Dimethyl sulfone for the treatment of chronic heat stress in poultry

Abstract

The present invention relates to dimethyl sulfone for use in the treatment of heat stress in poultry by oral administration, wherein the dimethyl sulfone is administered to chronically heat stressed poultry.

Description

Technical Field

[0001] The present invention relates to dimethyl sulfone and compositions for use in the oral administration of treatment for heat stress in chronically heat stressed poultry. Background Art

[0002] Animals are divided into two categories: cold-blooded (heterothermic) and warm-blooded (homeothermic). Birds are homeothermic because they have the ability to maintain a stable internal body temperature independent of external influences. However, their thermoregulatory mechanisms are effective only in the thermoneutral zone. The general characteristics of thermoregulation in birds are similar to those of other homeothermic animals. However, in contrast to homeothermic species, birds do not possess sweat glands. Furthermore, birds use feathers and fat for insulation, which acts as a good insulator in cold weather but inhibits heat loss in hot weather. Therefore, birds have difficulty dissipating heat through their feathered areas. This makes them more susceptible to stress in high temperatures. Specifically, birds have air sacs that allow inhaled air, which is typically cooler than body temperature, to penetrate deep into their abdominal cavity, allowing heat to be removed from the body when the bird exhales. Furthermore, during hot weather, birds use a panting mechanism (also known as gular flutter) to evaporate water from their throats, thereby lowering their body temperature. Panting is extremely effective in cooling birds. Furthermore, as endotherms, birds can regulate their body temperature by utilizing heat generated within their bodies. Heat is generated within the bird's body as a result of metabolism and muscle activity. The amount of heat produced in a bird's body is influenced by enzyme, vitamin, and hormone concentrations; physical activity; oxygen consumption; ambient temperature; and circadian rhythms. To maintain body temperature and avoid overheating, excess heat is dissipated to the surrounding environment through cellular conduction and vascular convection. Failure to dissipate heat can lead to metabolic disorders and, in extreme cases, death.

[0003] Therefore, high ambient temperatures can be devastating for commercial animals. Combined with high humidity, they can have even more detrimental effects. Heat stress interferes with animal comfort and inhibits the production performance, reproductive performance, economic traits and welfare of poultry. Heat stress can be chronic or acute. Sudden and short periods of extremely high ambient temperature and humidity can lead to acute heat stress. Long periods of elevated ambient temperature and increased humidity lead to chronic heat stress. If birds have difficulty achieving a balance between body heat production and body heat loss, they are "heat stressed." Heat stress can occur in poultry of all ages and in all types of poultry. The temperature comfort zone of birds depends on their age. Older birds are more sensitive to high temperatures. For example, a temperature of 21°C is ideal for poultry in the rearing period. However, in principle, it can be summarized that birds feel comfortable at approximately 21 to 24°C (75°F) and function normally when raised to approximately 27°C (80°F). Above 27°C (up to approximately 30°C), feed consumption decreases, while water intake increases. Feed conversion ratio (FCR) and weight gain decrease in broiler birds, and egg production decreases in layer and breeder flocks. At 30 to 32°C (86 to 95°F), a significant decrease in egg production and eggshell quality is observed. In layer birds, FCR based on egg mass and FCR per dozen eggs increases with increasing ambient temperature. When temperatures exceed approximately 35 to 37°C (96 to 100°F), birds attempt to reduce body heat by vigorous throat fluttering; however, temperatures within this range result in a high mortality rate. In addition, noticeable depression, nervous behavior, and symptoms such as trembling, staggering, and convulsions may also be observed.

[0004] At a temperature of 35°C (95°F) and a relative humidity of 40%, birds can dissipate 80% of their total body heat through evaporative heat loss (pharyngeal flutter), but at a temperature of 35°C (95°F) and a relative humidity of 50%, heat loss is reduced to only 50%. At a temperature of 35°C and a relative humidity of 100%, heat can no longer be lost from the body, resulting in severe stress, shock, and a high mortality rate. Temperatures of approximately 38°C (101°F) are lethal; at these temperatures, the risk of death increases and emergency measures are required.

[0005] Feed consumption and feed conversion factor are affected by high temperatures. Specifically, weight gain, egg production, especially egg size, egg quality and hatchability, and fertility are reduced in breeder flocks exposed to heat stress.

[0006] The article "Dietary methylsulfonylmethane supplementation and oxidative stress in broiler chickens" (MS Abdul Rasheed et al., Poultry Science, vol. 99, no. 2, 22 January 2020, pages 914-925) discusses the effects of dimethyl sulfone on oxidative markers in chickens stressed with oxidizing oil. However, no significant changes in the results were shown in the article. Furthermore, the article does not disclose any reduction in heat stress in broiler chickens.

[0007] Commercial poultry farming is a highly profitable business; however, any type of heat stress significantly reduces profitability. Solutions for preventing heat stress require multifactorial strategies involving the rearing environment, genetics, thermal regulation, and feeding, as well as nutrition. Many different strategies have been devised to mitigate heat stress in poultry. Briefly, such strategies can be divided into three groups of mechanical techniques, specifically the housing environment and thermal regulation; nutritional strategies involving restricted feeding and watering strategies, the use of phytochemicals such as lycopene, anthocyanins, gamma-glutamylethylamide, regulation of dietary electrolyte balance, particularly potassium bicarbonate, potassium chloride, sodium bicarbonate, sodium chloride and ammonium chloride in water and / or feed, vitamin supplementation, particularly vitamins C and E, and supplementation with vinblastine, betaine and prebiotics / probiotics, reduction of crude protein content using feed-grade amino acids, an increase of 3 to 5% in fat in the feed at the expense of carbohydrates without changing metabolizable energy, an increase of about 5 to 10% in digestible amino acids compared to what is normally used; and other management, such as on-farm activities, specifically not interfering with bird transportation during peak heat period, de-beaking, dubbing, etc.; early thermal conditioning, such as inducing heat tolerance, and litter management, such as litter that should be kept dry and not too wet. However, none of these strategies have proven successful in treating heat stress in poultry. In particular, none of these strategies have proven successful in reducing mortality in poultry exposed to heat stress. Therefore, there remains a need for methods for successfully reducing mortality in poultry exposed to heat stress, particularly chronic heat stress. Summary of the Invention

[0008] It was discovered that such a need could be met by orally administering dimethyl sulfone to poultry exposed to chronic heat stress.

[0009] Therefore, an object of the present invention is dimethyl sulfone for use in the treatment of heat stress in poultry by oral administration, wherein the dimethyl sulfone is administered to chronically heat stressed poultry.

[0010] Poultry that received dimethyl sulfone (DMS) administration showed increased heat tolerance and therefore reduced mortality compared to poultry that did not receive DMS administration. Specifically, the administration of DMS to poultry not only caused reduced panting and therefore reduced water loss, but also caused a lower rectal temperature. Specifically, the administration of DMS to poultry supports poultry to better adapt their metabolism to heat stress. For example, DMS has been shown to promote antioxidant function and redox homeostasis in poultry. This results in lower oxidative stress or higher antioxidant capacity in heat-stressed poultry. At the same time, the administration of DMS to the poultry diet did not negatively affect the overall performance of the poultry nor the meat quality; specifically, no significant white streaks or wooden breasts were observed in the carcasses. DETAILED DESCRIPTION

[0011] The term "poultry" is used in the context of the present invention to refer to any type of domesticated bird that is kept in captivity for its utility. Examples of poultry are domesticated birds, including chickens, turkeys, geese, quail, and ducks, that are raised for meat or eggs. Preferably, the term poultry in the context of the present invention refers to chickens.

[0012] In the context of the present invention, the term "heat stress" is used to define exposure to elevated ambient temperatures. Heat stress can be chronic or acute. In the context of the present invention, the term "chronic heat stress" is used to define prolonged periods of elevated ambient temperatures. In contrast, in the context of the present invention, acute heat stress is understood to define sudden, short periods of extremely high ambient temperatures. In principle, dimethyl sulfone according to the present invention is not restricted to chronic or acute heat stress. In the context of the present invention, and particularly in the context of chronic heat stress, the term "elevated ambient temperature" is used to define temperatures above the comfort level of poultry. As mentioned above, the thermal comfort zone of birds depends on their age. Older birds are more sensitive to high temperatures. Typically, birds are comfortable at temperatures between approximately 21 and 24°C (70 and 75°F) and function normally up to approximately 27°C (80°F). However, above 27°C (up to approximately 30°C), feed consumption decreases, while water intake increases. Feed conversion ratio (FCR) and weight gain decrease in meat-type birds, and egg production decreases in laying and breeding flocks. At temperatures between 30 and 32°C (86 and 95°F), a significant decrease in egg production and eggshell quality is observed. In laying birds, the FCR based on egg mass and the FCR per dozen eggs increase with increasing ambient temperature. When temperatures exceed approximately 35 to 37°C (96 to 100°F), birds attempt to reduce body heat by vigorous throat fluttering; however, temperatures within this range result in a certain degree of mortality. Preferably, in the context of the present invention, a temperature exceeding 27°C, in particular a temperature of at least 30°C, is an elevated ambient temperature. In particular, a temperature of from 30 to 40°C is an elevated temperature.

