Examination the effect of post-hatch heat-treatment and heat-stress in Transylvanian Naked Neck chicken


 Background

One of the most critical global problem nowadays is the increased environmental temperature. Agriculture is very susceptible to this adverse effect because the productivity of animals and poultry decreased. Although several studies reported the effects of heat-stress in chicken, the expression profile of heat-shock proteins and heat shock factors was not investigated in the gonads and germ cells of Transylvanian Naked Neck chickens.
Methods

In the first experiment, 24 hours after hatching 80 chicks were heat treated on 38.5oC ambient temperature with 60% humidity for 12 hours. After maturation, their primary productivity parameters, such as egg production, abnormalities in embryo development, sperm quantity, concentration, and motility were studied following two weeks of heat-stress on 30 °C room temperature. In the second experiment, the thermal manipulation of 60 chicks was the same but 15 treated and 15 control chicks were sacrificed immediately after the treatment. The other 15–15 chickens were raised to maturity. Expression levels for two heat-shock proteins and four heat shock factors were determined by real-time PCR in the gonads of heat-treated and heat-stressed chickens.
Results

We found that the heat-treated layers had significantly higher egg production than the control group in heat-stressed conditions. In cockerels, the sperm quality did not differ significantly between the heat-treated and heat-stressed group and the heat-stressed but not heat-treated group. We examined the expression pattern of HSPs and HSFs in the gonads. We found that the expression of HSP90 and HSF4 increased significantly (p < 0.05) in heat-treated female chick gonads but in adult females the expression of HSF2 and HSF3 were significantly lower compared to the control. In case of adult heat-treated males, the HSP70, HSF1 and HSF3 expression levels showed a significant increase in both gonads, compared to the control expression levels (P < 0.05).
Conclusion

Heat shock proteins and heat-shock factors protect cells against different stressors, including heat stress. Our findings show a significant effect on egg production but not on the sperm quality after post-hatch heat treatment in heat stress condition. The presented significant differences might be related to the increased expression level of HSP90 and HSF4 in heat-treated chickens.


Results
We found that the heat-treated layers had signi cantly higher egg production than the control group in heat-stressed conditions. In cockerels, the sperm quality did not differ signi cantly between the heat-treated and heat-stressed group and the heatstressed but not heat-treated group. We examined the expression pattern of HSPs and HSFs in the gonads. We found that the expression of HSP90 and HSF4 increased signi cantly (p < 0.05) in heat-treated female chick gonads but in adult females the expression of HSF2 and HSF3 were signi cantly lower compared to the control. In case of adult heat-treated males, the HSP70, HSF1 and HSF3 expression levels showed a signi cant increase in both gonads, compared to the control expression levels (P < 0.05).

Conclusion
Heat shock proteins and heat-shock factors protect cells against different stressors, including heat stress. Our ndings show a signi cant effect on egg production but not on the sperm quality after post-hatch heat treatment in heat stress condition. The presented signi cant differences might be related to the increased expression level of HSP90 and HSF4 in heat-treated chickens.

