The survey was to find the influence of dietetic insufficient protein on plasma amino acerb concentration in sows, seeable mammary secretory organ conformation, the look degrees of messenger RNA and protein of some impersonal amino acerb conveyance systems ; System A ; ATA2, System L ; LAT2 and System ASC ; ASCT1 in mammary tissues at peak lactation ( 500 18 of lactation ) . Eight multiparous lactating sows were provided limitation provender entree to one of two isocaloric diets, each 4 sows for deficient and normal protein diet group ( 8 and 18 % CP, as-fed footing, severally ) . The consequences showed that sows fed with deficient protein diet had important lessening in plasma amino acerb concentrations notably for impersonal and cationic amino acids ( P & lt ; 0.05 ) . The seeable mammary secretory organ conformation of sows fed with deficient protein diet seemed to be smaller when compared to those fed with normal protein diet. The quantitative messenger RNA looks of ASCT1 and LAT2 were down regulated significantly ( p & lt ; 0.05 ) for sows fed with deficient protein diet when compared to those fed with normal protein diet. In contrast, that of ATA2 was non changed ( P & gt ; 0.05 ) . Consequently, the protein look degrees of ATA2 and ASCT1 were non changed ( P & gt ; 0.05 ) but those of LAT2 was down regulated significantly ( p & lt ; 0.05 ) for sows fed with deficient protein diet when compared to those fed with normal protein diet. Therefore, the dietetic protein degree under peak lactation can modulate the different gene-protein look of some impersonal amino acid transporters in sow mammary secretory organ.
Dietary direction of the breastfeeding sow guaranting a diet sufficient in protein is an of import factor to accomplish the ability of the sows to bring forth equal milk back uping the rapid growing of their litters. The chief beginning of aminic acids or protein of suckling piggies is found in sow ‘s milk. Therefore, diet of breastfeeding sow need to be formulated to maximise milk production and keep organic structure status throughout lactation. Insufficient amino acid demands of the sows during lactation can take to diminish milk production and litter ablactating weights ( King et al. , 1993 ) . The surveies of Trottier et Al. ( 1997 ) and Nielsen et Al. ( 2002 ) reported that there were two different physiological mechanisms responsible for the visual aspect of aminic acids in milk, viz. mammary plasma flow and amino acid conveyance systems in mammary tissues. The degree of dietetic protein concentration is an of import factor affected aminic acerb concentration in sow ‘s milk via these two mechanisms ( Guan et al. , 2004a ) . As dietetic petroleum protein ( CP ) increased from deficient protein ( 7.8 % CP ) to normal protein ( 18.2 % CP ) degree, all indispensable amino acids taken up across mammary tissues were increased and reached upper limit in sows fed with the normal protein diet, and decreased in those fed with the extra CP diet. The mammary arteriovenous difference of aminic acids as amino acids which enter and leave the mammary secretory organs ( Trottier et al. , 1995 ) was increased with increasing dietetic CP concentration while the mammary plasma flow was non alteration ( P & gt ; 0.05 ) . Consequently, the mechanisms responsible to straight increase the arteriovenous difference may perchance be through types of amino acerb conveyance systems, look and localisation of amino acid transporters. Therefore, these assume that the amino acid transporter proteins may play the of import functions on amino acid conveyance non merely for sow ‘s milk production but besides milk composing and so impact their piggy growing.
