogen were measured. The plasma tryptophan:BCAA ratio was higher (P < .05) on d 1 postpartum than on d 107 of gestation and d 7 postpartum in the Control group. Dietary addition of BCAA (BC) reduced (P < .06) the plasma tryptophan: BCAA ratio during the early postpartum period compared with the Control group but did not affect the other plasma metabolites measured. Feed intake was lower (P < .08) between d 6 and 12 of lactation in sows fed the BC diet. Regression of d-1 plasma NEFA concentration on d-1 postpartum feed intake was significant (r2 = .41; P < .05). The PIC sows showed a tendency for positive lactation body weight response to the BC diet (P = .2), whereas the response of YD sows was not significant (P < .8).(ABSTRACT TRUNCATED AT 250 WORDS)

Adipic acid increases plasma lysine but does not improve the efficiency of lysine utilization in swine.

Adipic acid, upon catabolism, results in intermediates that bear a structural similarity to lysine degradation products. The objectives of this research were to determine whether adipic acid affects lysine concentrations in plasma and to evaluate whether adipic acid improves the efficiency of lysine utilization in pigs. In Exp. 1, nursery pigs (n = 14) were fed (for a period of 7 d) either a standard nursery diet or the same diet supplemented with 1% adipic acid to assess effects on plasma amino acid concentrations (plasma collected on d 7). In Exp. 2, nursery pigs (n = 56) were fed (for a period of 15 d) either a control diet or the same diet but deficient in either lysine, threonine, or tryptophan with or without supplemental adipic acid to assess the effects of adipic acid on the efficiency of amino acid utilization. The results from Exp. 1 showed that adipic acid increased plasma lysine (by 18%) but not alpha-amino adipic acid, an intermediate in lysine degradation. Experiment 2 demonstrated that adipic acid did not increase the efficiency of utilization of lysine, threonine, or tryptophan. The lack of effects on alpha-amino adipic acid in Exp. 1 and the lack of a positive effect on the efficiency of utilization of lysine, threonine, and tryptophan suggest that adipic acid does not inhibit the mitochondrial uptake of lysine and(or) its degradation in the mitochondrion. It is concluded that feeding adipic acid increases plasma lysine but does not improve the efficiency of lysine utilization.

Plasma amino acid uptake by the mammary gland of the lactating sow.

The objectives of this study were to measure arteriovenous concentration (A-V) differences and estimate uptake of amino acids across the mammary gland in lactating sows. Four sows were used in Trial 1 and nine sows in Trial 2. Cannulas were fitted in the right anterior mammary vein and the carotid artery around d 7 of lactation. Arteriovenous samples were obtained on d 11, 14, 17, and 20. Litters were separated from the sows for 90 min, after which the first blood samples were drawn. Samples were taken at 20-min intervals for the next two consecutive hours. Milk production measurements and milk samples were obtained between d 11 and 19. Liters of plasma to liters of milk ratio (conversion coefficient) was estimated with the Fick method using A-V difference for lysine and milk lysine concentration. The conversion coefficient and the daily plasma flow were 541.41 +/- 35.72 L of plasma per liter of milk and 4,275 +/- 386 L of plasma, respectively. The A-V differences and the plasma flow were used to quantify daily amino acid uptake by the mammary gland. Uptake estimates (grams/day) were 36.51 +/- 4.05 (leucine), 31.24 +/- 3.64 (arginine), 23.39 +/- 2.97 (lysine), 21.22 +/- 1.96 (valine), 18.36 +/- 1.92 (isoleucine), 15.9 +/- 1.9 (threonine), 15.46 +/- 1.58 (phenylalanine), 7.61 +/- 1.12 (histidine), and 6.54 +/- 2.01 (methionine). The uptakes of arginine, leucine, isoleucine, valine, phenylalanine, and threonine significantly exceeded output of these amino acids in the milk. These results indicate that the sow mammary gland retains specific amino acids above requirements for milk protein synthesis.

A technique for the venous cannulation of the mammary gland in the lactating sow.

