Effects of methionine supplementing on intestine, liver and uterus morphology, and on positivity and expression of calbindin-D28k and TRPV6 calcium carriers in laying quails in thermoneutral conditions and under thermal stress

The aim of this study was to provide support for the performance, localization and expression of the epithelial calcium transporter channels, calbindin-D28k (Calb) and TRPV6, and of the morphology of the digestive and reproductive system of laying quails under heat stress, and with methionine supplementation. Therefore, the present study characterized the positivity (immunohistochemistry) and expression (real-time PCR) of calcium channels (Calb and TRPV6) in the kidneys, intestine and uterus of 504 laying quails that were submitted to different methionine supplementation (100, 110 and 120%) and temperatures (20, 24, 28 and 32°C). The animals under thermal stress had lower villus height, villus:crypt ratio, and goblet cell index in the duodenum and jejunum, fewer secondary and tertiary uterine folds, smaller hepatic steatosis, and increased number of distal convoluted renal tubules (CT) positive to Calb (protein), and increased positivity in proximal CTs. The deleterious effects of heat stress were minimized with methionine supplementation for the following variables: duodenal crypts, number of goblet cells of the jejunum, number of uterine folds, decreased Calb positivity in intestines and kidney, increased positivity of Calb in the uterus and increased TRPV6 gene expression in the kidney. Calcium transporters were altered due to less need for calcium absorption and reabsorption due to more calcium available with the supplementation, increasing egg production and quality. Methionine supplementation further increased intestinal villus absorption area and height, increased steatosis, decreased Calb positivity in the intestine and kidney, increased uterine positivity and Calb expression, and increased TRPV6 expression in the uterus under thermoneutrality. This is the first study that describes the gene and protein expression of calcium transporters in the intestine, kidney and uterus of laying quails, and concludes that the use of methionine supplementation is justifiable in order to partially reverse the deleterious effects of thermal stress on the production.


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Quail laying farming has been growing in Brazil, mainly in the northeast region [1]. The raising of 49 quails is a very profitable activity and with broad perspectives, which induces the development of 50 research aiming at better production, perfecting techniques and alternatives to reach quality standards and 51 expansion throughout the territory. 52 In tropical climates, such as those found in most of Brazil, laying birds suffer a reduction in their 53 zootechnical indexes as well as an increase in mortality as a result of thermal stress, leading to productive 54 and economic losses in production [2]. This fact is mainly observed in laying quail farming, where the 55 first restrictive factor for eggshell formation is calcium, and calcium is negatively influenced by 56 temperature increase [3]. 57 Calcium comes mainly from intestinal absorption and bone resorption, which is mobilized from 58 the blood to the uterus very quickly [4,5]. Nascimento et al. [6] stated that 70% of the production cost is 59 based on food, and for this reason there is a need to develop balanced diets according to the needs of the 60 birds, enabling them to use the diet with maximum efficiency. 61 For birds subjected to heat stress, it is necessary to supplement the diets with glycogenic amino 62 acids, such as methionine, cystine and others [7]. Under such conditions of thermal stress, physiological 63 and behavioral changes occur in quails, which severely affect feed intake and cause structural changes in 64 the intestinal epithelium, reducing nutrient digestibility and absorption [8].    VH. Supplementation with 120% methionine even led to decreased VHs at the temperature of 32ºC. Such 214 event, without contextualize the crypt depth (CD), leads to believe in the reduction of intestinal area and 215 consequent lower contact with food, decrease of nutrient absorption and production.

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In the jejunum, the effects were similar; supplementation with 120% methionine promoted VH 217 increase at 20°C, maintained VH at 24°C, and also reduced VH at 28 and 32°C. Thus, methionine variable to be analyzed. Thus, the increase in CD can also predict an increase in VH when a trophic agent 241 is presented, because it is exactly in this region that the cells that will migrate are produced to ensure the 242 maintenance and/or increase of VH.