The transit of aminic acids from mammary arterial plasma across mammary cells by amino acerb conveyance systems is now recognized as an of import mechanism of amino acerb visual aspect in sow ‘s milk. Amino acids, absorbed from the digestive system, do non pervade through cell membranes and hence necessitate specialised conveyance proteins in order to traverse the cell membranes ( Christensen, 1990 ) . The breastfeeding mammary secretory organ takes up free amino acids from the blood in big measures to fulfill the demands of protein synthesis ( Guan et al. , 2004a ) and synthesis of incidental amino acids in mammary tissue protein ( Trottier et al. , 1997 ) . At least five amino acid conveyance systems have been characterized to transport aminic acids into the mammary secretory cells ( Shennen, 1997 ) . The major of import amino acerb conveyance systems for sows and their piggies that should be studied are: system A that prefers to transport impersonal amino acids, particularly short-chain aminic acids such as methionine ( Baumrucker, 1985 ) ; system L, which chiefly transports branched-chain amino acids that are taken up in surplus of their visual aspect in milk ( Baumrucker, 1985 ; Jackson et al. , 2000 ) ; system ASC that transports some impersonal aminic acids such as alanine serine and cysteine ( Christensen, 1990 ) . Nowadays, the demands for sow milk production are based entirely on the amino acerb composing in secreted milk protein. It may inaccurately foretell the sufficient demands ( Nielsen et al. , 2002 ) . Therefore, the appraisal of the amino acid demands should be assessed utilizing the amino acerb uptake across mammary tissues ( Trottier et al. , 1997 ) . Knowledge of the amino acerb conveyance systems is needed to be determined and corporately estimated the amino acid demands at peak lactation in sows. Most of the surveies have been conducted on mice, rats, guinea hogs, cowss, caprine animals, and worlds. Merely a research survey by Laspiur et Al. ( 2004 ) and our research lab were conducted on mRNA look of some amino acid transporter in sow lactating cells. Therefore, the aims of this survey were to find the consequence of dietetic protein degree on plasma amino acerb concentrations, seeable mammary secretory organ conformation, and non merely comparative copiousness messenger RNA look but besides comparative copiousness protein look of some impersonal amino acid transporters ; LAT2, ATA2, and ASCT1 at peak lactation ( d18 of lactation ) in mammary tissues of sows.
Methods and Materials
Experimental design and diets.
Animal processs used in this survey were approved by the Animal Care and Use commission, Faculty of Veterinary Science, Chulalongkorn University. Eight multiparous crossbred wet sows were indiscriminately selected based on their familial backgrounds, mammary secretory organ conformation and farrowing twenty-four hours. Sows were allocated into 2 groups ; each 4 sows for deficient and normal protein diet group ( 8 and 18 % CP, severally ) which were isocaloric ( 3.2 ME, Mcal/kg ) and balanced other foods, harmonizing to the recommendation of NRC ( 1998 ) . Sows were provided sufficient provender and free imbibing H2O.
Sows were moved to piging make 2 hebdomads prior to pig and housed separately in piging creates. After piging ( 500 0 ) , piggies were cross-fostered after birth to accomplish equal figure of piggy per sow. Their piggies were received sow ‘s milk throughout lactation period and given a starter diet ad libitum from 500 18 of lactation until weaned twenty-four hours on vitamin D 28 of lactation. Sows were weighed separately on vitamin D 1 postpartum, vitamin D 14, and d 28 of lactation.
Blood samples obtained from each sow on vitamin D 16 of lactation. Blood samples of sows were collected 4 H after feeding for 4 milliliters each sow, via anterior veins cava puncture and were so put into heparinized tubings. All blood samples were kept on ice until centrifugation. Plasma was separated by centrifugation at 3,000g for 15 min at 4A°C, so transferred to new tubing and stored at -20A°C until analysis of amino acid concentration harmonizing to method documented by Reverter et Al. ( 1997 ) .
Mammary tissue sampling.
On 500 18 of lactation, mammary tissue samples were obtained by incisional biopsy in five anterior secretory organs of the bag, after sows were feed backdown for 4 H at forenoon. The sows were anesthetized by i.m. disposal of Azaperone. The bag were locally infused by 2 % Lidocaine ( OLIC, Thailand ) at the scratch site which was performed about center ( 4 to 5 centimeter ) between the nipple and the upper line of the bag. The scratch was continued through the hypodermic tissues and fascia beds to expose the underlining mammary tissues. Approximately 15-mm egg-shaped scratch at a deepness of 5-10 millimeter, mammary tissues ( 1-5 g ) were collected and instantly placed in RNAlater tissue protection kitA® ( Qiagen, Hilden, Germany ) to forestall RNA debasement and stored at -20A°C for subsequent finding of gene-protein looks. The hypodermic tissues and tegument were sutured with coated VicrylA® ( Ethicon Division of Johnson & A ; Johnson, NSW, Australia ) .