The objective of this study was to develop a technique to cannulate the mammary venous system of the lactating sow in conjunction with the carotid artery. A total of 16 sows were subjected to surgery between d 3 and 6 of lactation. The dams were separated from their litter during the full surgical procedure and the post-surgical recovery period. The carotid artery was cannulated according to a previously described method. The mammary vein cannulation immediately followed the carotid artery cannulation. A small venous branch (approximately 4 mm in diameter) located on the lateral side of the thoracic region was used to obtain access to the mammary vein. It was isolated 4 to 5 cm above the plica lateralis, between the first and second anterior gland. The venous branch was exposed and a cannula inserted slowly for a distance of 16 cm or until the tip of the cannula would reach the most cranial point of the mammary vein. The cannula was allowed to follow the normal path of blood flow, running in a caudal to cranial direction. After recovery from surgery (1 to 2 h), the dams were returned to their respective litters and treated with antibiotics for a minimum of 6 d. All sows nursed and weaned normal litters. Cannulas were flushed with heparinized saline (20 U/mL) every 8 h and both arterial and venous blood samples (10 mL) were collected simultaneously at 0700, 1500, and 2300. Arterial and venous cannulas remained patent for a minimum of 15 d.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of feeding level and physiological status on total flow and amino acid composition of endogenous protein at the distal ileum in swine.

An experiment was conducted to investigate the effects of BW, feed intake, and the physiological condition of the animal on the loss and amino acid composition of endogenous protein in swine. Ten growing barrows and five multiparous sows were equipped with a T-cannula in the distal ileum for digesta collection. A protein-free diet was fed to all animals. The barrows were given free access to the experimental diet. The sows also were allowed to consume the diet on an ad libitum basis, and digesta were collected during lactation and in the following gestation period. In addition, digesta from the gravid sows were collected after restricting the sows to 2 kg of feed per day. For each animal group, the endogenous losses of protein and amino acids were calculated in relation to DMI, and the amino acid composition of endogenous protein was calculated. The total endogenous gut protein loss at the distal ileum of growing pigs, lactating sows, and gestating sows, given free access to feed, was 12.4, 9.4, and 11.2 g/kg DMI, respectively. These values were not different (P > .10). However, when gestating sows were fed only 2 kg/d, 17.8 g of endogenous protein was lost per kilogram of DMI, which was higher (P < .05) than for any of the other groups. This difference was mainly caused by higher (P < .05) losses of glycine, proline, and serine. There were no differences (P > .05) in amino acid composition of endogenous protein between growing pigs, lactating sows, and gestating sows given free access to feed, but restricted-fed gestating sows had an amino acid composition of endogenous protein that was significantly different from that of the other groups. The results from the experiment showed that age, BW, and the physiological condition of the animal have little or no effect on the amount of endogenous protein and amino acids lost at the distal ileum of hogs if calculated in relation to DMI. Likewise, the amino acid composition was not affected by the BW or physiological condition of the animal. However, DMI had a significant effect on endogenous protein losses in sows as well as on amino acid composition of endogenous protein.

Nitrogen balance and ileal amino acid digestibility in growing pigs fed diets reduced in protein concentration.

Two studies were conducted to assess the effect of dietary protein reduction on N utilization, N excretion, and AA digestibility in growing pigs. The objective was to determine whether pigs fed diets with a reduced CP concentration could maintain the same N retention as pigs fed an adequate diet. The second objective was to test whether reducing dietary CP concentration decreases AA digestibility. In each study, six barrows were allotted to one of six dietary treatments in a Latin square design. Treatments consisted of four corn-soybean meal-based diets containing 15, 12, 9, and 6% CP, a casein-based diet containing 15% CP, and a protein-free diet. Crystalline AA were included in the 12, 9, and 6% CP diets. The indispensable:dispensable AA ratio was maintained at 45:55 with the addition of L-glutamic acid to the 9 and 6% CP diets. The casein-based and protein-free diets were used to determine endogenous total tract N and ileal AA losses. In the first study, total N losses and N absorbed decreased linearly (P < 0.001) as dietary CP concentration decreased from 15 to 6%. Both a linear (P < 0.001) and a quadratic (P < 0.05) decrease in N retention were found with decreasing dietary CP concentration. Nitrogen retained as a percentage of intake and absorbed increased (P < 0.001) as dietary CP concentration was reduced from 15 to 6%. In the second study, six barrows were surgically fitted with a T-cannula at the terminal ileum to determine ileal AA digestibility. For all dispensable and most indispensable AA, apparent and standardized ileal digestibility increased linearly (P 0.01, and for arginine, P < 0.05) as dietary CP concentration decreased. These results indicate that dietary CP concentration can be decreased from 15 to 12% with crystalline AA supplementation to meet an ideal AA profile without adversely affecting N retention, and that decreasing dietary CP concentration from 15 to 6% increases both dispensable and indispensable AA ileal digestibility.

