Effects of dietary supplementation with L-arginine on the endogenous losses of amino acids in growing pigs exposed to heat stress.

Exposing pigs to heat stress (HS) provokes higher death of intestinal cells, resulting in elevated endogenous intestinal losses (EIL) of amino acids (AA) and damage to intestinal epithelia. Arginine (Arg) is precursor for the synthesis of polyamines, which are involved in proliferation of intestinal cells and restoration of the intestinal epithelia. Thus the effect of adding L-Arg to diets for HS pigs on the EIL of AA was analyzed. Twelve pigs (23.1 ± 1.1 kg body weight) implanted with T-type cannulas at the end of ileum were individually housed and allowed 15-days for surgery recovery under thermoneutral (TN) conditions (22 ± 2 °C). Following, the pigs were randomly assigned to one of three treatments: TN pigs fed a semi-purified, corn starch-3% casein basal diet (TN-B); HS pigs with the basal diet (HS-B); HS pigs consuming the basal diet supplemented with 0.20% L-Arg (HS-Arg). The experiment consisted of two 9-day periods; each period included 7-days of adaptation to their respective diet, followed by a 2-day ileal digesta collection period. Digesta was collected during 12 consecutive hours each day. The pigs were fed twice a-day. Ambient temperature (AT) inside the TN and HS rooms ranged from 18.6 to 27.6 °C and from 29.5 to 40.7 °C, respectively. Body temperature followed a pattern similar to that of AT. The daily EIL of indispensable AA increased (P < 0.01) in the HS-B pigs compared to both the TN-B and the HS-Arg pigs, however, there was no EIL difference between the TN-B and the HS-Arg pigs (P > 0.05). Likewise, with the exception of serine, daily losses of endogenous dispensable AA in the HS-B pigs were higher (P < 0.01) in comparison with those of TN-B and HS-Arg pigs. In summary, HS exposure compared to TN conditions increases the loss of endogenous AA, but dietary supplementation with L-Arg helped to counteract the negative HS effect.

Effect of dl-methionine supplementation above requirement on performance; intestinal morphology, antioxidant activity, and gene expression; and serum concentration of amino acids in heat stressed pigs.

The intestinal morphology and function can be compromised in pigs exposed to heat stress (HS), partly due to increased production of reactive-oxygen species. Because methionine (Met) functions as intracellular antioxidant, the requirement of Met may be increased in HS-pigs. The effect of dietary supplementation with dl-Met above requirement on performance, small intestine morphology, antioxidant enzymes activity, amino acid transporters expression, and serum concentration (SC) of free AA in HS-pigs was evaluated. A basal wheat-soybean meal diet was formulated to meet 100% Met requirement with the other indispensable AA exceeding at least 20% their requirement. Sixty individually housed pigs (23.0 ± 2.4 kg BW, 12 pigs per treatment) were randomly assigned to five treatments: TN100, thermal-neutral (22.7 °C) housed pigs fed the basal diet; HS100, HS120, HS140, HS160; HS-pigs (29.6 °C to 39.4 °C) fed the basal diet supplemented with dl-Met to contain 0%, 20%, 40%, and 60% dl-Met above the requirement, respectively. Pigs had free access to feed and water during the 21-d trial. Blood samples were collected on day 18 to analyze the absorptive AA-SC. The effect of ambient temperature (HS100 vs. TN100), as well as the linear and quadratic effects of increasing Met levels in the diets for HS-pigs were analyzed. The HS100 pigs gained less weight than TN100 and HS120 pigs (P < 0.01); gain:feed was also higher in HS120 pigs than in HS100 pigs (P ≤ 0.05). Feed intake of TN100 pigs was higher than that of HS-pigs fed the dl-Met supplemented diets (P < 0.05). Villi height reduced in pigs HS, but Met supplementation quadratically increased it (P < 0.05). Superoxide dismutase and catalase activities, reduced glutathione concentration, and relative expression of B0AT2 in ileum decreased (P < 0.05), but glutathione peroxidase activity increased in HS-pigs. dl-Met supplementation linearly affected catalase and glutathione peroxidase activities, as well as the relative expression of b0,+AT in jejunum (P < 0.05) of HS-pigs. The SC of Ile, Leu, Lys, Phe, and Val were higher in HS100 pigs than in TN100 pigs (P < 0.05). Graded levels of supplemental dl-Met in diets for HS-pigs linearly decreased SC of Ile, Leu, and Val (P < 0.05), tended to decrease His, Lys, and Thr (P < 0.10), and increased Met (P < 0.01). In conclusion, HS had negative effect on weight gain and intestinal morpho-physiology; however, it was ameliorated by adding 20% Met above the requirement in diets for growing pigs.