Background
The most important environmental stress factor is the increased average temperature caused by climate change. The agriculture is very sensitive to the climate variability and extreme weather [1]. Increasing number of articles were published in previous years about the effects of climate change in agriculture [2][3][4] and in animal husbandry [1,[5][6][7]. Animal exposure to hot environments deleteriously affects their reproductive functions [8]. The chickens are homeothermic animals and their body temperature is maintained in the range of 41 to 42 o C [9]. Higher temperature negatively impacts the feed intake, the reproductive function, the hatchability, and the meat and egg production [10][11][12][13][14][15]. In chickens, the high ambient temperature affects their endocrine system, reproductive and egg-laying performance, too [16].
Many researchers examined the effect of thermal manipulation in chicken. Two types of thermal manipulation are known: during the incubation period or after hatching. According to Al-Rubika et al. [17], the thermal manipulation during the late embryogenesis has not affected the hatchability, but the body weight of thermally manipulated embryos was higher than the control group. Walstra et al. [18] investigated the effect of temperature manipulation on the behaviour of layer chickens. The thermally manipulated embryos were incubated at 37.8 o C, but between the 14th and 18th embryonic day the embryos were exposed to 40 o C for 4 hours. The manipulated chicks preferred the lower ambient temperature, but no effect of thermal manipulation on behaviour and performance were observed [18]. The effect of thermal manipulation was not detected in case of body weight in broiler, but the manipulation induced the up-regulation of muscle grow factors and muscle marker genes [19].
Vinoth et al. [20] investigated the effect of thermal manipulation and thermal stress on HSP gene expression, DNA methylation in brain tissue of Naked Neck and Punjab Broiler-2 chicken breeds. Naked Neck gene is very common in the hot regions [21]. They found that the DNA methylation level was lower, and the gene expression was higher in case of heat stressed but not heattreated chickens, compared with other experimental groups (non-heat-treated but heat stressed; heat-treated and not heat stressed; heat-treated and stressed). Rajkumar and his collages [22] demonstrated, that the Naked Neck breeds have better growth performance in higher temperature than the normal siblings, and the Heterophil/Lymphocyte ratio was signi cantly lower, what is indicating that Naked Neck chickens were less stressed in higher temperature.
In case of heat stress, a lot of heat shock proteins (HSPs) and heat shock factors (HSFs) start to be expressed to protect the cells from the effect of heat stress. According to their molecular weights six HSP families are known (small HSPs, HSP40, HSP60, HSP70, HSP90 and HSP100) [23]. HSP70 and HSP90 are highly conserved ATP-dependent molecular chaperons which are essential for the eucaryote systems in unstressed conditions [24]. Of the many HSPs, the HSP70 correlates the best with thermotolerance [25]. The knockdown HSP70 mutant resulted an altered lens phenotype in zebra sh embryo [26]. The HSP90 chaperone is present in bacteria and all of eucaryotes. HSP90 participates in collaboration with the HSP70 chaperon system in protein folding and activation [27][28][29]. Increased HSP70 expression was detected in Japanese quail's myocardial tissue in case of isolation in darkness, loud noise and cold temperature [30]. These results denoted that the heat shock proteins are expressing in different types of stress. Under stress condition in poultries all heat shock factors are activated. In animals, four HSFs was known till 2018, when Saju and his collages found [31] the fth HSF, HSF5 in Danio rerio. This HSF5 is essential for the spermatogenesis in zebra sh. Two isoforms of HSF1 was discovered in gonads and liver of Danio [32]. Mezger and his colleagues [33] found that the HSF2 is essential in development of mouse, because they observed HSF2-like DNA binding activity at blastocyst stage. However, the HSF2 knock-out mutant mice was viable [34]. In chicken, the HSF1, HSF2 and HSF3 genes were isolated by cross-hybridization with mouse hsf1 cDNA probe [35]. HSF1 was mapped to the 2nd chromosome, the HSF2 to the 3rd, the HSF3 to the 4th, while the HSF4 gene to the 11th chromosome in chicken [36]. Zhang and his colleagues [11] examined the effects of acute heat stress in two different Chinese chicken breeds. They found that with increasing heat treatment time, both HSF3 and HSP70 expression rst decreased then showed a signi cant increase in both breeds. They found that the expression of HSF3 and HSP70 is species-speci c and tissue-speci c during heat treatment. HSF4 is an important heat shock factor for the formation of mammalian lens development [26]. Although both mouse HSF4a and HSF4b form trimers in the absence of stress, HSF4a acts as an inhibitor of the constitutive expression of heat shock genes, and HSF4b acts as a transcriptional activator. Furthermore, HSF4b complements the viability defect, but not HSF4a [37].
The transition during heat-stress has different effects on various poultry breeds. The broiler breeds are more sensitive, than the local breeds. The results of heat tolerance indexes suggested that with age the local breeds easily overcome heat stress, while the other chickens become increasingly vulnerable. These results have been con rmed by the high mortality rate observed in the commercial stock under heat stress, while there was no mortality among the local chickens [38].
In our study we investigated the effect of heat treatment and heat stress on the egg production, sperm quality, heat-stress protein and heat-shock factor expression pro le in Transylvanian Naked Neck Hungarian chicken breed. We found that the posthatch (24 hours after hatching) heat manipulation had an in uence mainly on the female reproductive parameters, while in adult animals, we found signi cant difference in heat-stress protein and heat-shock factor expressions in both genders.