The effect of litter size and day of lactation on amino acid uptake by the porcine mammary glands.

Twelve multiparous sows (PIC Camborough 15; parity >2) were used to investigate the relationship between litter size and day of lactation, and plasma amino acid (AA) arteriovenous differences (A-VD), AA uptake, and plasma flow across the mammary glands. Sows were assigned randomly to one of the following litter sizes: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 pigs per litter by cross fostering on d 2 postpartum. All sows were surgically fitted with catheters in the carotid artery and the main mammary vein. Matched arteriovenous blood samples were obtained on d 9, 12, 15, 18, 21, and 24 postpartum. Daily mammary uptake of AA was based on the product of plasma A-VD and daily mammary plasma flow (MPF). Daily MPF was estimated using the Fick method based on lysine conservation across the gland, and daily milk production. For the majority of AA, as litter size increased, A-VD did not increase, except for alanine (P < 0.05, linear and quadratic) and valine (P < 0.1; trend; linear and quadratic). As day of lactation increased, A-VD for the majority of AA increased (P < 0.05, linear and quadratic) except for arginine, lysine, and phenylalanine. As litter size increased, net daily mammary AA uptake increased for all indispensable AA (P = 0.001 to P < 0.05, linear and quadratic), excepting arginine. Milk production increased with increasing litter size (P < 0.001, linear) and with increasing day of lactation (P < 0.05, quadratic). Daily MPF increased (P < 0.05, linear) with increasing litter size, but did not change during the period measured from d 9 to 24. In conclusion, litter size appears to be a major determinant of net mammary AA uptake with daily mammary plasma flow a driving variable, whereas AA A-VD is a function of day of lactation and a major variable in determining net AA uptake with advancement of lactation.

Dietary protein concentration affects plasma arteriovenous difference of amino acids across the porcine mammary gland.

The objective of this study was to determine whether the porcine mammary gland responds to increasing dietary CP concentration through changes in AA arteriovenous difference (a-v). Sixteen Landrace x Yorkshire lactating sows were provided ad libitum access to one of four isocaloric diets varying in CP concentration (7.8, 13.0, 18.2, and 23.5 %; as-fed basis). Litters were adjusted to 11 pigs within 48 h of birth. Sows were fitted with catheters in the carotid artery and main mammary vein on d 4. On d 10, 14, 18, and 22 of lactation, arterial and venous blood samples were obtained every 30 min over 6 h. Milk yield was estimated on d 11 and 21 using the D2O dilution technique. Final litter sizes on d 21 were 10.3, 11, 9.5, and 11 piglets for sows fed the 7.8, 13.0, 18.2, and 23.5% CP diets, respectively. Piglet ADG tended (P = 0.088) to increase with increasing dietary CP concentration and were 186, 221, 220, and 202 g for sows fed the 7.8, 13.0, 18.2, and 23.5% CP diet, respectively. Daily total milk yield on d 21 (kg milk/d) tended (P = 0.099) to increase, and average milk yield per nursed piglet (kg of milk-pig(-1)d(-1)) increased (P < 0.05) with increasing CP concentration and were, on a per-piglet basis, 0.95, 1.19, 1.14 and 1.13 kg of milk/d for the 7.8, 13.0, 18.2, and 23.5% CP diets, respectively. As dietary CP increased from 7.8 to 23.5%, isoleucine and leucine a-v increased linearly only (linear, P < 0.01); all other AA a-v increased, reached a maximum in sows fed 18.2% CP, and decreased thereafter in sows fed 23.5% CP (quadratic, from P = 0.10 to P < 0.05). Amino acid uptake by the entire udder and by each gland increased (linear, P < 0.05) with increasing dietary CP. Arteriovenous differences response to increasing day of lactation varied among AA, from no change for histidine, isoleucine, lysine, methionine, tryptophan, and valine, to a linear trend increase for arginine (P = 0.055), leucine (P = 0.064), phenylalanine (P = 0.101), and threonine (P = 0.057). In summary, for the majority of AA, a-v increased with increasing dietary CP concentration from 7.8 to 18.2%, but decreased when CP concentration exceeded 18.2%. In contrast, mammary AA uptake, piglet ADG and milk yield per pig increased linearly with increasing dietary CP, suggesting a coordinated regulation between AA delivery and transport to meet the demand for milk yield.

Ammonia, volatile fatty acids, phenolics, and odor offensiveness in manure from growing pigs fed diets reduced in protein concentration.