The exposure of pigs to ambient temperature above their comfort zone affects several functions of the small intestine, especially those related with digestion of feed and absorption of nutrients, which in turn reduces the availability of nutrients for growth. Amino acids such as methionine are involved in multiple functions of intestinal cells. Thus, methionine supplementation may help pigs to overcome the negative impact of their exposure to high ambient temperature. Indeed, methionine supplementation to the diet increased growth rate and feed efficiency of pigs housed under heat stress, which was presumably associated with an improvement in the utilization of the absorbed amino acids.

Syndemic Conditions and Their Association with HIV/STI Sexual Risk Behaviors Among Transgender Women and Cisgender Men Sex Workers in Catalonia: The SexCohort Project.

BACKGROUND: Transgender women and cisgender men sex workers are vulnerable to HIV and sexually transmitted infections (STIs). This study aimed to explore in depth the prevalence of syndemic conditions and their association with the sexual risk behaviors for HIV/STI acquisition in cis men and trans women sex workers in Barcelona (Catalonia, Spain). METHOD: We conducted a study between 2014 and 2018 to determine whether syndemic conditions (frequent alcohol consumption and polydrug use (> 2) during sex with clients; experience of violence; and lack of healthcare access) are associated with HIV/STI sexual risk behaviors. A "syndemic index" was calculated based on the cumulative number of syndemic conditions (0 to 4). RESULTS: In the last year (2018), 78.8% of cisgender men and 68.1% of transgender women reported at least one syndemic condition. The most prevalent syndemic factor in both cisgender men and transgender women was violence (38.8% and 43.6% respectively). In multivariable analysis, an association was found between condomless anal sex and violence (aOR = 1.81), and frequent alcohol consumption and violence with reporting > 10 clients/week (aOR = 2.73 and 1.88, respectively). The higher the number of syndemic factors, the greater probability of having > 10 clients/week and reporting condomless anal sex with clients. CONCLUSION: Psychosocial conditions have a syndemic effect on risky sexual behaviors highlighting the need for a more holistic approach to HIV/STI prevention targeting these populations.

Apparent and standardised ileal amino acid digestibilities in heat-stressed pigs fed wheat-soybean meal diets supplemented with l-arginine and dl-methionine.

Heat stress (HS) exposure may damage the small intestine epithelia of pigs affecting the digestibility and absorption of amino acids (AA). Arg and Met can enhance antioxidant and intestinal cell proliferation activity, thus supplementing them in diets might alleviate epithelial damage and correct the reduced AA digestibility. The effect of adding extra l-Arg and dl-Met to diets on the apparent (AID) and standardised ileal digestibility (SID) of AA was analysed in a 10-day experiment conducted with 10 ileal-cannulated HS pigs (25.3 ± 2.4 kg body weight). The pigs were divided into two treatments: Control, wheat-soybean meal diet supplemented with l-Lys, l-Thr, dl-Met and l-Trp; and control diet plus 0.20% l-Arg and 0.20% dl-Met (Arg + Met). Following an 8-day diet adaptation period, ileal digesta was continuously collected for 12 h, starting at 0700, on Days 9 and 10. All pigs were daily exposed to 29.6-36.1°C; ileal temperature was continuously monitored at 15-min intervals. Feed was provided twice a day. The ileal temperature ranged from 40.3 to 41.5°C. Daily ileal outflow of His, Ile, Leu, Phe, Thr, Ser and Tyr decreased (p < 0.05), and that of Arg, Val and Pro tended to decrease (p < 0.10) in the Arg + Met pigs. The AID of Arg, His, Met, Thr and Tyr, and the SID of His, Met, and Thr increased in pigs fed the Arg + Met diet (p < 0.05). Thr and Val had the lowest AID values whereas Arg, Met, and Lys had the highest values. Arg (r = 0.64) and Met (r = 0.84) intake were highly correlated with their AID values; Met intake was highly correlated with its SID value (r = 0.72). Valine and Arg had the lowest whereas Arg had the highest SID values. In conclusion, supplementing l-Arg and dl-Met above the requirement decreases the ileal outflow of several AA, and increases the AID and SID of some essential AA in HS pigs.