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The histomorphometric analysis of CD in the duodenum showed that at thermoneutral 244 temperatures for quails, the CD was lower, that is, the temperature of thermal stress (32°C) and at the 245 lowest temperature (20°C), close to the thermal stress by low temperature, there is a higher need for cell 246 turnover. However, 120% methionine supplementation led to a decrease in CD at higher temperatures (28 247 e 32°C). It can be inferred that at high temperatures, methionine supplementation reduced the deleterious 248 effects of stress, reducing the need for cell proliferation in this region. The same result found for 249 duodenum in relation to methionine supplementation was found in the jejunum. Regarding temperature, 250 CD was different only at 20°C; it was lower at this temperature. The results show that methionine 251 supplementation at high temperatures leads to a decrease in CD, which leads to a greater villus:crypt 252 ratio, a variable used as an important marker of intestinal health, as it reveals a larger area of contact with 253 food, consequently increased absorption without the need for too much energy expenditure on crypt 254 turnover.
Crypt epithelium hyperplasia found at 32°C must have been induced to reestablish villus height, 256 and is considered a compensatory mechanism [29], since thermal stress by heat in broilers for four 257 consecutive days causes negative alterations in duodenum and jejunum crypts, including reduction in 258 villus height, in villus/crypt ratio, in absorption area, and increased crypt depth.

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The villus:crypt ratio (VCR) is related to the intestinal health of the animal, the higher the ratio,   (Table 3), which implies in a smaller area for the production of calcium carbonate, the main eggshell 299 compound [3], negatively influencing the egg production of the animals. High temperatures also 300 decreased eggshell production and thickness (Table 4). The highest indexes of uterine folds were found in 301 treatments at 20 and 24ºC. up to 5% probability.

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These results corroborate the egg production results (Table 4); the highest productive performance 311 was found at 24ºC, and the lowest performance was at 32°C. supplementation; however, these results did not interfere with egg production ( steatosis. This variable is important since the increase in hepatic steatosis is related to estrogen 324 production, that is, the higher the steatosis, the higher the estrogen production and the higher the egg production [14]. Thus, methionine supplementation (120%) at 24ºC would not only increase hepatic 326 steatosis but also egg production by these quails, which occurred in the present study, since the increase 327 in hepatic steatosis was reflected in a significant increase in production ( However, only at 24ºC, 110% methionine supplementation increased hepatic glycogen stores.

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These results demonstrate that at thermoneutrality, 110% supplementation maximizes energy storage in 337 the form of glycogen in the liver. Such a surplus can be transferred to production, in this case in egg 338 production. Thus, in heat stress methionine supplementation was not efficient.

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In modern strains of laying hens, which can be extrapolated to laying quails, the equivalent of 341 10% of total body calcium is transferred daily for deposition as eggshells [39-41]; the major sources of 342 calcium are through absorption from the diet at the intestinal level, renal resorption, and bone storage.

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Since calbindin-k28D is the carrier responsible for the absorption of calcium from the digestive system, it  was not positive (Fig 1). Positivity was more intense in the basal and more apical portion of the epithelium, since the middle portion was an area that had many enterocyte nuclei and the present marking  The decrease in positivity could be explained by the increased availability of calcium and 371 consequently less need for absorption, and increased eggshell quality, which actually occurred in the 372 present study. Although the performance model of calbindin-D28k has already been described in layers, 373 this is the first study in quails. In a study with methionine supplementation in diets with lower protein 374 levels in Thailand, a country with thermal similarities to that of northeastern Brazil, there was an increase 375 in laying production rates, including increased eggshell thickness [14]. In the aforementioned study, the increase in methionine must also have minimized the deleterious effect of heat stress and increased 377 calcium availability to improve production rates, as occurred in the present study. It can be imagined that 378 in this study the positivity of calbindin-D28k must also have decreased.  In the present study, at temperatures considered to be of higher thermal comfort for the quails (24 394 and 28ºC), these animals presented lower positivity of the cellular calcium transport in their duodenal 395 epithelia, exactly because they were in better thermal comfort, and did not need a higher absorption of 396 calcium (Fig 2). Literature provides studies [57] that show that the higher the ambient temperature and 397 thermal stress, the greater the need to supplement dietary calcium, as the animals will need more calcium gross values; however, these results were not significant (Fig 5), perhaps due to the small sample size 402 and/or the large standard deviation. Another similar fact was that methionine supplementation also decreased gene expression of this gene (Fig 6). Therefore, it is assumed that methionine supplementation 404 leaves more calcium available, which makes the need for lower intestinal calcium absorption necessary.

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The greatest positivity was at the temperature of thermal stress (32°C), when it is thought to have 406 less calcium available, which increases the need for calcium. Thus there were more calcium transporters 407 (calbindin-k28D) (higher positivity) to provide greater absorption to maintain the production. The 408 positivity at the temperature of 20°C is intermediate, because for quails, this temperature is already 409 relatively low, thus, the animal already feels some result of thermal stress, in this case for low 410 temperature, changing its physiology, and also needing more calcium. This explains the slightly higher 411 positivity at 20°C than that found in the 24 and 28°C treatments.