Methods
Eggs of Speckled Transylvanian Naked Neck (STN) chickens were from the National Centre for Biodiversity and Gene Conservation -Institute for Farm Animal Gene Conservation (NBGK-HGI), Gödöllő, Hungary. Hatching and heat treatment took place in the experimental hatchery, the comparative study under heat stress was done in the animal house of this institution.
All applied methods in NBGK-HGI were approved by the Directorate of Food Safety and Animal Health of the Government O ce of Pest County (License number: PE/EA197-4/2016) and by the Institutional Ethical Review Board.

Heat treatment
The eggs were incubated in a PL Machine 500 incubator in regular way. For the rst 24 hours after hatching, the chicks were placed under an infrared lamp at 32 °C on absorbent paper litter with ad libitum starter feed and water. Then the chicks were placed back to the hatcher for heat treatment. The temperature was set to 38.5 °C and the humidity was 60% for 12 hours. Drinking water and feed were ad libitum inside. Then in Experiment I. the animals were kept and raised up together with the control group, in Experiment II. chicks were immediately sacri ced for DNA analysis.

Heat stress
In Experiment I. 80-80 animals were in the treated and control groups. We randomly selected 31-31 layers with 3-3 males in the 24th life week for the further reproductive biology examinations. Both of them were kept on a wood chips / zeolite mixture litter with ad libitum layer feed and water under 16-hour lighting, with egg nests (10) and perches. 10-10 roosters were placed in the same air space, in individual cages, for semen examinations, with ad libitum rooster feed and water. Ventilation was provided 10 minutes per hour. The average temperature in the pen at the height of the birds' habitat was constantly around 30 °C.

Examination of embryonic abnormalities
Eggs were collected daily and marked with group number and date. Every week 20-20 eggs were candled on the 7th day of incubation and if any kind of developmental abnormality was observed they were opened and checked. The ratio of infertile eggs as well as the phenotype of dead or abnormal embryos was determined [39]. The other ones were incubated the regular way.

Semen collection and classi cation
Sperm-donor animals were selected on the basis of responsiveness to semen collection, followed by individual semen evaluation data. Semen collection was performed using Burrows and Quinn's [40] dorso-abdominal massage technique twice a week for 2 months -from 23 weeks of age to 34 weeks of age -following a two-week training period. Semen was classi ed weekly for the following spermatological parameters: • Volume (ml): determined with a pipette.
• Motility: determined by subjective estimation using a light microscope (Leica) on a scoring scale from 0 to 5 at 40x magni cation. The test was always performed by the same experienced person.
• Concentration: determined with a spectrophotometer (Accucell IMV, France). At the beginning of the experiment, the instrument was calibrated. A concentration curve was established by comparing the spectrophotometer data of the samples in a dilution series with the concentrations determined using the Makler chamber.
• Type of morphological abnormalities and live / dead cell ratio: the study was performed using eosin-aniline blue vital staining [41].

Collection of gonadal tissues
The chickens were euthanized by cervical dislocation after the experiment. The tissue samples were collected in sterile plastic dishes. Small pieces from each gonad were placed into RNAlater™ Solution (Invitrogen, Thermo Fisher Scienti c). We collected thigh muscle samples from each chicken for DNA analysis. The samples were transferred into TRizol™ reagent after two days.
The samples were incubated in TRizol™ for 10 minutes on room temperature than stored on -80 o C.