Effect of arginine supplementation on the morphology and function of intestinal epithelia and serum concentrations of amino acids in pigs exposed to heat stress.

The exposure of pigs to heat stress (HS) appears to damage their intestinal epithelia, affecting the absorption of amino acids (AAs). Arg is involved in the restoration of intestinal epithelial cells but HS reduces Arg intake. The effect of dietary supplementation with Arg on the morphology of intestinal epithelia, AA transporter gene expression, and serum concentration (SC) of free AAs in HS pigs was analyzed. Twenty pigs (25.3 ± 2.4 kg body weight) were randomly assigned to two dietary treatments: Control (0.81% Arg), wheat-soybean meal diet supplemented with l-Lys, l-Thr, dl-Met, and l-Trp, and the experimental diet where 0.16% free l-Arg was supplemented to a similar Control diet (+Arg). All pigs were individually housed and exposed to HS, fed ad libitum with full access to water. The ambient temperature (AT), recorded at 15-min intervals during the 21-d trial, ranged on average from 29.6 to 39.4 °C within the same day. Blood samples were collected on day 18 at 1600 hours (AT peak); serum was separated by centrifugation. At the end of the trial, five pigs per treatment were sacrificed to collect samples of mucosa scratched from each small intestine segment. The expression of AA transporters in intestinal mucosa and the SC of AAs were analyzed. Villi height (VH) was higher (P < 0.01) in the duodenum, jejunum, and ileum but the crypt depth did not differ between the Control and the +Arg pigs. Supplementation of l-Arg increased the mRNA coding for the synthesis of the cationic AA transporter b0,+ (P < 0.01) and the neutral AA transporter B0 (P < 0.05) in the duodenum by approximately 5-fold and 3-fold, respectively, but no effect on mRNA abundance was observed in the jejunum and ileum. The supplementation of l-Arg increased serum Arg, His, Met, Thr, Trp, and urea (P < 0.05) and also tended to increase Val (P < 0.10) but did not affect Ile, Lys, Leu, and Phe. These results indicate that supplementing 0.16% l-Arg to the Control diet may help to improve the function of the small intestine epithelium, by increasing the VH, the abundance of AA transporters, and the SC of most indispensable AAs in pigs exposed to HS conditions. However, the lack of effect of supplemental Arg on both Lys SC and weight gain of pigs suggests that increasing the Lys content in the +Arg diet might be needed to improve the performance of HS pigs.

Dietary protein-bound or free amino acids differently affect intestinal morphology, gene expression of amino acid transporters, and serum amino acids of pigs exposed to heat stress.

Pigs exposed to heat stress (HS) increase body temperature in which can damage the intestinal epithelia and affect the absorption and availability of amino acids (AA). Protein digestion and metabolism further increase body temperature. An experiment was conducted with six pairs of pigs (of 47.3 ± 1.3 kg initial body weight) exposed to natural HS to assess the effect of substituting dietary protein-bound AA by free AA on morphology and gene expression of intestinal epithelial and serum concentration (SC) of free AA. Treatments were: high protein, 21.9% crude protein (CP) diet (HShp) and low protein, 13.5% CP diet supplemented with crystalline Lys, Thr, Met, Trp, His, Ile, Leu, Phe, and Val (HSaa). The HShp diet met or exceeded all AA requirements. The HSaa diet was formulated on the basis of ideal protein. Pigs were fed the same amount at 0700 and 1900 hours during the 21-d study. Blood samples were collected at 1700 hours (2.0 h before the evening meal), 2030 hours, and 2130 hours (1.5 and 2.5 h after the evening meal). At the end, all pigs were sacrificed to collect intestinal mucosa and a 5-cm section from each segment of the small intestine from each pig. Villi measures, expression of AA transporters (y+L and B0) in mucosa, and SC of AA were analyzed. Ambient temperature fluctuated daily from 24.5 to 42.6 °C. Weight gain and G.F were not affected by dietary treatment. Villi height tended to be larger (P ≤ 0.10) and the villi height:crypt depth ratio was higher in duodenum and jejunum of pigs fed the HSaa diet (P < 0.05). Gene expression of transporter y+L in jejunum tended to be lower (P < 0.10) and transporter B0 in the ileum was lower (P < 0.05) in HSaa pigs. Preprandial (1700 hours) SC of Arg, His, Ile, Leu, Thr, Trp, and Val was higher (P < 0.05), and Phe tended to be higher (P < 0.10) in HShp pigs. At 2030 hours (1.5 h postprandial), serum Lys, Met, and Thr were higher in the HSaa pigs (P < 0.05). At 2130 hours (2.5 h), Arg, His, Ile, Phe, and Trp were lower (P < 0.05); Met was higher (P < 0.05); and Lys tended to be higher (P < 0.10) in HSaa pigs. In conclusion, feeding HS pigs with low protein diets supplemented with free AA reduces the damage of the intestinal epithelia and seems to improve its absorption capacity, in comparison with HS pigs fed diets containing solely protein-bound AA. This information is useful to formulate diets that correct the reduced AA consumption associated with the decreased voluntary feed intake of pigs under HS.