[0013] In one embodiment of the dimethyl sulfone according to the present invention, the poultry is exposed to a temperature exceeding 27°C, preferably to a temperature of at least 30°C.

[0014] In the context of the present invention, the term "prolonged period" or "prolonged period of elevated ambient temperature" is used to define a period of at least 5 hours per day. Preferably, an extended period or a prolonged period of elevated ambient temperature refers to a period of 5 to 24 hours per day, in particular at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or even up to 24 hours per day.

[0015] In one embodiment of the dimethyl sulfone according to the present invention, the poultry are exposed to a temperature exceeding 27°C, preferably to a temperature of at least 30°C, for at least 5 hours per day.

[0016] Typically, the elevated temperatures to which the poultry are exposed follow a cyclical course: the temperature reaches a minimum at night, then steadily increases during the day until it reaches a maximum, and from this maximum, the temperature decreases again at night to the minimum. If this temperature course extends over several days or weeks, it is also referred to in the context of the present invention as chronic cyclical heat stress.

[0017] In a preferred embodiment of the dimethyl sulfone according to the present invention, the chronic heat stress is chronic periodic heat stress.

[0018] In many cases, especially in southern countries, particularly in the subtropics, tropics, and generally those countries near the equator, rising temperatures are accompanied by higher relative humidity. In the context of the present invention, the term "higher relative humidity" is used to mean an average relative humidity of at least 40%. Preferably, in the context of the present invention, the relative humidity is on average 40% to 70%, in particular on average 50% to 60%.

[0019] In another embodiment of the dimethyl sulfone according to the present invention, the poultry is exposed to an average relative humidity of at least 40%.

[0020] The life cycle of poultry in captivity can be divided into three stages: brooding (starter), growing (grower) and fattening (finisher) stages. For example, the entire life cycle of a chicken is 39 days, wherein the days (d) from d-0 to d-10 are called the brooding stage, the days from d-10 to d-21 are called the growing stage, and the days from d-21 to d-39 are called the fattening stage. In principle, the dimethyl sulfone according to the present invention administered to chronic heat stressed poultry is not limited to any specific stage or period in the life cycle of poultry. Therefore, dimethyl sulfone can be administered to poultry under chronic heat stress in or during any of the following stages, i.e., at any conceivable time point in or during the brooding, growing and / or fattening stages. Nevertheless, it is preferred that dimethyl sulfone be administered to poultry under chronic heat stress in or during the fattening stage. In the context of the present invention, the term "in" with respect to the stage is used to indicate a selected time point, such as hours, days or weeks, which do not necessarily have to be a continuous period. In contrast, the term "during" with respect to a phase is used in the context of the present invention to denote a continuous period of hours, days or weeks.

[0021] In a further embodiment of the dimethyl sulfone according to the invention, dimethyl sulfone is administered to the poultry in or during the brooding, growing and / or finishing phase.

[0022] Preferably, dimethyl sulfone according to the invention is administered to the poultry from the beginning of the brooding phase until slaughter, or from the beginning of the finishing phase until slaughter.

[0023] Nevertheless, it appears that birds supplemented with DMS in the preceding feeding period are better prepared, thereby resulting in improved feed conversion in the subsequent feeding period, to cope with the upcoming heat stress. Specifically, it appears that providing birds with supplemented DMS in the finishing period supports the birds in better coping with the upcoming heat stress in the subsequent fattening period.

[0024] In a preferred embodiment of the dimethyl sulfone according to the present invention, dimethyl sulfone is administered to the poultry during the fattening period.

[0025] Preferably, dimethyl sulfone according to the present invention is administered to poultry together with and / or in conjunction with a low-protein diet and / or a diet with a low amount of digestible lysine. Preferably, the diet still has an ideal amino acid profile in order to avoid or significantly reduce any negative impact on the weight gain of the poultry.

[0026] The dimethyl sulfone according to the present invention administered to chronically heat-stressed poultry can be part of any matrix, preferably a liquid and / or solid matrix. For example, the dimethyl sulfone can be administered to chronically heat-stressed poultry via their drinking water, their diet, or a combination of both. It has been found that a concentration of only 0.05% by weight of dimethyl sulfone, based on the total weight of the liquid and / or solid matrix, administered via the matrix, has a beneficial effect on chronically heat-stressed poultry.

[0027] Preferably, dimethyl sulfone is administered to the poultry in a concentration of at least 0.05% by weight, based on the total weight of the liquid and / or solid matrix.

[0028] It is further preferred that dimethyl sulfone is administered to the poultry in a concentration of 0.05 to 0.5 wt%, 0.05 to 0.4 wt% or 0.5 to 0.3 wt%, based on the total weight of the liquid and / or solid matrix.

[0029] Therefore, another object of the present invention is also a composition comprising dimethyl sulfone for use in the treatment of heat stress in poultry by oral administration, said composition comprising at least 0.05% by weight of dimethyl sulfone, based on the total weight of said composition, wherein said composition is administered to poultry suffering from chronic heat stress.

[0030] In one embodiment, the composition according to the invention comprises dimethyl sulfone in a concentration of 0.05 to 0.5% by weight, based on the total weight of the composition.

[0031] Preferably, the composition comprises dimethyl sulfone in a concentration of 0.05 to 0.4 wt.-% or 0.5 to 0.3 wt.-%, based on the total weight of the liquid and / or solid matrix.

[0032] Chronic heat stress is characterized by prolonged periods of elevated temperatures. As explained in detail above, in the context of the present invention, temperatures exceeding 27°C, preferably temperatures of at least 30°C, preferably temperatures from 30°C to 40°C are elevated temperatures. Furthermore, prolonged periods of elevated temperatures are understood to mean periods of at least 5 hours per day, preferably 5 to 24 hours per day, in particular at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or even up to 24 hours per day.

[0033] In a further embodiment of the composition according to the invention the poultry are exposed to a temperature above 27°C, preferably to a temperature of at least 30°C.

[0034] In one embodiment of the composition according to the invention, the poultry are exposed to a temperature above 27°C, preferably to a temperature of at least 30°C, for at least 5 hours per day.

[0035] In a preferred embodiment of the composition according to the invention, the heat stress is chronic periodic heat stress.

[0036] In another embodiment of the composition according to the invention, the poultry are exposed to a relative humidity of at least 40% on average. Preferably, in the context of the present invention, the relative humidity is from 40 to 70% on average, in particular from 50 to 60% on average.

[0037] In a further embodiment of the composition according to the invention, the composition is administered to poultry in or during the brooding, growing and / or fattening phase.

[0038] Preferably, the composition is a liquid and / or solid matrix.

[0039] When the composition according to the invention is a solid matrix, it is preferred that the composition is a diet for poultry.Preferably, the diet is a brooding, growing and / or fattening diet for poultry, in particular a fattening diet.

[0040] When the composition according to the invention is a diet for poultry, a low protein diet and / or a diet with a low amount of digestible lysine is also preferred. Preferably, the diet still contains an ideal amino acid profile in order to avoid or at least significantly reduce a negative impact on the weight gain of the poultry.

[0041] When the composition according to the invention is a liquid matrix, it is preferred that the composition is drinking water for poultry, the drinking water being supplemented with at least 0.05 wt. % of dimethyl sulfone, based on the total weight of the drinking water.

[0042] When the ambient temperature rises, birds will drink more water. This will also increase the amount of liquid excrement in the pen and therefore also increase the moisture in the litter. However, wet litter has poor quality and leads to poor food footpad health. Therefore, wet litter is considered to have a negative impact on the welfare of birds. It has been found that the administration of dimethyl sulfone according to the present invention and / or the administration of a composition comprising dimethyl sulfone according to the present invention helps to reduce the amount of liquid excrement in the pen and therefore reduce the overall moisture in the litter.

[0043] Therefore, a further object of the present invention is also: dimethyl sulfone and / or a composition comprising dimethyl sulfone for use in reducing moisture in litter, wherein the dimethyl sulfone according to the invention and / or a composition comprising dimethyl sulfone according to the invention is administered to poultry suffering from chronic heat stress - preferably, dimethyl sulfone according to the invention and / or a composition comprising dimethyl sulfone according to the invention for use in reducing moisture in litter, wherein the dimethyl sulfone according to the invention and / or a composition comprising dimethyl sulfone according to the invention is administered to poultry suffering from chronic heat stress.