DNA isolation and Sex determination
The thigh muscle samples were digested using 0.1% Proteinase-K Lysis Buffer solution and incubated at 55 o C for 3 hours. After the incubation, the Proteinase-K was inactivated at 99 o C for 10 minutes. Total DNA was extracted using the Phenol-chloroform DNA isolation protocol. The isolated DNA was quanti ed by measuring the absorbance at 260 nm using a NanoDrop One Spectrophotometer (Thermo-Scienti c, USA). The purity was assessed by determining the ratio of the absorbance at 260 and 280 nm.
The sex of treated and control chickens were determined using CHD1 (Chromosome Helicase DNA binding protein 1) primer set ( Table 1). The isolated DNA was diluted to 25 ng/µl for the PCR and gel electrophoresis. PCR was performed using MyTaq Red Mix. 13 µL of the reaction solution was used: 6.75 µL MyTaq Mix; 0.5 µL reverse CHD1 primer (10 µmol); 0.5 µL forward CHD1 primer (10 µmol); 4.25 µL sterile water and nally 1 µL DNA sample. The cycling parameters were 95 o C for 1 min. 28 cycles of 95 o C for 15 s followed 30 s at 48 o C and 72 o C for 10 sec. It was nally melting at 72 o C for 5 min. The PCR products were separated by electrophoresis, using 1.5% agarose gel stained with ethidium bromide, at 100 V for 30 minutes. The DNA bands were visualized under UV illumination and photographed.

RNA isolation, cDNA writing, and Real-time qPCR
Total RNA extraction and puri cation from cells collected in the TRIzol Reagent was following the manufacturers' protocol. The RNA was quanti ed by measuring the absorbance at 260 nm using a NanoDrop One Spectrophotometer (Thermo-Scienti c, USA), and the purity was assessed by determining the ratio of the absorbance at 260 and 280 nm. Total RNA (15 µL) was reverse transcribed using a cDNA synthesis kit (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems). SYBR Green PCR master mix was applied for the qPCR as a double-stranded uorescent DNA-speci c dye according to the manufacturer's instructions (Applied Biosystems, Life Technologies, Carlsbad, US). The primers used for real-time PCR are displayed in Supplementary Table 1 We tested the expression of two housekeeping genes (GAPDH and ß-Actin) [12]. We compared the average Ct values of GAPDH and ß-Actin in control and heat-treated samples. We decided to use GAPDH as we found lower Ct values in the case of GAPDH, and the standard deviation were higher using ß-Actin ( Supplementary Fig. 1A). There was not signi cant difference between the control and heat-treated samples (p = 0.523) comparing the average GAPDH Ct values. We used chicken embryonic broblast as reference sample [12,42,43]. All reactions were performed in triplicate. The number of used samples is indicated in

Statistical analysis
To evaluate and analyse the collected data RStudio (1.0.136), R (R-3.2.2), GeneEx (6.0) and Excel (Microsoft O ce) software were used. For the data obtained from the qPCR runs, expression changes of the target genes were calculated compared to the expression of the housekeeping gene with the standard 2^(−ΔΔCt) method, where Ct = cycle threshold; ΔCt = Ct (target gene) − Ct (housekeeping gene) and ΔΔCt = ΔCt (test sample) − ΔCt (control sample).
The mean values of the different sample groups were compared with t-tests, furthermore the categorical data was tested with Chi-squared tests. Signi cance levels were set as follows: * p < 0.05, * * p < 0.01, and * * * p < 0.001.

Results
1. Analysis of the effect of heat stress on reproductive parameters of heat-treated chickens

Spermatological analysis in roosters
The reproductivity parameters of heat-treated and heat-stressed (HTHS) Transylvanian Naked Neck roosters were examined compared to the heat-stressed but not heat-treated (HS) ones. In the spermatological analysis, 10 HTHS and 10 HS rooster were used. Four parameters (quantity, concentration, motility and live-dead ratio of sperm was determined (Fig. 1.). The volume of the semen was measured by pipetting. We could not nd signi cant difference between the two experimental groups (p = 0.5075). In the collected ejaculate, the sperm concentration was de ned by a calibrated spectrophotometer. According the sperm concentration, no signi cant difference was found between the HTHS and HS groups (p = 0.1077). No signi cant difference was found in the motility rate between the two groups (p = 0.6972). Finally, in the live-dead sperm ratio no signi cant difference was found (p = 0.8816) between the two experimental groups. In summary, we can conclude that the pre heat-treatment has no effect on sperm quantity and quality.