The objective of this study was to investigate whether reducing dietary CP concentration decreases fecal VFA, manure ammonia (NH3) emission and odor, and urinary phenolic metabolites. Six barrows were allotted to one of six dietary treatments in a Latin square design. Treatments consisted of four corn-soybean meal based diets containing 15, 12, 9, and 6% CP, a casein-based diet containing 15% CP, and a protein-free diet (0% protein). Crystalline AA were included in the 12, 9, and 6% CP diets. The casein-based and protein-free diets were used to determine basal endogenous contribution of VFA, phenolics, NH3, and manure odor. Pigs were housed individually in metabolism cages to allow total collection of feces and urine. Feces and urine were collected and pooled within pig and period. Feces and urine were analyzed for VFA and phenolic metabolite concentrations, respectively. Feces and urine were then mixed, stored, and fermented at room temperature for 30 d. For NH3 determination, headspace air was sampled from manure slurries at 24, 48, and 72 h after fermentation. Slurry samples were placed into vials, capped, and randomized before odor panel evaluation. Odor offensiveness was classified on severity: 1 = non-offensive; 2 = mildly offensive; 3 = moderately offensive; 4 = strongly offensive; and 5 = extremely offensive. Reducing dietary CP increased (P < 0.05) fecal VFA concentrations but did not affect phenolic concentrations in urine. Manure NH3 emission was reduced (P < 0.05) as dietary CP concentration decreased from 15 to 0%. The 15% diet had the least offensive manure slurry with odor qualitative ranking of 2.58 (i.e., mild-moderately offensive). Compared with the 15% CP diet, manure from the 9 and 6% CP diets was found to be more offensive (P < 0.05), with qualitative rankings of 2.92 and 3.10, respectively. Odor qualitative rank for the 12% CP, protein-free diet, and casein-based diet did not differ from that of the 15% CP diet. These results indicate that reduction in dietary CP concentrations decreases manure NH3 emission, but it does not diminish manure odor offensiveness and fecal VFA concentrations.

Transcript abundance of hormone receptors, mammalian target of rapamycin pathway-related kinases, insulin-like growth factor I, and milk proteins in porcine mammary tissue.

Prolactin, glucocorticoids, and insulin are commonly used to induce milk protein synthesis in bovine mammary cell cultures. In addition, administration of GH increases milk yield in dairy cows, likely via the mammalian target of rapamycin (mTOR) pathway and IGF-I synthesis. As such, the hypothesis of this study was that mRNA abundance of hormone receptors, mammalian target of mTOR pathway-related kinases, IGF-I, and milk protein-encoding genes increases in the porcine mammary gland in response to greater lactation demand. Selected genes included those encoding for receptors of GH (GHR), insulin (INSR), glucocorticoid (NR3C1), prolactin (PRLR), IGF-I (IGF-I), mTOR (FRAP1), and p70S6 kinases (RPS6KB1), and the milk proteins α-lactalbumin (LALBA) and ß-casein (CSN2). Mammary tissue was biopsied from 4 sows on d 110 of gestation (prepartum), d 5 and 17 of lactation, and d 5 after weaning (postweaning), and gene expression was quantified by reverse-transcription quantitative PCR. Compared with prepartum, d 5 of lactation increased (P < 0.001) NR3C1, tended to increase (P = 0.06) GHR, and decreased (P < 0.001) PRLR mRNA abundance. Compared with d 5 of lactation, d 17 of lactation increased PRLR (P < 0.001) and decreased GHR (P < 0.01). Expression of INR and FRAP1 did not differ when comparing either prepartum or d 17 of lactation with d 5 of lactation. Compared with d 17 of lactation, postweaning decreased (P < 0.001) PRLR, did not affect INSR, and increased both IGF-I and GHR (P < 0.05) mRNA abundance. From prepartum to d 17 of lactation, NR3C1 mRNA abundance was positively correlated with CSN2 (r = 0.85; P < 0.001) and LALBA mRNA abundance (r = 0.79; P = 0.002), whereas mRNA abundance of GHR tended to be positively correlated with that of IGF-I (r = 0.46; P = 0.06). In conclusion, expression of the genes NR3C1, PRLR, GHR, and IGF-I changed in the porcine mammary gland during the prepartum to postweaning periods, but only NR3C1 mRNA abundance was positively correlated with expression of CSN2 and LALBA.

Characterization of L-lysine transport across equine and porcine jejunal and colonic brush border membrane.