[0044] The present invention is further illustrated by the following figures and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The course of the relative humidity (upper line, right Y-axis) and the course of the temperature (lower line, left Y-axis) from d-0 until the end of the experiment are shown.

[0046] Figure 2 The course of the relative humidity (upper line, right Y-axis) and the course of the temperature (lower line, left Y-axis) from d-24 until the end of the experiment are shown.

[0047] Figure 3 is a scatter plot representation of ADG for each treatment for the period from d-10 to d-21 with mean ± deviation.

[0048] Figure 4 is a scatter plot representation of FG of each treatment for the period from d-10 to d-21 with mean ± deviation.

[0049] Figure 5 Shown are the progression of body weight for each treatment and for the male Ross 308 Performance Objectives 2019 and as expected for the finishing phase.

[0050] Figure 6Shown are the feed intake per bird per day during the 6-hour period of heat stress (lower 3 lines) and during the rest of the day (upper 3 lines) for the respective treatments over the period from d-24 until d-39.

[0051] Figure 7 The percentage of total feed intake and water intake consumed during hours of heat stress for the period from d-24 until d-39 (6 hours per day) for each treatment for all pens together are shown.

[0052] Figure 8 Daily death cases and treatments for the period from d-24 to d-39 are shown.

[0053] Figure 9 and Figure 8 Similar, but excluding pen 24 (T2) with 5 deaths in the period from d-24 to d-39; the number of deaths in this period was 14, 10, and 8 for T1, T2, and T2, respectively.

[0054] Figure 10 based on Figure 9 The data were plotted using Kaplan-Meier to show the percentage of birds surviving during the heat stress period (d-24 to d-39; day 1 and day 16 of heat stress correspond to d-24 and d-39, respectively). A log-rank test for trend, p = 0.159, indicated that there were no statistical differences in the survival curves. Figure 10 and Figure 3 Similarly, pen 24 (T2) with 5 deaths during the period from d-24 to d-39 is not included. For T1, T2, and T3, the number of deaths during this period is 14, 10, and 8, respectively.

[0055] Figure 11 Shown are the deaths + exclusions and treatments for each day from d-24 to d-39 (eliminations are dashed blocks). For T1, T2, and T3, the number of deaths + exclusions in this period were 16, 17, and 8, respectively.

[0056] Figure 12 Shown are the deaths + exclusions and treatments for each day from d-24 to d-39 (exclusions are dashed blocks). Figure 11 Similar, but excluding pen 24 (T2) with 5 deaths from d-24 to d-39. The number of deaths + culls during this period was 16, 12, and 8 for T1, T2, and T3, respectively.

[0057] Figure 13 based on Figure 12The data were expressed using Kaplan-Meier to show the percentage of birds surviving during the heat stress period (d-24 to d-39; day 1 and day 16 of heat stress correspond to d-24 and d-39, respectively). The log-rank test for trend, p=0.087, indicated that there were no statistical differences in the survival curves. Figure 13 and Figure 3 Similar, but excluding pen 24 (T2) with 5 deaths from d-24 to d-39. The number of deaths in this period was 14, 10, and 8 for T1, T2, and T3, respectively.

[0058] Figure 14 Box plots + Tukey whiskers of rectal temperatures of birds during heat stress for each treatment.

[0059] Figure 15 Box plots + Tukey whiskers of rectal temperatures of birds sampled on d-23, d-25 and d-39.

[0060] Figure 16 Shown are the concentrations of T3, malondialdehyde and glutathione and the T3 / T4 ratio in the blood of male broiler birds subjected to chronic periodic heat stress sampled at d-25 according to the time after the start of heat stress.

[0061] Figure 17 Shown is the relationship between malondialdehyde and glutathione in the blood of male broiler birds subjected to chronic cyclic heat stress sampled at d-39.

[0062] Figure 18 Shown is the effect of DMS dosing on litter moisture.

[0063] experiment

[0064] 1. Materials and Methods

[0065] 1.1 Experimental Design

[0066] The experimental design consists of a control group (T1) and two other groups, each of which receives a control diet supplemented with increasing levels of DMS (dimethyl sulfone), specifically 0.1% DMS (T2) and 0.2% DMS (T3). In a completely randomized block design, treatment was repeated in each of 12 enclosures. Block refers to the physical distribution in the experimental facility. All animal experiments were carried out in accordance with the requirements of the Ethics Committee of the Faculty of Veterinary Medicine (University of Ghent, EC2018-71).

[0067] 1.2 Poultry and breeding environment

[0068] Experiment was carried out at the experimental facility of the Department of Animal Sciences and Aquatic Ecology of Ghent University, Ghent University, Melle, Belgium. A total of 792 male broilers (Ross 308, Aviagen Group) were provided by Vervaeke-Belavi hatchery (Tilde, Belgium). Since males are more sensitive to heat stress (HS) (Cahaner and Leenstra, 1992), a greater therapeutic effect was expected with males. The experiment consists of three feeding stages: brooding (from d-0 to d-10), breeding (from d-10 to d-21) and fattening (from d-21 to d-39). One-day-old chicks were weighed and evenly distributed in the fence according to their body weight (BW). The fence was randomly assigned to the treatment group. At the beginning of the experiment, the initial average animal BW was 41.3 ± 0.31g. The newly selected chicks were placed in a separate climate-controlled room containing 36 enclosures, each housing 22 animals. Each enclosure had a size of 0.80 x 1.70 m and a concrete floor covered with wood shavings. The climate-controlled room was equipped with a liner drinker (two nipples per enclosure), and each enclosure had a tubular metal feeder. The temperature during the brooding and growing phases was set to linearly decrease from 34 ° C at d-0 to a base temperature of 22 ° C at d-24. During the period from d-0 to d-7, the lights in the control room were turned on for 23 h, then turned on at 4:00 and turned off at 22:00 (18 L:6 D) during the remainder of the experiment.

[0069] 1.3 Heat stress protocol

[0070] The fattening stage starts from d-21, and from d-24 until d-39, a chronic periodic heat stress (HS) scheme has been applied. From d-24, the temperature is increased to 34 ℃ by 22 ℃, and continues 6 hours every day (from 9:00 to 15:00), wherein the relative air humidity (RH) is between 52% and 58%. From 8:00 to 9:00, the temperature is increased to 34 ℃ by 22 ℃, and from 15:00 to 16:00, this temperature is reduced to 22 ℃ by 34 ℃. The temperature of the rest of the day is 22 ℃. The control room is equipped with a heater and an air circulator with a thermostat to be able to maintain required temperature and RH, and enough ventilation and air conditions are provided. Periodically, by manually spraying water on a stable wall, RH is regulated. Use a dry-bulb hygrometer (Escort RH iLog, EscortVerification Technologies Inc., Buchanan, the U.S.) to record this temperature and this RH at intervals of 10 minutes.

[0071] 1.4 Dietary therapy

[0072] According to Ross 308 recommendation or slightly lower to avoid excessive mortality in the fattening stage, a feed based on wheat and soy flour was formulated to meet the nutritional needs of broilers. The diet does not contain anticoccidial agents, synthetic antioxidants, or emulsifiers. The final formula was approved by the committee. The ingredients and nutritional composition of all diets are given in Tables 1 and 2. In all feeding stages, the test substance dimethyl sulfone was added to T2 and T3 at the top with 0.1% and 0.2% respectively. The diet was processed and passed to Ghent University by Research Dietary Services (RDS, Wijk bij Duurstede, the Netherlands). During all feeding stages, all diets were provided in the form of pellets. During the entire experimental period, the chickens were fed ad libitum and had free access to water.

[0073] 1.5 Performance and Health Records

[0074] For all performance indicators, the pen is the experimental unit. Total feed intake (FI) was recorded for each feeding period. In addition, the FI during HS was recorded every day, and the feeders were weighed every day before (at 8:00) and after (at 16:00) HS. This allows the calculation of the relative FI during the period with HS relative to the total FI every day during HS (d-24 to d-39). The total body weight (BW) of each pen was recorded at d-0, d-10, d-21 and d-39, and divided by the number of chickens in the pen on that day to obtain the average BW of each pen. Average daily gain (ADG, equation 1), average daily feed intake (ADFI, equation 2), the ratio of feed amount to gain (F:G, equation 3) and mortality (MR, equation 4) are calculated using the following formula:

[0075] (1)

[0076] Among them ADG P is ADG (g / d) at a given feeding period, BW 第一天 is the mean BW (g) for that pen on the first day of the study rearing period, where day 1 = 0, 10, or 21 for the brooding, growing, and finishing phases, respectively, and BW 最后一天 is the mean BW (g) for that pen on the last day of the study rearing period, where last day = 10, 21 or 39 for the brooding, growing and finishing phases, respectively.