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This is the first study to cite calbindin-d28k protein expression in the intestine of laying quails, 413 and it is also the first to demonstrate the influence of high temperature heat stress on this calcium 414 transport. large renal blood vessels (Fig 2). The renal corpuscle, as well as the glomerulus (capillaries), were not 422 positive for anti-calbindin-d28k. cortex. This feature can be explained by the fact that these areas have blood with a higher amount of 429 calcium, which has not yet been reabsorbed.

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The positivity is lower at 24 and 28ºC compared to at 20 and 32ºC. In the treatment at 32ºC, the 431 amount of positive DCT increased, always in greater numbers near the great renal veins (Fig 2). At 28 432 and 32ºC, DCT were more positive when compared to previous temperatures.  and 28ºC (lower positivity). In animals with some degree of thermal stress, such as at 32ºC (high 447 temperature) and at 20ºC, theoretically because it is a temperature below the thermal conformation for the 448 species, they would have greater need to reabsorb more calcium (higher positivity).

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Although it is well known that thermal stress by high temperatures decreases the presence of 450 calbindin-D28k in the ileum, cecum, colon and uterus of birds, causing deterioration of eggshell quality

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[56], there was no information in literature on the influence of this calcium transport at renal level for thermal stress by high or low temperatures, as seems to occur at a temperature of 20°C. Thus, this is the first report on the influence of heat stress on such a transport in the kidney, which, like the intestine, has 454 the opposite effect to that found for other organs in other experiments with layers [56].

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In the case of high temperature heat stress treatment, more DCT were positive for anti-calbindin-456 d28k, which shows that under such a situation not even increased positivity was enough to reabsorb the 457 calcium needed for the production of these birds; in addition to the increase in the expression of this 458 transport, the increase in the number of DCT that expressed such transport was needed (Fig 3). Positivity to anti-calbindin-D28K was high in the uterine glands, since these are the sites of 474 calcium carbonate production and secretion, which is produced and released for eggshell production in 475 the uterus, and is influenced by increased circulating estrogen [46], and modulates eggshell production 476 and quality. Uterine gland cells transport calcium from their basal portion to the apical surface during 477 calcium carbonate production, the more calcium carbonate, the faster the egg production and/or better eggshell quality. The epithelium (ciliated pseudostratified) is not positive for anti-calbindin-D28k except 479 for a thin layer on the apical portion of this epithelium.

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The positivity pattern was higher at 24 and 28°C, lower in the treatment in which the animals were 481 submitted to 32°C, and intermediate at 20°C (Fig 4). Methionine supplementation by 120% increased  temperatures. Such an increase in this gene increases calcium reabsorption, making more of the mineral 536 available for egg production, specifically in the release by the uterus for eggshell production. by up to 5% error probability; **,* and ns Indicate, respectively, significant differences up to 1%, up to 544 5% and not significant by the F test.

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These results corroborate studies that already cited the gene expression of both genes (TRPV6 and  For uterine tissue, Calbindin-D28K and TRPV6 gene expression also occurred in all treatments, 561 and as for intestine, it was poor for TRPV6. Temperature increase and methionine supplementation did 562 not influence gene expression of calbindin-D28k or TRPV6. Thus, the uterine tissue is not altered under 563 these conditions either, thus not justifying the use of methionine supplementation.

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The results described and observed in this study show the gene expression of TRPV6 and .

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Through analysis of variance, it was possible to verify the interaction between temperature and 570 supplementation for both genes in the kidney (p≤0.01), supplementation for calbindin-D28k in the 571 intestine (p≤0.01) and temperature for TRPV6 in the uterus (Table 5). 575 *, **, and ns indicate, respectively, significant differences up to 5%, up to 1%, and not significant by the F test.

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In addition, the responses of calbindin-D28k and TRPV6 in the kidney, intestine and uterus of 577 Japanese quails supplemented with methionine + cystine (100% and 120%) at different temperatures showed a strong positive correlation (r = 0.90*) in the kidneys and moderately positive (r = 0.69 *) in the 579 intestines between Calb 28 and TRPV6 gene expressions (Fig 6), indicating that both calbindin-D28k and 580 TRPV6 (mRNA) act synergistically, modulating resorptive (kidney) and absorptive (intestine) capacity, 581 and subsequent calcium deposition by the uterus. The correlations between the other variables were weak 582 and not significant.