Examination of the egg production and fertilization rate in hens
In the females, two parameters, the egg production and the percent of unfertilized eggs were measured (Fig. 2.). The daily egg production of heat-treated chickens (HTHS) was signi cantly higher than the non-heat-treated (HS) hens on high environmental temperature (30 o C) (p = 0.00002) (Fig. 2.A). Altogether, 1654 eggs were collected. The HTHS group produced 890 (54%) eggs, while in case of the HS group, we could collect 764 (46%) eggs (Fig. 2.B). We also determined the ratio of unfertilized eggs. In the HTHS group, 108 eggs, while in the HS group, 172 eggs were analysed. The eggs were incubated till 3rd day of embryonic development, then we opened the eggs, and analysed the embryonic development. In the case of HTHS group from 108 eggs, 4 (3.7%) was unfertilized, while in the HS group from 172 eggs 19 (11.05%) did not contain embryo. We found all together 257 fertile eggs. 52% of fertile eggs were derived from heat-treated group, the remaining 48% originated from control group (Fig. 2.C). immediately after the heat-treatment (38.5 o C for 12 hours, in 2-days-old chickens) and in adulthood. We isolated RNA from the collected gonads. We performed qPCR to analyse the expression pro le of two heat shock protein (HSP70, HSP90) genes and four heat stress factor (HSF1, HSF2, HSF3, HSF4) genes in male left and right gonads, and female genital ridges. To determine whether there is any difference in the expression pro le of heat treated and control groups, we pooled the RNAs of the individual samples group by group. As we found differences in the expression pattern compared to the heat treated and control groups, we performed qPCR runs from the individual samples to prove, whether these differences are statistically different or not. We  Figure 1). Comparing the Delta Ct values calculated in the individual samples, we found signi cant difference between the chicks and adults (Fig. 3.1, 1A-6A). Signi cant difference was determined between the Delta Ct values of HSP70 (p = 0.0289), and HSF3 (p = 0.0482) expressions in the heat-treated and control samples in case of adult male left gonads (Fig. 3.1 1A and  Fig. 3.2 5A). In case of the right gonads, signi cant differences were found in HSP70 (p = 0.023); HSF1 (p = 0.0007) and HSF3 (p = 0.0013) (Fig. 3.1 1B and 3B; Fig. 3. 5B) between the control chicks and control adults. Only the HSP70 showed a signi cant difference comparing the values of control and heat-treated right gonads in adults (p = 0.0136) (Fig. 3.1 2A).
In case of the female gonads, only HSF4 Delta Ct values de ned signi cant difference (p = 0.0016) between the control chicks and control adults (Fig. 3.2 6C). Signi cant differences were found between the control and heat-treated chickens in case of HSP90 (p = 0.0355) (Fig. 3.1 2C) and HSF4 (p = 0.0342) values (Fig. 3.2 6C). In other cases, we could not nd signi cant differences between the groups. The Delta Ct values were analysed with Chi-squared tests.

Comparison of the relative expression pro le
To get more detailed information whether there is any signi cant difference in relative expression of HSPs and HSFs in control and treated samples, we calculated the relative expression values using the GenEx (6.0) software. The relative expression was determined using 2 −ΔΔCt method. The mean values of the different sample groups were compared using t-tests. The HSP90 (p = 0.0094) and HSF4 (p = 0.0387) expression was signi cantly higher in the heat-treated female chicken gonads than in the control (Fig. 4. E). However, in the adult female gonads all of HSPs and HSFs decreased compared to the control groups, the HSF2 (p = 0.0181) and the HSF3 (p = 0.0011) were signi cantly lower in treated samples. (Fig. 4. F). In the case of chicks, there was no signi cant difference between the male left and right gonads compared to the control group (Fig. 4. A.; Fig. 4. C). However, in the left gonads of adults, the HSP70 (p = 0.0002); HSF1 (p = 0.0013); HSF2 (p = 0.0217) and HSF3 (0.0014) relative expression levels showed signi cant increase compared to the controls (Fig. 4. D). Analysing the male right gonads in adult samples we found that the expression of all HSPs and HSFs increased. The expression of HSP70 (p = 0.0052); HSF1 (p = 0.0333); HSF3 (p = 0.0332) and HSF4 (0.0498) showed signi cant increase compared to the control (Fig. 4. B).