[0077] (2)

[0078] Among them, ADFI p is the ADFI (g / d) per bird at a given feeding period, TFI p is the total FI(g) for that pen during a given feeding period, and AD p is the number of animal-days during the feeding period (days).

[0079] (3)

[0080] Where F:G p ADFI is the ratio of feed amount to increase amount (g / g) at a given feeding period. p is the ADFI (g / d) per chicken, AD p is the number of animal-days (d) during the feeding period, TBW 第一天 is the total BW (g) for the pen on the first day of the study rearing period, where day 1 = 0, 10, or 21 for the brooding, growing, and finishing phases, respectively, and TBW 最后一天is the total BW (g) of the pen on the last day of the study rearing period, where last day = 10, 21 or 39 for the brooding, growing and finishing phases, respectively, and BW 死亡 is the total BW (g) of birds that died during the study period, including birds that were sampled in some cases.

[0081] All dead birds were collected and their weights recorded daily. MR was calculated for each feeding period using the following ratio:

[0082] (4)

[0083] Among them, MR p is the MR (%) at a given time period, # birds 第一天 is the number of animals present in the pen on the first day of the period, where day 1 = 0, 10 or 21 for the brooding, growing and finishing periods respectively, # birds 最后一天 is the number of animals present in that pen on the last day of the period, where last day = 10, 21 or 39 for the brooding, finishing and finishing phases respectively. Birds sampled on d-23 and d-25 were excluded from the respective calculations.

[0084] Finally, the European Productivity Efficiency Factor (EPEF, Equation 5) is calculated as follows:

[0085] (5)

[0086] Where EPEF is the European production efficiency factor, MR is the MR of the total period (%), BW d-39 is the average BW (kg) at d-39 and F:G is the F:G (g / g) over the total period.

[0087] 1.6 Sampling and physiological measurements

[0088] Rectal temperature and wheezing frequency (video analysis) (measured for 2 selected birds per pen on the following 3 days: d-24, d-31 and d-38, after 4 h of heat stress on the day)

[0089] Sampling of one bird per pen (with a weight close to the average weight of the pen) on d-23, d-25, and on d-39 (sampling started on d-25 and d-39 after >3 h of heat stress on that day; 108 birds in total)

[0090] Malondialdehyde; HSP70 (chicken heat shock protein 70, Hsp-70 ELISA Kit) Cusabio Code CSB-E11196Ch), HSP90 (chicken heat shock protein 90, HSP-90 ELISA Kit; Cusabio Code CSB-E12873C), GPx, SOD in plasma; GSH / GSSG in erythrocytes; and T3, T4, and nitric oxide in serum

[0091] -Breast muscle and liver: malondialdehyde, AOX (GPx, SOD), GSH / GSSG

[0092] After an overnight fast on d-40 (no heat stress that day), two birds per pen were sampled (with a weight close to the average weight of the pen)

[0093] - Carcass yield, breast percentage, thigh percentage, drumstick percentage and belly fat

[0094] - Carcass yield: with skin, blood, without viscera, without feet, without head, without neck, without belly fat; relative to the weight of the bird

[0095] Breast percentage: skinless, boneless; relative to the bird's weight

[0096] Thigh percentage: with skin, with bone; relative to the weight of the bird

[0097] Percentage of leg meat: with skin, with bone; relative to the weight of the bird

[0098] Belly Fat Percent: Belly fat; relative to the bird's body weight

[0099] -Breast meat quality

[0100] - Chest muscle disease score (lignified chest and white stripes)

[0101] Lignified chest: Normal, 0; Mild, 1; and Severe, 2

[0102] White streaks: Normal, 0; Mild, 1; and Severe, 2

[0103] - pH and color, both at time 0 h and 24 h (after sampling at time 0 h, breasts were stored at 4° C. until 24 h)

[0104] - Drip loss, determined for the period 0-24 h and for the period 0-72 h (after sampling at time 0 h, the breasts were stored at 4° C. until 72 h and weighed at 24 h and 72 h without exudate)

[0105] - compression loss, measured at 24 h (after sampling at time 0 h, the breasts were stored at 4° C. for up to 24 h, and then compression loss was measured)

[0106] - Oxidative stability (TBARS after simulated retail display)

[0107] 1.7 Statistical analysis

[0108] All data were checked for abnormalities and outliers (see explanation of results). Next, data for each dependent variable were tested for normal distribution (Kolmogorov-Smirnov) and homogeneity of variance (Levene's test) across treatments. Data showing mortality and mortality + culling rates were not normally distributed. Therefore, unless otherwise stated, data for this endpoint were evaluated using the nonparametric Kruskal-Wallis test, while other endpoints were tested using the GLM procedure.

[0109] The data were analyzed using the following statistical models:

[0110] Y j =μ+D j +ε j

[0111] where Y j represents the mean of treatment j (T1, T2 and T3), μ i is the grand mean, Dj is the fixed effect of treatment j, and ε j is the error term. If significant, block is also included as a random factor. In some cases, day is included (e.g., FI HS , rectal temperature) as a within-subject factor (repeated measures). Orthogonal contrasts were applied to test the linear and quadratic effects of increasing levels of DMS supplementation in the diet. For all analyses, the pen was considered the experimental unit. Means are given as estimated marginal means (least square means). Tukey post hoc tests were used to separate means. Differences were considered significant at p < 0.05 and trends were considered significant at p < 0.1.

[0112] Statistical evaluation of lignified breast and white stripes was performed using the chi-square test. Differences were considered significant at p < 0.05, and trends were considered significant at p < 0.1.

[0113] In terms of physiological measurements, if appropriate, the time of sampling (representing the number of minutes between the start of heat stress and the exact moment of sampling of the birds on that day) was also included in the model as a covariate, if significant. Orthogonal contrasts were applied to test the linear and quadratic effects of increasing levels of DMS supplementation in the diet. Means are given as estimated marginal means (least square means). Means were separated using Tukey's post hoc test. Differences were considered significant when p < 0.05, and trends were considered significant when p < 0.1.

[0114] Table 1 Composition of wheat-soybean based basal diets used for brooding (d-0 to 10), growing (d-10 to 21) and finishing (d-21 to 39) stages.

[0115]

[0116]

[0117]

[0118] (*) Premix Artikel 252 provides per kg of diet: Vitamin A (retinyl acetate), 10,000 IU; Vitamin D3 (cholecalciferol), 2,500 IU; Vitamin E (dl-α-tocopheryl acetate), 50 mg; Vitamin K3 (menadione), 1.5 mg; Vitamin B1 (thiamine), 2.0 mg; Vitamin B2 (riboflavin), 7.5 mg; Niacin, 35 mg; D-pantothenic acid, 12 mg; Vitamin B6 (pyridoxine-HCl), 3.5 mg; Vitamin Vitamin B12 (cyanocobalamin), 20μg; folic acid, 1.0mg; biotin, 0.2mg; choline chloride, 460mg; Fe (FeSO4.H2O), 80mg; Cu (CuSO4.5H2O), 12mg; Zn (ZnO), 60mg; Mn (MnO), 85; I (Ca(IO3)2), 0.8mg; Co (Co2CO3(OH)2), 0.77mg; Se (Na2O3Se), 0.15mg.

[0119] (**) Dig M / Dig M+C in brooding, growing and finishing diets were 66%, 63% and 62% respectively

[0120] Dig = digestible, ME = metabolizable energy

[0121] 2. Results

[0122] 2.1 Diet formulation and analysis

[0123] The diet was suitably prepared into pellets and delivered to the experimental facility. The samples were analyzed for approximate composition and amino acids by Evonik Nutrition & Care GmbH (Table 2). With regard to the brooding diet, it can be concluded that the supplementation levels of LYS, MET, THR and VAL were consistent with the feed formulation and very similar between all nutrients in the various treatments. In addition, the crude protein, ether extract and ash analyzed showed slightly lower than expected. Taking into account the uncertainty of 3% for all amino acids, all analytical values for amino acids were shown to be consistent with the formulated values. Due to the use of different MEs, the consistency of metabolizable energy (ME) could not be evaluated. Similar observations can be made for the growing diet. Therefore, the formulated crude protein was lower again (between 0.3% and 0.5%). Finally, the fattening diet was again shown to have no difference in the various treatments. The difference in the crude protein content in T2 relative to T1 and T3 could not be confirmed by the difference in amino acid levels because the amino acid content of all fattening diets was very identical. Nevertheless, the amount of crude protein was lower than formulated again, similar to slightly lower ether extracts and ash.

