1. Introduction
Japanese quails are good models for investigating testosterone regulation in sexual behavior, especially, in captive situation (Ball & Balthazart, Reference Ball and Balthazart2010). However, these quails are susceptible to heat stress conditions during the dry season in the tropics (Tuleun et al., Reference Tuleun, Adenkola and Yenle2013). Heat stress (which could be induced in poultry birds during the dry season) activates the hypothalamus–pituitary–adrenal (HPA) axis and elevates cortisol level due to elevated adrenocorticotropic hormone activity (Mormède et al., Reference Mormède, Andanson, Aupérin, Beerda, Guémené, Malmkvist and Richard2007). The increase in adrenocorticotropic activities depresses hypothalamus–pituitary–gonad axis and lowers testosterone in Japanese quails (Tsutsui et al., Reference Tsutsui, Ubuka, Bentley and Kriegsfeld2012). Antioxidant supplementation is used to ameliorate the adverse effects of heat stress on reproductive parameters of the quail (Abd El-Hack et al., Reference Abd El-Hack, Abdelnour, Khafagad, Arif, Ayasan and Abdel-Daim2020). In addition, some male sexual traits, such as size of cloacal gland and testes are useful indicators of reproductive potentials of male Japanese quails (Mohan et al., Reference Mohan, Moudgal, Sastry, Tyagi and Singh2002).pt
2. Objective
Ascorbic acid (AA) is an antioxidant which scavenges stress-induced free radicals in poultry birds (Ismail et al., Reference Ismail, Al-Busadah and El-Bahr2015). Betaine (BET) exerts methyl-donor, osmolyte (Amerah & Ravindran, Reference Amerah and Ravindran2015) and antioxidant properties in poultry birds (Alirezaei et al., Reference Alirezaei, Gheisari, Ranjbar and Hajibemani2012). However, there is paucity of information on the effects of AA and BET supplementation on steroid activities and some sexual traits in male Japanese quails. The study is aimed at investigating effects of AA and BET supplementation on stress responses, testosterone levels, and some sexual traits in male Japanese quails during the dry season.
3. Methods
3.1. Experimental site
The experiment was performed during the dry season at the Animal House of Department of Veterinary Parasitology and Entomology, University of Nigeria, Nsukka (6° 52' 0" N; 7° 23' E), in the Derived Savannah Ecological zone (Uguru et al., Reference Uguru, Baiyeri and Aba2011).
3.2. Ethical matters
The methods of handling protocol were in accordance with the Animal Use and Care Committee of University of Nigeria, Nsukka, Nigeria. All quails were managed in accordance with the Guide for the Care and Use of Laboratory Animals (Committee on Care and Use of Laboratory Animals, 1996).
3.3. Animals and experimental protocol
A total of 240 male Japanese quail chicks; 14 days old and weighing 55.7 ± 1.06 g, were sourced commercially. A total of 240 male Japanese quail (14 days old; weighing 55.7 ± 1.06 g) were used and randomly assigned to four groups, each group has three replicate (n = 20; Al-Salhie et al., Reference Al-Salhie, Sabah and Rabia2017). Birds in each replicate were housed in spaces (91.44 cm length × 76.2 cm width × 91.4 cm height) within windowed animal pen and exposed to the natural environmental conditions. Birds were fed basal diets supplemented with AA (200 mg/kg; Sahin et al., Reference Sahin, Sahin and Yaralıoglu2002), BET (2 g/kg de Jong et al., Reference de Jong, Berg, Butterworth and Estevéz2012), and a combination of AA at 200 mg/kg and BET at 2 g/kg (AA + BET) during the 49 days of study, whereas the control birds were administered basal diet only. AA used (Kempex Holland BV, Volkel, Netherlands) had a minimum purity of 99%, whereas BET (Sigma-Aldrich, St. Louis, MO) was ≥99.0% purity. Daily dietary supplementation with AA and/or BET commenced after baseline serum testosterone values were established (when birds were 21 days old), and lasted 49 days (at 70 days old). Quails were given access to feeds and water ad-libitum.
3.4. Measurements
Thermal environmental conditions, measured as ambient temperature (AT) and relative humidity (RH) were recorded daily at 08:00 hr, 13:00 hr, and 17:00 hr throughout the study period. Blood collection was performed from six quails randomly selected and identified from each group at 21, 28, 49, and 70 days old. Blood samples were centrifuged at 3,000g for 10 min (El-Tarabany, Reference El-Tarabany2016) sera were harvested and immediately analyzed. Indicators of stress such as serum catalase (KU/L) and reduced glutathione (nMoles/ml) were determined spectrophotometrically, but cortisol (μg/dl); using enzyme-linked immunosorbent assay technique. Testosterone levels were determined using the microplate enzyme immunoassay kit (Monobind Incorporated, Lake Forest, CA). At 28, 49, and 70 days of age, sexual traits of quails randomly selected from each group were determines by measuring cloacal gland size (n = 12) and indices of testicular development (n = 4) such as paired testicular weight, left testicular area, and combined testicular volume. Index of gland size (or area) was determined by the product of cloacal gland width and height, and expressed in cm2 (Siopes & Wilson, Reference Siopes and Wilson1975). The formulas described by Chaturvedi et al. (Reference Chaturvedi, Bhatt and Phillips1993) was used to calculate cloacal volume, expressed in cm3:
4/3 × 3.5414 × a × b 2 (where a = 0.5 × long axis and b = 0.5 × short axis).