Extra dietary protein-bound or free amino acids differently affect the serum concentrations of free amino acids in heat-stressed pigs1.

Pigs exposed to heat stress (HS) reduce feed intake and consequently the consumption of AA. Adding extra protein-bound or free AA to the diet may correct the reduced AA intake of HS pigs. However, extra protein-bound AA may further increase the body heat load, whereas extra free AA does not affect the heat load of HS pigs. Two experiments were conducted. In Exp. 1, the performance depression because of HS, compared with thermal neutrality, was determined with 30 pigs (31.1 ± 1.2 kg BW) fed diets with AA only as protein or as a mix of protein and free AA. Heat stress pigs consumed 18 to 25% less Lys and Thr than thermal neutral. In Exp. 2, the effect of extra dietary protein-bound or free AA on performance and serum concentration of AA in 25 HS pigs (33.6 ± 0.65 kg BW) was evaluated. Treatments were as follows: CON, wheat-soybean meal-free Lys-Thr-Met diet; xP diet, 26% more protein than the CON diet; xAA diet, 24% or more of each AA than the recommended level. Pigs were fed ad libitum. Blood samples were collected between 1600 and 1700 h, when pigs were exposed to the highest ambient temperature (around 41.3 °C). Body temperature ranged daily from 39.9 to 41.1 °C. The performance data were reported already. Pigs fed the xP diet consumed more of all indispensable AA and dispensable AA than the CON pigs (P < 0.05), and more Arg, Ile, Asp, Glu, Gly, and Ser (P < 0.05) than the xAA pigs. Except for Arg, xAA pigs consumed more indispensable AA than the CON pigs (P < 0.05). Serum Arg, His, Lys, Phe, Thr, Trp, and Val, was higher (P < 0.05) in xP than in CON pigs. Except for Ile serum, indispensable AA were higher in xAA than in CON pigs (P < 0.05). Serum Ile, Leu, Thr, and Val were higher (P < 0.05), and Met tended to be higher (P < 0.10) in xAA than in xP pigs. The difference of Ile, Leu, Met, Thr, and Val between the CON and the xAA pigs was larger than that between the CON and the xP pigs (P < 0.05). Serum Asn and Tyr were higher, and Cys and Glu were lower (P < 0.05) in xP than in CON pigs. Serum Cys tended to be lower in xAA than in CON pigs (P < 0.10). Asp and Glu were higher (P < 0.05) in xAA pigs than in xP pigs. In conclusion, these serum AA results combined with the reported performance data indicate that extra free AA in diets for HS pigs may help to correct the reduced AA availability and performance of HS pigs, although higher levels of specific AA such as Ile and Met might be needed.

Serum concentrations of free amino acids in growing pigs exposed to diurnal heat stress fluctuations.