Entire RNA was extracted from mammary tissues with AurumTM ; Total RNA Fatty and Fibrous Tissue Kit ( BioRad, Hercules, USA ) harmonizing to the maker ‘s protocol.
Rearward Transcription-PCR and Sequence of oligonucleotide primers
The entire RNA samples were synthesized the first strand complementary DNA utilizing iScriptTM Select complementary DNA Synthesis kit ( BioRad, Hercules, USA ) . A 20-ml of rearward written text reaction mixture, incorporating 10 mM random-primer, 1 pg to 1mg entire RNA sample and 11 ml nuclease-free H2O, was consecutive incubated at 65A°C for 5 min, at 25A°C for 5 min. 5x iScript selected reaction mix ( incorporating dNTPs, Mg chloride, and stabilizers ) and iScript contrary RNA polymerase were added for 4 milliliters and 1 milliliter in each reaction and incubated consecutive at 25A°C for 5 min, at 42A°C for 30 min, at 85A°C for 5 min. The synthesized complementary DNA merchandises were so amplified by PCR by utilizing IQTM Supermix kit ( BioRad, Hercules, USA ) . The PCR elaboration in a concluding volume of 50-ml composed of 25 milliliters IQTM Supermix ( incorporating 100 mM KCl, 40 millimeter Tris-HCl, 1.6 millimeter dNTPs, iTaq DNA polymerase, 50 units/ml, 6 millimeter MgCl2, and stabilizers ) , 0.5 milliliter of each primer, and 2 milliliter complementary DNA templet. The PCR primers were designed from the published human LAT2, rat ATA2, and human ASCT1. The primer braces used in the subsequent PCR and the expected size of the PCR merchandises were summarized in Table 1.
Table 1. Sequence of oligonucleotide primers used for PCR elaboration
5 ‘ 3 ‘
GenBank accession no.
Expected size ( bp )
TTTCCAGGAACCTGACATCG ( F )
ACATTGCAGTGACATAAGCG ( R )
AATCTGACCAATGCGATTGTG ( F )
AATAAAGACCCTCCTTCGTTG ( R )
GGACTCTTTCCTCGACCTG ( F )
ATCTCAGTGCCTATGGGGATCT ( R )
CCGCGGTTCTATTTTGTTGGTTTT ( F )
CGGGCCGGGTGAGGTTTC ( R )
Relative messenger RNA copiousness of SNAT2/ATA2, LAT2 and ASCT1 in sow mammary cells by the Semiquantitative Reverse Transcription-PCR technique.
The comparative copiousness of some impersonal amino acid transporters was determined by semiquantitative RT-PCR utilizing IQTM Supermix kit. In multiplexed PCR, 18S rRNA was used as an internal control. Entire RNA samples were rearward transcribed into complementary DNA utilizing random-primer. The cDNA merchandise concentration of each sample, which were diluted to 140 times, was measured the concentration utilizing SPECTRONICO GENESYSTM spectrophotometer ( Spectronic Instruments, INC, . N.Y. ) at a wavelength of 260 nanometers ( in the UV spectrum ) . The concluding DNA concentration of each sample was adjusted and equalized by diluted DNA sample to 1 mg/ml with autoclaved extremist pure H2O. Multiplexed PCR reactions were so set up with a gene-specific primer braces and 18S rRNA primer to let coincident elaboration of the cistron of involvement and the internal control in a individual tubing. Preliminary experiments were conducted to find both the optimum sum of internal control primers and interested cistron primers ratio and the figure of PCR rhythms that would enable detected PCR elaboration within the additive scope of the PCR reaction for both groups of sows fed with deficient and normal protein diet. The protocol of multiplexed PCR reaction consisted of 3 proceedingss at 95A°C, followed by 33 rhythms of denaturing at 94A°C for 30 seconds, tempering at 52A°C for 30 seconds, extension at 72A°C for 60 seconds, and eventually, halt reaction at 72A°C for 10 proceedingss. The attendant PCR merchandises were size-fractionated in 1.8 % agarose gels, stained with ethidium bromide. The PCR merchandises were loaded for 18 ml/well. The electromotive force of cataphoresis was set up at 70 electromotive force for 70 min. The strengths of both the amino acid transporter and 18S rRNA set for each sample were measured by densitometry utilizing Scion Image package plan ( www.scioncorp.com ) . The information of look degrees of each transporter cistron were reported as values normalized to the 18S rRNA.