[0124] Table 2 For brooding, growing and experimental fattening (d-21 to 39) 1,2 Analytical composition (in %) of the corn-soybean based basal diet.

[0125]

[0126] 1 T1: control diet, T2: control diet + 0.1% DMS, and T3: control diet + 0.2% DMS

[0127] 2 Unless otherwise stated, nutrients were analyzed by wet chemistry and are given in %.

[0128] 2.2 Room temperature and relative humidity during the study

[0129] The temperature during the brooding and rearing phases was set to decrease linearly until d-24, with the final temperature being 22°C ( Figure 1 On d-5, the infrared lamps were turned off, which resulted in a 3°C drop in temperature, but the temperature was still acceptable for these older birds and was restored shortly thereafter. RH was initially low, but gradually increased towards the beginning of the fattening phase when diurnal variations became greater due to the HS protocol. Humidity was not controlled during the brooding and finishing phases and may depend on the age of the birds, temperature, ambient humidity (rain, snow), ventilation rate and volume, litter quality, etc. Starting on d-24, the HS protocol ( Figure 1 and Figure 2However, 7, 5, and 5 deaths were observed on d-28, d-29, and d-31, respectively ( Figure 3 In order to prevent a significant bias in the data due to excess mortality, it was decided to step down the heat stress protocol slightly by having 32°C during the heat stress period from d-32 to d-37, which was increased again to 33°C on days 38 and 39. Figure 3 Mortality rates were shown to decrease with decreasing temperatures during HS. RH exhibited significant diurnal variation during the HS period. When the stable temperature was raised to 34°C to induce HS, humidity tended to decrease. However, a target RH of 50-58% was maintained by regular water misting, if necessary. RH showed significant fluctuations during the rest of the day, occasionally reaching over 80% during nighttime hours on some days.

[0130] Typically, when starting a HS regimen, birds tend to reduce movement, stop feeding, then become immobile, spread their wings, increase ground contact, and ultimately pant heavily. The effect on water intake is unclear; birds may increase water consumption due to losses through evaporation and urination, however, water consumption is reduced relative to feed intake. During the remainder of the day, birds resume normal behavior and (compensatory) feed intake.

[0131] 2.3 Animal performance

[0132] Birds were found to be healthy throughout the study and performed very well overall, with final weights exceeding the 2019 male Ross 308 performance index. The various endpoints showed that during the period from day 24 to day 39, Pens 1 (T2) and Pen 2 (T1) (the stable edge) did not experience the same heat stress as the other pens; i.e., no or minimal panting was observed. Feed intake during the heat stress period was higher relative to total daily intake, approaching 33.3%, which is equal to the ratio of the daily duration of heat stress (6 hours) to the total light period (18 hours). Therefore, it was concluded that Pens 1 and 2 were not included in the statistical evaluation of any endpoints for the fattening phase and the total period. Furthermore, Pen 24 (T2) showed excessive mortality during the fattening phase, i.e., 5 deaths, equating to 22.7%, which skewed the performance index calculations. Therefore, it was concluded that Pen 24 was not included in the statistical evaluation of any endpoints for the fattening phase and the total period. Figure 3-6This is also highlighted. Table 3 summarizes the performance data of broilers for all rearing stages. During the brooding period, no significant effects were found on either endpoint. The body weight at the end of the brooding period was approximately 360 g. In contrast, during the growing period, the final body weight and ADG showed a linear increase (P < 0.05) with a higher content of DMS in the diet. Compared to the control group (T1), the ADG during this period was higher in T2 and T3 by 2.6% and 3.4% (both P < 0.05), further supported by the fact that the DMS content in the diet was 0.05. Figure 3 This was explained by a significant improvement in feed conversion ratio, with F:G ratios decreasing by 6 and 7 points at T2 and T3, respectively, compared to T1 (P < 0.05 for both). Figure 4 The scatter plot of F:G in this stage is shown. The fattening stage comprises two sub-stages: d-21 to d-24 is that there is no heat stress scheme, but in the period of d-24 to d-39, the heat stress scheme prevails, thereby reducing the potential growth of poultry. Unexpectedly, none of the performance indicators are affected by the treatment. However, it can be clearly observed that the numerical value of the mortality rate is reduced by supplementing DMS, and the mortality rate in T1, T2 and T3 is 6.3%, 4.7% and 3.1% respectively. From these data, it is concluded that supplementing DMS has improved the heat tolerance of animals, and therefore reduced the mortality rate. When the chronic periodic heat stress model is applied in the facilities used, a mortality rate of 6.3% is usually observed, but it is rarely found that the mortality rate numerical value observed in T2 and T3 is reduced. This highlights the importance of numerical reduction here. Consistent with the fattening stage, overall performance is not affected by the diet, but the numerical difference in mortality rate is once again worthy of full attention. The fact that DMS improved performance in the growing phase but not in the finishing phase may be related to the large reduction in formulated digestible LYS in the growing diet compared to the brooding diet (1.05% vs. 1.24%), which may be far too low when considering Ross 308 nutrient recommendations. The subsequent reduction in the finishing diet was smaller (0.99 vs. 1.05%). It is possible that DMS is more effective in a low-protein / low-digestible-LYS (while maintaining an ideal amino acid profile) diet.

[0133] Table 3. During the fattening phase 123 Dietary effects on BW, ADG, ADFI, F:G, mortality and EPEF in male broiler chickens subjected to chronic cyclic heat stress (n=10-12, i.e. 10-12 pen replicates per treatment).

[0134]

[0135]

[0136] 1Broilers were fed a wheat-soybean starter diet from d-0 to d-10, a finisher diet from d-10 to d-21, and a finisher diet from d-21 to d-39. A chronic cyclic heat stress model was implemented from d-24 to d-39.

[0137] 2 Values with different superscripts within a row are significantly different at p < 0.05

[0138] 3 T1: control diet, T2: control diet + 0.1% DMS, and T3: control diet + 0.2% DMS

[0139] 4 Standard deviation of the mean

[0140] 5 The percentage of total feed intake consumed during the hours of heat stress (6 h per day) from day 24 to day 39 was included as a within-subject factor (repeated measures) for the statistical analysis.

[0141] 6 Data across treatments were not normally distributed, so the nonparametric Kruskal-Wallis test was used to compare treatments.

[0142] 7 The F:G for the total period was corrected to a final body weight of 3050 g by assuming a 1.76 point gain per 100 g of body weight (based on male Ross 308 performance indicators 2019).

[0143] 8 European productivity factor,

[0144] Figure 5 Better explain the effect of the chronic periodic heat stress on the growth of poultry. It shows the growth from d-19 to d-39, thus highlighting the growth in the late rearing and fattening stages. First, the growth according to the male Ross 308 performance index 2019 (all black line) is given. Obviously, the T1-T3 (grey line) treatment shows higher growth, but as mentioned above, no difference between the treatments was found in terms of body weight. Next, an estimated growth (dashed black line) is established, indicating the growth under thermoneutral conditions. This is accomplished by linking the average weight of all treatments at d-21 (the end of the rearing stage, heat stress has not yet been established) to the estimated weight at d-39 in the absence of heat stress. This estimate is based on the following assumptions, which are based on a meta-analysis of 43 published papers (83 challenges) including studies on thermoneutral and heat stress treatments:

[0145] - Meta-analysis showed that for each °C above thermoneutrality, ADG decreased by 2.1 g / d,

[0146] - For the chronic periodic heat stress model, the difference between thermoneutrality and thermophilia is halved, in this case (34-22) / 2=6°C,

[0147] - In this experiment, chronic cyclic heat stress was performed for 15 days.

[0148] - In conclusion, it can be calculated that the weight loss on d-39 is equal to 189 g.

[0149] For the T1-T3 treatments, the mean observed body weight on day 39 was 3083 g, which means that in the absence of heat stress, the estimated body weight on day 39 would have been 3272 g. In other words, implementation of this chronic periodic heat stress model resulted in a reduction in final body weight of 189 g or 5.8%.