Discussion
El-Tarabany [44] reported the impact of high temperature humidity index. They found that the control groups had signi cantly greater fertility and hatchability, than the heat stressed group. It was published that in the broiler chickens the genetically lean breed is more resistant to the higher ambient temperature, than the fat counterpart [21]. Laine and her collages [45] studied the effect of higher temperature in the hypothalamic-pituitary-gonadal-liver axis of Great Tit (Parus major). They found that the zona pellucida glycoprotein 4 (ZP4) is differently expressed before and after the onset of egg-laying [45]. We could not detect difference in spermatological parameters between the heat-treated and control groups in heat-stress condition analysing four spermatological parameters; quantity, concentration, motility and live-dead ratio of sperm (Fig. 1). On the other hand, analysing the female reproductive performance, we found signi cantly higher egg production and fertility rate (Fig. 2). The reason why we did not nd any difference among the spermatological parameters might be the effect of the Transylvanian Naked Neck breed. However, Végi and her colleagues found that the spermatological parameters decline after heat-treatment compared with the control group in Transylvanian Naked Neck chicken breed [46]. It was published that the Naked Neck chickens show better body temperature regulation and higher radiation rates from the naked neck than the covered neck breeds if they are kept in 35 o C [47].
Mezquita et al. [48] investigated the HSP70 expression in adult chicken testes. They found that the HSP70 was highly expressed in the left and right testes at higher temperatures (44 or 46 o C). However, at normal internal temperature the HSP70 is not expressed in the left gonad but it is present in the regressed (right) gonad [48]. We could detect low HSP70 expression level, but we found signi cantly higher HSP70 expression at adult age in roosters both in left and right gonads when they were heattreated. Interestingly, we found that HSF3 expression level increased parallel with HSP70 expression. Zhang and his colleagues [11] found that the level of HSP70 declined in the heart 6 hours after the heat stress, but the HSF3 expression remained high. Tarkhan and his colleagues [49] examined the HSP70 and HSF3 expression levels in cold stress. They found decreased expression levels in the liver in case of both genes.
In AA Broiler breed (from China) the HSP90 mRNA level increased in the liver, heart and kidney after 2 hours of high temperature. The HSP90 expressed in the endothelium cells and the blood vessel walls, which in uences the regulation of the blood ow [50]. Hao and Gu examined the expression of HSP90 on pectoralis major in broiler breed after acute heat stress. They found that the HSP90 expression is positively correlate with corticosterone and superoxide dismutase, but negatively correlate with the pH in pectoralis major [51].
We found high HSP90 expression in chickens compared to the HSP70 expression level. We could observe signi cant differences only in case of female chickens between the control and heat-treated HSP90 expression level.
It was reported that numerous transcripts in the testes expressed differentially between the heat-stressed broiler-type and layertype chickens [52]. We found in the left gonads of the adult heat-treated males that the HSP70 HSF1, HSF2 and HSF3 relative expression levels showed signi cant increase compared to the controls. Whether these expression patterns associate with the heat-tolerance require a further investigation. It was found that after 2 hours of heat treatment the expression of HSP27, HSP90 and HSP70 increased in a Taiwanese country chicken rooster, but the mRNA of CDH5, CIRBP, SLA and NTF3 were downregulated in the testes [52]. Wang et al. [53] published that in the heat-stressed chicken testes the proteins that involve in autophagy and the major HSPs (HSP90α, HSPA5, HSPA8) were upregulated but the proteins that negatively regulate apoptosis were downregulated. In the future, we plan to check the expression level of these factors in our heat-treated samples, too.
Furukawa et al. [54] shows that the HSF1 is a very important regulator in the ovarian differentiation of Medaka. They made a HSF1 knock-out animal and found that HSF1 protects the female germ cells under heat stress. We could detect signi cantly higher HSF1 expression in heat-treated roosters, in both left and right gonads, compared to the control, but there was no signi cant difference in the level of HSF1 in treated and control females.
HSF2 is very important in the development of brain and reproductive organs, but the fundamental rule is not identi ed yet [55]. In HSF2 knock-out B Lymphocyte cells they found that the KO line was more sensitive to the heat stress than the wild type [56]. We found higher HSF2 expression in heat-treated gonads in adult roosters, but signi cantly lower expression in adult females.
The mutation of HSF4 gene may cause a congenital or senile cataract in human. We found signi cantly higher HSF4 expression in heat-treated female chickens parallel with high HSP90 expression. In case of males, we could not detect difference in the expression pro le between the heat-treated and the control ones. According to these ndings, we propose that the increased HSP90 and HSF4 levels could eliminate somehow the effect of heat stress, but further analysis is needed to nd the molecular pathways responsible for this effect.