In nonruminant herbivores, microbially derived AA could contribute to whole-body AA homeostasis and thus decrease predicted AA requirements. However, postileal capacity of AA uptake is currently unknown. Therefore, to test the hypothesis that Lys is transported across the large colon mucosal apical membrane with capacity similar to that of the small intestinal mucosa in the pony and pig, we examined Lys transport in vitro using brush border membrane vesicles (BBMV). Mucosa was collected from the distal jejunum (DJ) and proximal large colon (PLC) of growing pigs (n = 3) and ponies (n = 4), flash frozen in liquid nitrogen, and stored at -80°C. Jejunal and colonic BBMV were manufactured by Mg(2+) precipitation and used to determine initial rates and kinetics [the maximal transport rate (V(max)) and the Michaelis constant (K(M))] of l-Lys transport into apical epithelia by rapid filtration technique in Na(+)-gradient incubation buffer. Initial rates of total l-Lys uptake did not differ between the PLC and DJ in either the pig or the pony, or between the pony and the pig, at each l-Lys concentration. In the pig, compared with the DJ, l-Lys transport V(max) in the PLC did not differ (121 ± 26 and 180 ± 26 pmol•mg of protein(-1)•s(-1), respectively; P = 0.14) and l-Lys K(M) in the PLC tended to be greater (0.23 ± 0.22 and 0.89 ± 0.22 mM, respectively; P = 0.09). In the pony, compared with the DJ, l-Lys transport V(max) in the PLC was greater (62 ± 25 and 149 ± 25 pmol•mg of protein(-1)•s(-1), respectively; P = 0.04) and l-Lys K(M) in the PLC was greater (0.08 ± 0.22 and 1.05 ± 0.22 mM, respectively; P = 0.02). l-Lysine diffusion was not different between segments; however, total intestinal diffusion was greater (P = 0.03) in the pony than in the pig (115 ± 10 and 73 ± 10 pmol·mg of protein(-1)•s(-1), respectively). These results demonstrate that the large colon is capable of l-Lys transport across the apical epithelial membrane with greater capacity and less affinity than the jejunum, indicating that the large colon may play a significant role in l-Lys absorption and homeostasis in hindgut fermenters.

Effect of amino acids supply in reduced crude protein diets on performance, efficiency of mammary uptake, and transporter gene expression in lactating sows.

To test the hypothesis that reduction in dietary CP concentration coupled with crystalline AA inclusion increases the efficiency of AA use for milk production, mammary AA arteriovenous concentration differences (A-V), AA transport efficiency (A-V/A × 100), and transcript abundance of AA transporters and milk protein genes were determined in lactating sows fed 1 of 3 diets containing 9.5% (Deficient), 13.5% (Ideal), or 17.5% (Standard) CP, with a similar profile of indispensable and dispensable AA. On d 7 and 18, arterial and mammary venous blood and mammary tissue were sampled postfeeding. Transcript abundance of AA transporters b(0,+)AT (SLC7A9), y(+)LAT2 (SLC7A6), ATB(0,+) (SLC6A14), CAT-1 (SLC7A1), and CAT-2b (SLC7A2) and milk protein ß-casein (CSN2) and LALBA (α-lactalbumin) were determined using reverse transcription quantitative PCR. Piglet ADG increased curvilinearly (linear and quadratic, P < 0.03) with increasing percent CP from Deficient to Standard. On d 7, Lys and Arg A-V and transport efficiency increased quadratically (P < 0.05) with increasing percent CP. On d 18, Lys A-V tended to increase (linear, P = 0.08) with increasing percent CP. Increasing CP increased Ile and Val A-V on d 7 (linear, P = 0.05 and P = 0.08, respectively) and Leu and Val on d 18 (linear, P = 0.07 and P = 0.04, respectively). On d 7, plasma concentrations of branched chain AA (BCAA):Lys decreased quadratically (P < 0.05). Expression of genes SLC7A9, SLC7A6, SLC6A14, SLC7A1, SLC7A2, CSN2, and LALBA was unaffected by diet. In conclusion, decreasing the dietary CP from 17.5% to 13.5% with inclusion of crystalline AA did not affect piglet ADG, AA transporter, or milk protein gene expression but increased mammary transport efficiency and A-V of Lys and Arg on d 7 of lactation. This increase was associated with a decrease in plasma concentration of BCAA:Lys, suggesting a competitive mechanism between cationic and BCAA for transport of AA across mammary cells.

Protein quality and utilization of timothy, oat-supplemented timothy, and alfalfa at differing harvest maturities in exercised Arabian horses.