[0150] Table 3 shows that during the fattening period, the parameters ADFI and FI HS There were no significant differences among the different treatments. HS It represents the percentage of the total feed intake consumed during the hours of heat stress (6 h per day) during the period from d-24 to d-39. Figure 6 and Figure 7 More details are given regarding daily feed and water consumption in the period from d-24 until d-39. Figure 6 The absolute feed intake per bird per day is shown during the 6 h heat stress period and during the rest of the day. During the 15 day heat stress regime, the intake during the rest of the day shows a more or less steady increase, which corresponds to the normal increase in feed intake when birds become heavier. The intake during the 6 h heat stress period shows more inconsistent fluctuations, with an almost constant intake until d-31, after which the intake increases and then decreases again on d-37 and d-38. It should be noted that this reflects the actual temperature in stabilization almost perfectly. As mentioned above (see Figure 2 ), the initial high temperature was set at 34°C, but by d-32 it was reduced to 32°C and then increased again to 33°C on d-38. It is very clear that during this high temperature period, the actual room temperature had a direct effect on feed intake. It is important to observe that on most days, feed intake during the 6 h heat stress period was higher in the supplemented group compared to the control group, resulting in a linear trend only on d-26. This may indicate that the supplemented birds were less affected by the increase in calories due to feed consumption. When referring to Figure 7This is even clearer when the heat stress period is 6 hours. Relative feed intake during the heat stress period fluctuates between 22% and 30%, with a linear trend observed over several days with increasing levels of DMS in the diet. To understand this figure, the light schedule is 18L:6D, meaning the lights are on for 18 hours per day, with 6 hours of heat stress. Therefore, theoretically, feed intake during heat stress could be equivalent to one-third of total intake. Figure 7 It is clearly shown that this is much lower (between 22% and 30%), thus indicating a reduced feed intake caused by the heat stress regimen. As can be seen in Table 3, the FI of the supplemented group was significantly lower than that of the control group. HS The results are numerically lower, thus indicating a higher heat tolerance. Not surprisingly, the relative water intake during the 6-hour heat stress period (measured for all pens together) was much higher than a third, in fact exceeding 35%, and rising to 42% on some days. Thus, during a heat stress event, birds reduce their feed intake while significantly increasing their water consumption.

[0151] As highlighted above, DMS supplementation appears to improve heat tolerance, thereby reducing mortality. According to Table 3, mortality during the finishing period accounted for 6.3%, 4.7%, and 3.1% of the total mortality in T1, T2, and T3, respectively. The following figure details all cases of mortality and culling during the finishing period, with or without the exclusion of pen 24 ( Figure 8-13 As an example, discuss Figure 9 In this figure, the number of deaths for T1, T2 and T3 were 14, 10 and 8 respectively. Besides the fact that the number was lower in the DMS supplemented group, it is interesting to note that the deaths in T1 occurred mainly at the beginning of the heat stress period, while the dead birds in T3 were found mainly in the second half of the period. T2 had deaths throughout the day. This is Figure 10 This is further illustrated in the figure by plotting survival curves for periods of heat stress with implementation. This lends itself to the speculation that during the acute phase of a heat wave (the first few days), DMS supplementation confers the greatest benefit to birds, whereas in birds with more chronic heat stress, DMS supplementation may not add an advantage. Similarly, Figure 12 and 13 The mortality rate + culling rate is given. The number of cullings is limited by only in T1 and T2. Here, Figure 13The statistics above show a trend toward differences in the survival curves. It's also possible to speculate whether this difference in the timing of deaths could affect the calculation of ADFI and F:G during the fattening period. For example, as birds age, F:G increases, so birds with more deaths at the end of the fattening period would overestimate F:G. In other words, it's plausible that the F:G for fattening at T1 is underestimated and the F:G for fattening at T3 is overestimated. If this were the case, the true F:G would favor T3 and not be higher than T1, as shown in Table 3. However, the authors found no way to correct for this and recalculate F:G in one way or another.

[0152] Table 4 shows the effects of treatment on rectal temperature and wheezing on days 24, 31, and 38 of the study. Both parameters were measured during the last 2 hours of a 6-h heat stress event and illustrate the high ambient temperatures to which the birds were subjected. With regard to rectal temperature, supplementation with DMS resulted in numerically lower rectal temperatures on all days, supporting the notion that heat tolerance was improved. Figure 14 These effects were shown. A linear effect was observed on day 38 (P < 0.05). Overall, rectal temperature was significantly reduced by 0.2% DMS in the diet compared to the control group (P < 0.05). Notably, rectal temperatures in thermoneutral birds fluctuated between 40.5 and 41.5°C. Panting was severe during heat stress events and increased significantly as the birds aged, although it was no higher on day 38 than on day 31, likely due to room temperatures of 33°C and 34°C on these days, respectively. A significant treatment effect was observed on day 24, indicating that 0.2% DMS reduced panting frequency compared to the control group, and the effect was linear on day 24 (P < 0.05). The values on days 31 and 38 were not significant, but numerically lower panting frequency was again observed for overall panting frequency. The respiratory rate of birds in thermoneutrality will be between 40 and 80, depending on age and metabolism. The fact that the differences in both rectal temperature and panting were more pronounced (and significant), particularly on day 24, may indicate that the DMS-supplemented birds were better prepared during the previous feeding period, thereby contributing to improved feed conversion during the rearing period, to cope with the upcoming heat stress. It also appears that the enhanced heat tolerance was less pronounced at the end of the heat stress period. This is consistent with the findings regarding mortality mentioned above.

[0153] Table 4. During the fattening phase 123 Effects of feed on rectal temperature and wheezing in male broiler chickens subjected to chronic cyclic heat stress (n=10-12, i.e. 10-12 pen replicates per treatment)

[0154]

[0155] 1 Broilers were fed a wheat-soybean starter diet from d-0 to d-10, a finisher diet from d-10 to d-21, and a finisher diet from d-21 to d-39. A chronic cyclic heat stress model was implemented from d-24 to d-39.

[0156] 2 Values with different superscripts in a row are significantly different at p < 0.05

[0157] 3 T1: control diet, T2: control diet + 0.1% DMS, and T3: control diet + 0.2% DMS

[0158] 4 Standard deviation of the mean

[0159] 5 Statistics were completed for all days, including day as a within-subject factor (repeated measures)

[0160] 2.4 Carcass yield and breast meat characteristics

[0161] No significant effects were found on carcass yield and portion (Table 5). The effects on breast meat properties are shown in Table 6. The pH of breast meat at slaughter and 24 h post-mortem was not altered by the diet. Regarding color, consistent effects on L* and b* can be expected, although significance was only seen for b* at 24 h post-mortem.

[0162] Table 5. After an overnight fast on d-40, during the fattening phase 123 Effects of feed on carcass yield and fraction in male broiler chickens subjected to chronic cyclic heat stress (n=10-12, ie 10-12 pen replicates per treatment).

[0163]

[0164] 1 Broilers were fed a wheat-soybean starter diet from d-0 to d-10, a finisher diet from d-10 to d-21, and a finisher diet from d-21 to d-39. A chronic cyclic heat stress model was implemented from d-24 to d-39.

[0165] 2 Values with different superscripts in a row are significantly different at p < 0.05

[0166] 3T1: control diet, T2: control diet + 0.1% DMS, and T3: control diet + 0.2% DMS

[0167] 4 Standard deviation of the mean

[0168] Table 6 After overnight fasting on day 40, during the fattening stage 123 Effects of dietary factors on breast characteristics in male broiler chickens subjected to chronic cyclic heat stress (n=10-12, ie 10-12 pen replicates per treatment).

[0169]

[0170] 1 Broilers were fed a wheat-soybean starter diet from d-0 to d-10, a finisher diet from d-10 to d-21, and a finisher diet from d-21 to d-39. A chronic cyclic heat stress model was implemented from d-24 to d-39.

[0171] 2 Values with different superscripts in a row are significantly different at p < 0.05

[0172] 3 T1: control diet, T2: control diet + 0.1% DMS, and T3: control diet + 0.2% DMS

[0173] 4 Standard deviation of the mean

[0174] 5 Lignified thorax: normal, 0; mild, 1; and severe, 2. The counts for each level were given in order of severity, and statistical evaluation was done by chi-square test (n=22-24).

[0175] 6 White streaks: normal, 0; mild, 1; and severe, 2. The counts for each level were given in order of severity, and statistical evaluation was performed by chi-square test (n=22-24).

[0176] 2.5 Physiological measurements in birds sampled on d-23, d-25, and d-39

[0177] At d-23, d-25 and d39, one bird (with a weight close to the average weight of the fence) was sampled in each fence. At d-25 and d-39, sampling was started after the heat stress of >3h on the same day. Therefore, a total of 108 chickens were sampled, and different endpoints were determined in several tissues. Table 7 records the body weight and rectal temperature of the birds sampled. Recall that the fattening period starts from d-21, and the heat stress program is performed from d-24 onwards. This means that d-23 is the last day before heat stress, and d-25 is the second day that birds are subjected to high temperature. Compared to those reported at d-21 (Table 3), the body weight (Table 7) at d-23 and d-25 is naturally higher, but not affected by treatment. The body weight of the birds sampled at d-39 is very close to the body weight at d-39 shown in Table 3, which confirms that birds with a weight close to the average weight of the fence have been selected. Again, supplementation with DMS did not affect this. Rectal temperatures of birds sampled on day-23 represented values under thermoneutral conditions and therefore ranged from 41.1°C to 41.3°C with no treatment differences ( Figure 15 ). However, rectal temperature at d-25 was substantially elevated and significantly affected by diet (P<0.05). With the higher amount of DMS in the diet, rectal temperature decreased linearly, with both supplemented groups differing from the control group, with the rectal temperature at T3 being 0.5°C lower than that of the control group. As shown in Table 4, the rectal temperatures were slightly higher than those at d-24, at least for the T1 and T2 groups, indicating an increased disturbance of body temperature homeostasis in these groups. With the higher amount of DMS in the diet, rectal temperature at d-39 decreased again, but did not reach significance. Nevertheless, these observations are consistent with the findings at d-38 (Table 4).