Conclusion
The average global temperature has increased over the century. Heat shock proteins and heat-shock factors play an essential role in normal cellular physiology and protection against different stressors, including heat stress. In chicken, HSP and HSF levels are increased in almost all the tissues in response to heat stress. This increased HSP level protects cellular proteins from heat-stress induced damage. Our ndings show a signi cant effect on egg production but not on the sperm quality after posthatch heat treatment. The egg production is more complex, longer and energy intense process than the spermiogenesis and that could be one of the reasons why we could not nd any difference in the sperm quality between the control and heat-stressed group. The found signi cant differences might be related to the increased expression level of HSP90 and HSF4 in heat-treated female chickens.  In this gure the results of heat-treated and heat stressed (HTHS) and only heat stressed but not treated (HS) mature cockerel's sperm parameters are summarized. Signi cant difference between the HT and HTHS group was not found. A: The quantity of sperm (ml) collected with dorso-abdominalis massage from cockerels. The sperm amount measured by pipetting. No signi cant difference was determined between the HTHS and HS group. (p=0.5075). B: The concentration of sperm (106/ml) in the collected ejaculations (the spectrophotometer was calibrated before the experiment). No signi cant difference was determined between the HTHS and HS group (p= 0.1077). C: The sperm motility was measured by subjective estimation. The estimation was in 40x magni cation. The sperm motility classi es in 0-5 scale. Every estimation was made by the same researcher. No signi cant difference was determined in the motility data between the HTHS and HS group (p=0.6972). D: Finally, the live-dead ratio of sperm was measured in the collected ejaculates. Eosin-aniline vitality staining was used. The results of live-dead ratio represented in %. No signi cant difference was determined in the live-dead ratio of HTHS and HS group (p= 0.8816).

Figure 1
In this gure the results of heat-treated and heat stressed (HTHS) and only heat stressed but not treated (HS) mature cockerel's sperm parameters are summarized. Signi cant difference between the HT and HTHS group was not found. A: The quantity of sperm (ml) collected with dorso-abdominalis massage from cockerels. The sperm amount measured by pipetting. No signi cant difference was determined between the HTHS and HS group. (p=0.5075). B: The concentration of sperm (106/ml) in the collected ejaculations (the spectrophotometer was calibrated before the experiment). No signi cant difference was determined between the HTHS and HS group (p= 0.1077). C: The sperm motility was measured by subjective estimation. The estimation was in 40x magni cation. The sperm motility classi es in 0-5 scale. Every estimation was made by the same researcher. No signi cant difference was determined in the motility data between the HTHS and HS group (p=0.6972). D: Finally, the live-dead ratio of sperm was measured in the collected ejaculates. Eosin-aniline vitality staining was used. The results of live-dead ratio represented in %. No signi cant difference was determined in the live-dead ratio of HTHS and HS group (p= 0.8816).

Figure 2
Demonstration of the daily egg production and the ratio of fertilized eggs in heat-treated and heat stressed (HTHS) and only heat stressed (HS) group. The eggs were collected every day, and were incubated to determinate the fertilized egg ratio. A: Illustration of the daily egg production of HTHS and HS hens. In case of HTHS hens, signi cantly higher egg production was observed than in the HS group (p=0.0000235). B: We collected 1654 eggs all together. 54% derived from HTHS group, while 46% from HS group. C: 280 eggs were incubated all together. We found 257 fertilised eggs. 52% derived from HTHS group and 48 % from the HS group.