To evaluate the protein quality and postgut N utilization of full-bloom timothy hay, oat-supplemented timothy-hay diets, and alfalfa hay harvested at different maturities, apparent whole tract N digestibility, urinary N excretion, and serum AA profiles were determined in light to moderately exercised Arabian horses. Six Arabian geldings (16.0 ± 0.3 yr; 467 ± 11 kg of BW) were randomly allocated to a 6 × 6 Latin square design. Diets included full-bloom timothy grass hay (G), G + 0.2% BW oat (G1), G + 0.4% BW oat (G2), mid-bloom alfalfa (A1), early-bloom alfalfa (A2), and early-bud alfalfa hay (A3). Forages were fed at 1.6% of the BW of the horse (as-fed). Each period consisted of an 11-d adaptation period followed by total collection of feces and urine for 3 d. Blood samples were taken on d 11 for analysis of serum AA concentrations. During the 3-d collection period, urine and feces were collected every 8 h and measured and weighed, respectively. Approximately 10% of the total urine volume and fecal weight per period was retained for N analyses. Fecal DM output was less (P < 0.05) in A1, A2, or A3 compared with G, G1, or G2. Apparent whole tract N digestibility was greater (P < 0.01) in A1, A2, and A3 compared with G, G1, or G2, and was greater (P < 0.05) in G1 and G2 compared with G. Nitrogen retention was not different from zero, and there were no differences (P > 0.05) in N retention among diets. Urinary N excretion and total N excretion were greater (P < 0.05) in A1, A2, and A3 compared with G, G1, or G2. Plasma concentrations for the majority of AA increased curvilinearly in response to feeding G, A1, A2, and A3 (quadratic, P < 0.05), with values appearing to maximize 2-h postfeeding. Although alfalfa N digestibility increased with decreasing harvest maturity, N retention did not differ and urinary volume and N excretion increased, indicating that postabsorptive N utilization decreased. In contrast, inclusion of oats at either 0.2 or 0.4% of the BW of the horse to timothy hay markedly enhanced N digestibility without increasing N excretion, indicating improvement in postgut N utilization. These findings indicate that feeding oat-supplemented timothy hay is more environmentally sustainable than feeding alfalfa to the horse at maintenance or under light to moderate exercise.

Cationic and neutral amino acid transporter transcript abundances are differentially expressed in the equine intestinal tract.

To test the hypothesis that AA transporter transcripts are present in the large intestine and similarly expressed along the intestinal tract, mRNA abundance of candidate AA transporter genes solute carrier (SLC) family 7, member 9 (SLC7A9), SLC7A1, SLC7A8, and SLC43A1 encoding for b(0,+)-type AA transporter (b(0,+)AT), cationic AA transporter-1 (CAT-1), L-type AA transporter-2 (LAT-2), and L-type AA transporter-3 (LAT-3), respectively, was determined in small and large intestinal segments of the horse. Mucosa was collected from the equine small (jejunum and ileum) and large intestine (cecum, left ventral colon, and left dorsal colon), flash frozen in liquid nitrogen, and stored at -80 degrees C. Messenger RNA was isolated from tissue samples, followed by manufacture of cDNA. Relative quantitative reverse transcription-PCR was conducted using the 2(-DeltaDeltaCT) method, with glyceraldehyde-3-phosphate dehydrogenase serving as the housekeeping gene. Compared with the jejunum, cationic and neutral AA transporter SLC7A9 mRNA abundance was similar in the ileum, cecum, and large intestinal segments. Compared with the jejunum, cationic AA transporter SLC7A1 mRNA abundance was similar in the ileum and decreased in the cecum, left ventral colon, and left dorsal colon (P < 0.001). Neutral AA transporter SLC7A8 mRNA abundance decreased from the cranial to caudal end of the intestinal tract (P < 0.001). Neutral AA transporter SLC43A1 mRNA abundance was similar in the ileum and left dorsal colon and increased in the cecum (P < 0.01) and left ventral colon (P < 0.1) compared with the jejunum. Cationic and neutral AA transporter SLC7A9 mRNA abundance was similarly expressed in the large compared with small intestine, whereas cationic AA transporter SLC7A1 was of low abundance in the large intestine; neutral AA transporters SLC7A8 and SLC43A1 were differentially expressed with decreased abundance of SLC7A8 and increased abundance of SLC43A1 in the large intestine. Results indicate that the large intestine might contribute to both cationic and neutral AA uptake and absorption predominantly via transporters LAT-3 and b(0,+)AT.