[0178] Table 7. During the fattening phase 123 Dietary effects on body weight and rectal temperature in male broiler chickens subjected to chronic cyclic heat stress, sampled at 4 h and 6-7 h between the corresponding days of heat stress on d-23, d-25 and d-39 (n=10-12, i.e. 10-12 pen replicates per treatment)

[0179]

[0180] 1 Broilers were fed a wheat-soybean starter diet from d-0 to d-10, a finisher diet from d-10 to d-21, and a finisher diet from d-21 to d-39. A chronic cyclic heat stress model was implemented from d-24 to d-39.

[0181] 2 Values with different superscripts in a row are significantly different at p < 0.05

[0182] 3 T1: control diet, T2: control diet + 0.1% DMS, and T3: control diet + 0.2% DMS

[0183] 4 Standard deviation of the mean

[0184] Table 8. During the fattening phase 123 Dietary effects on parameters in blood of male broiler chickens subjected to chronic cyclic heat stress, sampled at 4 h and 6 h on d-23, d-25 and d-39 between the corresponding days of heat stress (n=10-12, i.e. 10-12 pen replicates per treatment).

[0185]

[0186]

[0187]

[0188] 1 Broilers were fed a wheat-soybean starter diet from d-0 to d-10, a finisher diet from d-10 to d-21, and a finisher diet from d-21 to d-39. A chronic cyclic heat stress model was implemented from d-24 to d-39.

[0189] 2 Values with different superscripts in a row are significantly different at P < 0.05

[0190] 3 T1: control diet, T2: control diet + 0.1% DMS, and T3: control diet + 0.2% DMS

[0191] 4 Standard deviation of the mean

[0192] 5 Many values were below the detection limit of 0.42 μg / dL, so statistical analysis could not be completed.

[0193] 6 The sampling time significantly negatively affects the variable

[0194] The parameters in blood are given in Table 8. In some cases, the time after the start of the day of heat stress linearly affected the response to the results ( Figure 16 ). Specifically, T3, T3 / T4, and glutathione decreased over time, regardless of treatment (all P < 0.05). For clarity, malondialdehyde was not affected by time after the onset of heat stress, but was used as an example in Figure 16The figure also shows that only a small part of the variance is explained by the time after the onset of heat stress, r 2 Between 0.14 and 0.19. However, the decrease in T3 and the consistent decrease in T3 / T4 can be considered as an adaptation to reduce body metabolism and the heat production of metabolism so far. This view may be supported by the fact that T3 values dropped sharply when compared to d-23, d-25 and d-39 (Table 8). This means that when heat stress lasts for 2 weeks, a continuous decrease in T3 and its activation from T4 can be observed. Since thermoneutral controls were not included in the study, the influence of age cannot be ruled out. However, the decrease is strong, and T4 does not seem to be affected by the number of days. In addition, glutathione decreases with time after the start of heat stress, which may be caused by the following: a higher demand in other tissues such as the small intestine and respiratory system to cultivate antioxidant function and redox homeostasis; or due to reduced synthesis, mainly in the liver, when heat stress persists. Interestingly, glutathione in erythrocytes also seemed to be severely affected by the heat stress protocol, since the values at d-23 were between 0.91 and 1.25 μmol / L, while from d-25 onwards, these values did not exceed 0.70 μmol / L (Table 8). Furthermore, since GSSG / GSH remained fairly stable during the sampling time, no redox imbalance was expected, thus affecting only glutathione synthesis.

[0195] Heat shock protein 70 and heat shock protein 90 were measured in plasma using competitive inhibition enzyme immunoassay techniques (HSP70, chicken heat shock protein 70, HSP-70 ELISA Kit; Cusabio Code CSB-E11196Ch) and quantitative sandwich enzyme immunoassay techniques (HSP90, chicken heat shock protein 90, HSP-90 ELISA Kit; Cusabio Code CSB-E12873C), respectively. For HSP-90, very low absorbance was observed, and in most cases, it was below the lowest standard dilution. However, the values were generally less than 0.39 ng / mL, as mentioned in the user manual as the lowest detectable dose. Therefore, data on HSP-90 cannot be reported here. In contrast, HSP-70 functioned normally, with values above the sensitivity of 0.125 ng / mL. Apart from a linear decreasing trend on day 23, no treatment effect on HSP-70 was detected on any of the sampling days. It also showed that plasma HSP-70 levels showed a slight decrease during acute heat stress (day 25), but increased significantly two weeks after heat stress (day 39). It is important to note that significant differences between samples were observed on day 39. Thyroxine (T4) was undetectable on day 23, while higher values were found on days 25 and 39. A linear trend of increase was observed on day 39 with higher DMS doses. Triiodothyronine (T3), a derivative and the most active form of T4, was reduced by feeding 0.2% DMS on day 23 compared to the control group (P < 0.05). On day 39, the trend suggests that the increase in T3 by DMS was actually a result of higher T4 levels, as the T3 / T4 ratio was unaffected. This suggests that the diet did not alter conversion efficiency. Regardless of treatment, circulating T3 levels decrease dramatically with age in birds, thus demonstrating prolonged heat stress. As mentioned above, this can be seen as an adaptation to reduce body metabolism and the production of heat from metabolism to date, and it is clear that birds fed DMS appear to be less affected. There were no differences in nitric oxide between the different treatments on any sampling day. In addition, interesting changes were found in malondialdehyde, a marker of lipid peroxidation. First, a linear decrease in this metabolite was found on both d-25 and d-39 when fed DMS (both P < 0.05), suggesting lower oxidative stress and / or higher antioxidant capacity. It is noteworthy that in T1, there was a large increase in malondialdehyde from d25 to d-39, which did not occur in chickens fed DMS. The antioxidant enzymes glutathione peroxidase and superoxide dismutase were not altered by the diet and therefore cannot explain the effect on malondialdehyde.In contrast, glutathione (GSH) increased on all days of the diet with DMS supplementation, with a linear increase on day 23 (P < 0.05), a trend toward a linear increase on day 25, and a linear increase on day 39 (P > 0.05), indicating that 0.2% DMS resulted in higher GSH levels in erythrocytes compared to the control group. As described above, erythrocyte glutathione appeared to be significantly affected by the heat stress regimen, as values on day 23 ranged from 0.91 to 1.25 μmol / L, whereas from day 25 onward, these values did not exceed 0.70 μmol / L. Furthermore, since the GSSG / GSH ratio remained fairly stable during the sampling period, no redox imbalance was expected, thus affecting only glutathione synthesis. Therefore, higher GSH in DMS-fed chickens promotes antioxidant function and redox homeostasis. However, there was no relationship between plasma malondialdehyde and erythrocyte glutathione. Figure 17 ).

[0196] In the liver, only glutathione peroxidase activity differed between treatments (Table 9). This was confirmed to be a linear decline (P<0.05), although the differences were small. Furthermore, it is noteworthy that GSH levels decline in aging birds, which has been repeatedly shown in heat stress models. This means that the synthesis and rescue of GSH - the liver is the main site of synthesis - becomes limiting, and / or that the output to tissues requiring GSH increases. Both hypotheses are possible. First, as can be seen in Table 9, the ratio of GSSG to GSH increases sharply from d-25 to d-39. This suggests that rescue, i.e., the reduction of GSSG to GSH by glutathione reductase, is hindered, leading to a redox disorder. Second, Table 10 shows that GSH levels in breast muscle increase with age, from 1.03-1.10 to 1.39-1.56 μmol / g (d-23 to d-39). Given that breast muscle mass increases significantly during this period of growth, this must mean that a large amount of GSH is needed to supply the muscle. The high demand of the breast muscle may negatively affect the levels in the liver. Interestingly, in Table 10, DMS at d-25 linearly increased GSH (P < 0.05), and the trend showed the same on d-39. Surprisingly, a decrease in malondialdehyde was found on d-23 (P < 0.05), but not on other days.