Relative protein copiousness of SNAT2/ATA2, LAT2 and ASCT1 in sow mammary cells by the western smudge technique
The frozen sow mammary tissue was homogenized in lysis buffer incorporating peptidase inhibitor cocktail ( Sigma, USA ) and centrifuged, so the supernatant was collected. The sum of protein was measured with modified Lowry ‘s check utilizing commercial trial kit ( Biorad Laboratories ) . Samples ( 50 – 75 milligrams protein/lane ) were resolved by 9 % SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes in Tris-glycine transportation buffer. Blotted membranes were so blockd with 5 % nonfat powdered milk in Tris-buffered saline for 4 hours at room temperature. For designation of proteins, membranes were washed and incubated overnight at 4oC with the primary antibodies diluted in 1 % milk. The primary antibodies were polyclonal anti-SNAT2/ATA2, anti-LAT2, anti-ASCT1 ( Santa Cruz, USA ) , and anti-b-actin ( Sigma, USA ) . Following the primary antibody incubation, the membranes were washed and so incubated in horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hr. This incubation measure was terminated with several washes and the immunoreactive protein sets were visualized utilizing chemiluminescence technique ( ECL Plus ; Amersham Biosciences ) . Membranes were exposed to movie ( Hyperflim-ECL ) for clip adequate to visualise chemiluminescent sets. Differences in determined by scanning densitometry in proportion to b-actin immunoreative sets ( Scion Image ; Scion Corporation, USA ) . All samples were repeated in extra.
All checks were conducted in extra or triplicate. Statistic significance of comparative differences in mRNA look was presented as meansA±SE. Differences between groups were analyzed by unpair t-test utilizing the commercially computing machine plan GraphPad Prism ( Prism3 ) at p-value P & lt ; 0.05.
Plasma amino acid concentration.
The plasma amino acerb concentrations in sows fed with dietetic protein lack, most indispensable amino acids were significantly decreased such as arginine ( 1.11 % to 0.47 % ) , threonine ( 0.61 % to 0.30 % ) , tyrosine ( 0.43 % to 0.18 % ) , isoleucine ( 0.62 % to 0.26 % ) , leucine ( 1.27 % to 0.60 % ) , valine ( 0.74 % to 0.38 % ) , and phenylalanine ( 0.65 % to 0.29 % ) compared to those fed with dietetic protein sufficiency ( p?0.05 ) . In contrast, most of plasma incidental amino acerb concentrations were invariably remained in response to dietetic protein lack ( p & gt ; 0.05 ) .
Visible mammary secretory organ conformation.
The seeable mammary secretory organ conformation of sows fed with deficient protein diet seemed to be smaller when compared to those fed with normal protein diet.
The comparative messenger RNA copiousness of some impersonal amino acid transporters in sow mammary tissues.
Sows fed with deficient protein diet expressed less copiousness of ASCT1 and LAT2 than those fed with normal protein diet ( p & lt ; 0.05 ) . The mRNA copiousness of an amino acid transporter: ATA2 in breastfeeding mammary tissues was non different between both groups of sows fed with normal and lacking protein diets. ( p & gt ; 0.05 ) .
The comparative protein look of some impersonal amino acid transporters in sow mammary tissues.
Sows fed with deficient protein diet expressed less copiousness of LAT2 protein than those fed with normal protein diet ( p & lt ; 0.05 ) . The protein look of ATA2 and ASCT1 in breastfeeding mammary tissues were non different between both groups of sows fed with normal and lacking protein diets. ( p & gt ; 0.05 ) .