In areas where the ambient temperature (AT) is above the thermo neutral (TN) zone of pigs, significant changes within a 24-h period occur, differently affecting the availability of amino acids (AA) within the same day. An experiment was conducted to analyze the serum concentrations (SC) of free AA in pigs exposed to diurnal variations in AT. Six pigs (27.1 ±1.3kg body weight) implanted with a thermometer to register the body temperature (BT) at 15-min intervals were used. Blood samples were collected on the last 3 d of the 14-d study, at 0700h (lowest AT), 1200h (mild HS), and 1600h (severe HS). The pigs received 1.2kg/d of an AA-supplemented, wheat-soybean meal diet, in two equal meals (0700 and 1900h). The AT and BT, recorded at 0700, 1200, and 1600h was: 30.6, 38.6, 41.1°C, and 38.2, 39.5, 40.3°C, respectively. The BT was significantly correlated (P < 0.001) with the AT. The SC (µM/mL) of Ile, Lys, Met, Val, Ala, Asn, and Pro were higher (P ≤ 0.01); Arg, Phe, Glu, and Tyr tended to be higher (P ≤ 0.10); but Cys was lower (P < 0.05) at 1200h than at 0700h. Lys was higher, Cys and Tyr were lower (P < 0.05), and Ile and Val tended to be higher (P ≤ 0.10) at 1600h than at 0700h. Serum Arg, Ile, Phe, Ala, Asn, Gln, Pro, Ser, and Tyr were lower (P < 0.05), and Leu and Val tended to be lower at 1600h than at 1200h. These data demonstrate that AT directly alters the BT of pigs, and that diurnal variations in AT differently affect their SC and availability of AA for growth.

Effects of antiproteolytic agents on corneal epithelial viability and matrix metalloproteinase-2 and metalloproteinase-9 activity in alkali-burned corneas of rats.

OBJECTIVE: To evaluate the effects of agents on corneal re-epithelization and metalloproteinase-2 and metalloproteinase-9 (MMP-2 and MMP-9) activities in corneas of rats submitted to ulceration. ANIMALS STUDIED: Ninety eight healthy rats. PROCEDURES: Corneal ulcers were created using 1N NaOH in their left eye. Eyes were treated every 6 h with 1% ethylenediaminetetraacetic acid (EDTA), 3% chondroitin sulfate (CS), 10% N-acetylcysteine NAc and saline (S) at 6-h intervals. Corneas were stained with fluorescein and photographed at the same time points. Following 20 h and 40-42 h of corneal injury, corneas were processed for scanning electron microscopy (SEM) to quantify microvilli density, and MMPs activities were analyzed using zymography. RESULTS: The percentage of wound area and the time in hours for corneal re-epithelization did not differ significantly among treatment groups (P > 0.05). In first and the second moments, latent MMP-2 was significantly elevated in the eyes treated with NAC and CS (P < 0.001). Active MMP-2 did not change significantly among treatment groups in the first moment (P > 0.05); significantly higher activity was observed in the second moment in the eyes treated with CS (P <0.001). In the second moment, latent MMP-9 decreased significantly in eyes treated with EDTA and S (P < 0.01). Microvilli corneal density did not change significantly between healthy subjects and treatment groups (P > 0.05). CONCLUSION: Any of the studied substances did not accelerate corneal re-epithelization and did not add protection to the corneal microvilli. Significant higher levels of active form of MMP-2 in 3% chondroitin sulfate-treated group may indicate that the agent acts as substrate for such enzyme. At the end of the experiment, 1% EDTA was the most efficient agent to inhibit significantly the latent form of MMP-9. However, any of the substances add benefit over saline on reducing the proteolytic activity in the cornea of rats after alkali injury.

Cloning of the bovine beta-carotene-15,15'-oxygenase and expression in gonadal tissues.

Beta-carotene-15,15'-oxygenase (betaCO), found mainly in intestinal mucosa and liver, is the enzyme responsible for cleaving beta-carotene into retinal, which can be used or stored at these sites or carried by the bloodstream to different target cells within the body. We isolated the cDNA for bovine betaCO and demonstrated its expression in gonadal tissues. A cDNA of 2130 base pairs (bp) was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), using degenerate oligonucleotides; the deduced protein shared an identity of 75% with its homologues from other mammalian species. In order to evaluate the expression of this enzyme, we performed RT-PCR and in situ hybridizations in the ovary and testis of bovines. RT-PCR showed the expression of betaCO in testis, ovary, and cultured granulosa cells. In situ hybridization of complete ovary and testis revealed expression in granulosa cells and the corpus luteum in the ovary and in germinal and interstitial cells in the testis. These results suggest that beta-carotene could act as a local source of retinoids, which have been shown to be important during proliferation, differentiation, and maturation of both female and male germinal cells.