[0197] Table 9 During the fattening stage 123 Dietary effects on liver parameters in male broiler chickens subjected to chronic cyclic heat stress, sampled at 4 h and 6 h on d-23, d-25 and d-39 between the corresponding days of heat stress (n=10-12, i.e. 10-12 pen replicates per treatment).

[0198]

[0199] 1 Broilers were fed a wheat-soybean starter diet from d-0 to d-10, a finisher diet from d-10 to d-21, and a finisher diet from d-21 to d-39. A chronic cyclic heat stress model was implemented from d-24 to d-39.

[0200] 2 The values with different superscripts in the same row are significantly different at P < 0.05.

[0201] 3 T1: control diet, T2: control diet + 0.1% DMS, and T3: control diet + 0.2% DMS

[0202] 4 Standard deviation of the mean

[0203] Table 10. During the fattening phase 123 Dietary effects of breast muscle parameters in male broiler chickens subjected to chronic cyclic heat stress, sampled on d-23, d-25 and d-39 at 4-6 h between the corresponding days of heat stress (n=10-12, i.e. 10-12 pen replicates per treatment).

[0204] In breast muscle samples, the content of oxidized glutathione (GSSG) was below the detection limit (0.05 μmol / g) and therefore not reported here.

[0205]

[0206] 1 Broilers were fed a wheat-soybean starter diet from d-0 to d-10, a finisher diet from d-10 to d-21, and a finisher diet from d-21 to d-39. A chronic cyclic heat stress model was implemented from d-24 to d-39.

[0207] 2 Values with different superscripts in a row are significantly different at p < 0.05

[0208] 3 T1: control diet, T2: control diet + 0.1% DMS, T3: control diet + 0.2% DMS

[0209] 4 Standard deviation of the mean

[0210] 2.6 Mattress moisture

[0211] Furthermore, it was investigated whether the administration of dimethyl sulfone and / or compositions comprising dimethyl sulfone also has an effect on the moisture content of the litter in the pen.

[0212] For this purpose, two treatments were completed (T1: control diet and T2: control diet + 0.05 wt% DMS), each with 13 replicate pens of 10 birds per pen. Collection of litter was completed on day 28 and day 42 to determine litter moisture. Litter sampling was completed at five different points in each pen (each corner and the center of each pen) and then homogenized to form a representative pen sample. The moisture content in the litter was determined by weighing the sample beforehand and placing it in an oven at 103°C for 24 hours. The calculation was completed according to the following formula:

[0213]

[0214] The experiment showed that the moisture content in the litter was significantly reduced at d-42, after the birds from the first experimental run were killed and the pens were filled with new birds.

[0215] 3. Conclusion

[0216] DMS supplementation did not affect performance during the brooding phase. In contrast, during the rearing phase, final body weight and ADG increased linearly with higher levels of DMS in the diet, being 2.6% and 3.4% higher at 0.1% and 0.2% DMS, respectively, compared to the control. This was associated with a significant improvement in feed conversion. F:G ratios decreased by 6 and 7 points for 0.1% and 0.2% DMS, respectively, compared to the control. During the finishing phase, no performance indicators were affected by treatment. Nevertheless, a clear numerical reduction in mortality was observed with DMS supplementation, with mortality rates of 6.3%, 4.7%, and 3.1% in the control group and 0.1% and 0.2% for DMS, respectively. In addition to the lower number of deaths in the DMS-supplemented groups, it is interesting that most deaths in the control group occurred at the beginning of the HS period, while in the 0.2% DMS group, most deaths were found in the latter half of the period. The 0.1% DMS treatment showed deaths on all days. This was further supported by reduced panting frequency and rectal temperature in birds fed DMS during heat stress. Furthermore, the higher tolerance to heat stress can be correlated with the following observations: 1 / a linear increase in serum T3 on d-39 with higher DMS doses, which may indicate better metabolic adaptation to HS; 2 / a linear decrease in plasma malondialdehyde on both d-25 and d-39, which suggests lower oxidative stress and / or higher antioxidant capacity; 3 / increased erythrocyte glutathione (GSH) on all days with the addition of DMS to the diet, which promotes antioxidant function and redox homeostasis; and 4 / a linear increase in GSH in breast muscle on d-25 with DMS, with the trend on d-39 suggesting the same.

[0217] Thus, DMS supplementation improves performance in the growing period, with a possible carryover effect in the finishing phase, which supports heat tolerance and substantially reduces mortality when HS is applied. Various physiological observations support this finding.

Claims

1. Use of dimethyl sulfone in preparing a medicament for treating chronic heat stress in poultry, wherein the dimethyl sulfone is administered orally.

2. The method according to claim 1, wherein the poultry is exposed to more than 27 ℃ temperature.

3. The use according to claim 1, wherein the poultry is exposed to a temperature of at least 30°C.

4. The use according to claim 1, wherein the poultry is exposed to a temperature of 30°C to 40°C.

5. The use according to claim 1 or 2, wherein the poultry is exposed to a temperature exceeding 27°C for at least 5 hours per day.

6. Use according to claim 1 or 2, wherein the poultry is exposed to a temperature exceeding 27°C for a period of 5 to 24 hours per day.

7. The use according to claim 1 or 2, wherein the chronic heat stress is chronic periodic heat stress.

8. The use according to claim 1 or 2, wherein the poultry are exposed to an average relative air humidity of at least 40%.

9. The use according to claim 8, wherein the relative air humidity is on average 40% to 70%.

10. The use according to claim 1 or 2, wherein the dimethyl sulfone is administered to poultry in the brooding, growing and / or fattening stages.

11. The use according to claim 1 or 2, wherein the dimethyl sulfone is administered to poultry from the beginning of the brooding stage until slaughter or from the beginning of the finishing stage until slaughter.

12. The use according to claim 1 or 2, wherein the dimethyl sulfone is administered to poultry in the fattening stage.

13. The use according to claim 1 or 2, wherein the dimethyl sulfone is administered to poultry together with and / or in conjunction with a low protein diet and / or a diet with a low amount of digestible lysine.

14. The use according to claim 1 or 2, wherein The dimethyl sulfone is administered to the poultry at a concentration of at least 0.05% by weight based on the total weight of the liquid and / or solid matrix.

15. The use according to claim 1 or 2, wherein The dimethyl sulfone is administered to the poultry at a concentration of 0.05% to 0.5% by weight based on the total weight of the liquid and / or solid matrix.

16. Use of a composition comprising dimethyl sulfone in the preparation of a medicament for treating chronic heat stress in poultry, wherein: The composition comprises at least 0.05 wt% of dimethyl sulfone based on the total weight of the composition, and the composition is administered orally.

17. The use according to claim 16, wherein The composition comprises dimethyl sulfone at a concentration of 0.05 wt % to 0.5 wt % based on the total weight of the composition.

18. The use according to claim 16 or 17, wherein The composition is administered to poultry exposed to temperatures exceeding 27°C.

19. The use according to claim 16 or 17, wherein The composition is administered to poultry exposed to temperatures of at least 30°C.

20. The use according to claim 16 or 17, wherein The poultry were exposed to temperatures between 30°C and 40°C.

21. The use according to claim 16 or 17, wherein The composition is administered to poultry exposed to temperatures exceeding 27°C for at least 5 hours per day.

22. The use according to claim 16 or 17, wherein The composition is administered to poultry exposed to temperatures exceeding 27°C for 5 to 24 hours per day.

23. The use according to claim 16 or 17, wherein the chronic heat stress is chronic periodic heat stress.

24. The use according to claim 16 or 17, wherein the composition is administered to poultry exposed to an average relative air humidity of at least 40%.

25. The use according to claim 16 or 17, wherein the composition is administered to poultry exposed to an average relative air humidity of 40% to 70%.

26. Use according to claim 16 or 17, wherein the composition is administered to poultry in the brooding, finishing and / or fattening stages.

27. Use according to claim 16 or 17, wherein the composition is administered to poultry from the beginning of the brooding stage until slaughter or from the beginning of the finishing stage until slaughter.

28. Use according to claim 16 or 17, wherein the composition is a liquid and / or solid matrix.

29. The use according to claim 16 or 17, wherein the composition is a diet for poultry.

30. Use according to claim 29, wherein the diet is a brooding, finishing and / or fattening diet for poultry.

31. Use according to claim 29, wherein the diet is a low protein diet and / or a diet with a low amount of digestible lysine.

32. The use according to claim 16 or 17, wherein the composition is an aqueous formulation.

33. The use according to claim 16 or 17, wherein the composition is drinking water for poultry, the drinking water being supplemented with at least 0.05% by weight of dimethyl sulfone, based on the total weight of the drinking water.

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