Discussion and Decision
Deficient protein diet had negative effects on organic structure weight alteration of lactating sows which accordingly affected growing public presentation of their piggies by diminishing in organic structure weight and mean day-to-day addition, particularly at last period of lactation ( unpublished information ) . These consequences agree with old survey of Guan et Al. ( 2004a ) , sows fed with deficient protein diet ( 7.8 % CP ) lost their weight for 25.8 kilogram. Breastfeeding sows frequently lost their organic structure proteins to back up milk production. Nutritional inadequacy can do an inordinate organic structure protein loss and accordingly idiots litter growing rate. Main consequence of decrease in piggy growing is sow ‘s milk production and composing. Severe protein limitation during lactation decreased milk production of sow ( Jones and Stahly, 1999 ) and besides decreased proteins composed in sow ‘s milk ( Guan et al. , 2004a ) . Consequently, piggies did non have sufficient foods including aminic acids for organic structure protein synthesis to back up rapid growing at early phase of life. The consequence of decrease in piggy growing may be confirmed by the degree of plasma amino acerb concentration found in plasma of sows. The probe of plasma amino acerb concentrations may be used as an index of available plasma amino acid supply to mammary secretory organ at peak lactation accounting from 500 15-21 of lactation ( Trottier et al. , 1997 ) . The plasma indispensable amino acid concentrations in sows fed with dietetic protein lack, whereas most of plasma incidental amino acerb concentrations were invariably remained. The incidental amino acids can be mobilized from the organic structure protein, peculiarly skeletal musculus ( Jones and Stahly, 1999 ) . Further supported by Guan et Al. ( 2004a ) , they found that the arterial plasma indispensable amino acerb concentrations were lower in sows fed with deficient protein diet ( 7.8 % CP ) compared with those fed with normal protein diet ( 18.2 % CP ) .
In relation to the significantly lessening of plasma concentrations of some indispensable amino acids in sows fed with deficient protein diet, the quantitative messenger RNA and protein looks of some impersonal amino acid transporters were besides affected by dietetic protein inadequacy. In this survey, the quantitative messenger RNA looks of ASCT1 and LAT2 as the same look of LAT2 protein were down regulated significantly ( p & lt ; 0.05 ) in response to dietetic protein lack meanwhile the quantitative messenger RNA looks of ATA2 as the same look of ASCT1 and ATA2 protein did non differ between two experimental groups ( P & gt ; 0.05 ) . The possible account might be caused of amino acid substrates of LAT2 ( System L ) such as branched-chain amino acids were significantly decreased in plasma of sows fed with deficient protein diet. Consequently, substrate handiness of sow ‘s mammary secretory organ was decreased, which so affect on the decreasing in gene-protein look of this amino acid transporter. In contrast to LAT2 gene-protein look, ATA2/SNAT2 of system A was non change in response to dietetic protein lack when compared to normal protein diet group. This phenomenon was likely to happen in order to counterbalance for amino acid by modulating through adequate capacity of this transporter as supported by plasma amino acerb concentrations of most impersonal amino acid substrates such as methionine, glycine, proline were non significantly decreased in plasma of breastfeeding sows fed with deficient protein diet.
The consequence can be concluded that dietetic protein inadequacy had the effects on damage of sow mammary secretory organ conformation during peak lactation, lessening in plasma concentrations of indispensable amino acids, and down modulating gene-protein look of some impersonal amino acid transporters. Consequently, these effects are an of import cause to retard growing public presentation of piggies such as organic structure weight at weaned twenty-four hours. To the best of our cognition, it is utile for farther surveies on substrate dietetic amino acid or protein supplementation to optimise aminic acerb composing in sow ‘s milk. In add-on, the consequence of dietetic protein lack on look of other cistron and protein amino acid transporters in breastfeeding porcine mammary cells remains to be investigated.
This work was supported by the Ratchadaphiseksomphot Endowment Fund, the Research and development centre for farm animal production engineering, and the alumnus thesis grant, Graduate School, and Faculty of Veterinary Science, Chulalongkorn University.