Figure 2
Demonstration of the daily egg production and the ratio of fertilized eggs in heat-treated and heat stressed (HTHS) and only heat stressed (HS) group. The eggs were collected every day, and were incubated to determinate the fertilized egg ratio. A: Illustration of the daily egg production of HTHS and HS hens. In case of HTHS hens, signi cantly higher egg production was observed than in the HS group (p=0.0000235). B: We collected 1654 eggs all together. 54% derived from HTHS group, while 46% from HS group. C: 280 eggs were incubated all together. We found 257 fertilised eggs. 52% derived from HTHS group and 48 % from the HS group.  In case of the male left gonads, signi cant differences were determined in the expression of HSP70 between the CTRL chicks and CTRL adults (p=0.014), furthermore in case of adults between the CTRL and HT samples the differences were signi cant (p=0.0289). 1B: In male right gonads between the CTRL chicks and CTRL adult samples (p=0.0228), and in case of the adults, between CTRL and HT was signi cant difference (p=0.0136). 1C: Comparing the female gonads no signi cant differences were determined. 2A: In case of the male left gonads, signi cant difference was determined in the expression of HSP90 between the CTRL chicks and CTRL adults (p=0.0072). 2B: No signi cant differences were determined in male right gonads. 2C: Between the CTRL and HT chicks' signi cant difference (p=0.0355) was determined in the female gonads. 3A: The HSF1 expression of male left gonads were signi cantly different (p=0.0002) compared to the CTRL chicks and CTRL adult group. 3B: Signi cant HSF1 differences (p=0.0007) were determined in the male right gonads between the CTRL chicks and CTRL adults. 3C: In the female gonads, no signi cant differences were found. 4A: In case of male left gonads, we found signi cant differences between the CTRL chicks and the CTRL adult samples in HSF2 expression (p=0.0417). 4B: In the male right gonads there was no difference between the two groups. 4C: Comparing the female gonads signi cant difference was not found. 5A: Signi cant difference was found in the HSF3 expression in male left gonads between the CTRL chicks and CTRL adults (p=0.0002), furthermore between the adult CTRL and adult HT samples (p=0.0482), too. 5B: In the male right gonads, between CTRL chicks and adult group was signi cant difference (p=0.0013), furthermore between CTRL and HT group in case of adult samples was signi cant differences (p=0.0349) too. 5C: In case of female gonads, we did not nd a signi cant diversion. 6A: In the left male gonads between the CTRL chicks and CTRL mature groups were signi cant differences (p=0.0113) in the HSF4 expression. 6B: In case of male right gonads there was no difference. 6C: Comparing the female gonads between CTRL chicks and CTRL adult a signi cant difference was found (p=0.0016), furthermore between CTRL and HT was signi cant difference (p=0.0342) in chicks. ( * p<0.05, * * p<0.01 and * * * p<0.001) Figure 4 The bar charts show the relative expression values in female and male chick and adult samples in control and heat-treated gonads relative to chicken embryonic broblast sample. The relative expression values of Heat Shock Protein (HSP70 and HSP90) and Heat Shock Factor (HSF1, HSF2, HSF3, HSF4) genes were determined in female and male gonads. GAPDH was chosen as reference gene. A: Relative expression values in right male chick gonads. No signi cant differences were found compare the heat-treated and control groups. B: Relative expression values in right male adult gonads. In adult HT male right gonads, all of the heat-shock related markers increased. Signi cant increase was observed in HSP70 (p=0.0052); HSF1 (p=0.0333); HSF3 (p=0.0332) and HSF4 (p=0.0498). C: In the left gonads of male chicks, we couldn't recognise any signi cant difference between HT and CTRL groups. D: Relative expression values in left male adult gonads. HSP70 (p=0.0002); HSF1 (p=0.0013); HSF2 (p=0.0217) and HSF3 (p=0.0014) expressions showed signi cant difference. E: Relative expression values in female chicks. In case of HT female chick gonads, the HSP90 (p=0.0094) and HSF4 (p=0.0387) values were signi cantly different from CTRL. F: Relative expression values in female adult gonads. Comparing the relative expression values in the HT and CTRL adult female gonads only the HSF2 (p=0.0181) and HSF3 (p=0.0011) showed signi cant difference.