Measurements of length and width of left and right testes were taken to the nearest 0.01 mm using digital vernier caliper (Shanghai Techway Industrial Co., Ltd., Shanghai City, China). Bissonett’s formula was used to evaluate testicular volume: 4/3πab 2 (a = half of the long axis and b = half of the short axis), and expressed in cm3 (Banerjee & Chaturvedi, Reference Banerjee and Chaturvedi2017).
3.5. Statistical analysis
Data obtained are expressed as mean ± standard error of mean. The values were subjected to statistical analysis and compared using analysis of variance (ANOVA), followed by Tukey’s post-hoc test. Values of p < .05 were considered significant (Snedecor & Cochran, Reference Snedecor and Cochran1994). GraphPad Prism (GraphPad Software, Incorporated, San Diego, CA) version 6.0 was for analysis.
4. Results
4.1. Thermal environmental conditions
The range values of AT were 25.0–37.0°C, whereas those of RH were from 48.0 to 92.0% during the experimental period (Figure 1).
Figure 1. Boxplot describing micro-environmental conditions within the poultry house during the study period. DBT, dry bulb temperature (
$ {}^{\circ} $
C); RH, relative humidity (%).
4.2. Indicators of stress
All serum catalase, reduced glutathione, and testosterone levels were higher (p < .05) in the groups treated with AA ± BET, whereas cortisol levels were lower (p < .05) when compared with the control group (Table 1).
Table 1. Indicators of stress in Japanese quails reared during the dry season (n = 6)
Abbreviations: AA, ascorbic acid; AA + BET, combination of ascorbic acid and betaine included in diets; BET, betaine; Control, basal diet.
a,bMean values with different superscript letters within the same row are significantly different (p < .05).
4.3. Serum testosterone levels
Serum testosterone concentrations were not detected at 21 days old in all the groups. In all groups (control and AA ± BET), serum testosterone levels were increased as the birds grew older. This trend is observed in higher (p < .05) serum testosterone levels in birds at 49 and 70 days old when compared with when they were 28 days old in each of the experimental groups. At 28 and 49 days old, serum testosterone levels in quails that are fed with either enriched AA, BET, or AA + BET were higher (p < .05) when compared with values recorded in control group. Testosterone levels increased (p < .05) in birds fed either AA or AA + BET in their diets when compared with control at 70 days old (Figure 2).
Figure 2. Changes in serum concentration of testosterone with age of Japanese quails during the study period. Control, basal diet; AA, ascorbic acid; BET, betaine; and AA + BET, combination of ascorbic acid and betaine included in diets. a,b,1,2Mean values with different superscript letters and numbers between and within groups, respectively, are significantly different (p < .05).
4.4. Sexual traits of male quails
Cloacal gland area increased (p < .05) in the treated quails when compared with those of the control, especially, in 28 days old birds. In addition, cloacal volume was higher (p < .05) in the treated quails when compared with values obtained in the control, especially at 28 and 49 days of age. Furthermore, quails of the treated group recorded increased (p < .05) paired testicular weight when compared with birds of control group at 28 days old. Testicular area and combined volume were higher (p < .05) at 28 and 49 days old, when compared with the corresponding values obtained in the control group (Table 2).
Table 2. Variations in sizes of cloacal gland and testes in male Japanese quails during the dry season
Abbreviations: AA, ascorbic acid; AA + BET, combination of ascorbic acid and betaine included in diets; BET, betaine; Control, basal diet.
a,b,1,2Mean values with different superscript letters and numbers between and within groups, respectively, are significantly different (p < .05).
5. Discussion
5.1. Thermal environmental conditions
The results of the present study indicate that the recorded AT and RH fluctuated widely and predominantly exceeded the thermoneutral zone for Japanese quails. The thermoneutral zone for quails are 23.8 ± 0.7°C for AT and 58.5 ± 5.7% for RH (El-Tarabany, Reference El-Tarabany2016). Thus, the results of the current study suggest that the natural environmental conditions during the dry season are potentially stressful for Japanese quails. The findings of the present study agree with those of Minka and Ayo (Reference Minka and Ayo2013), who showed that the dry season in the Northern Guinea Savannah, with mean AT and RH of 32.5 ± 1.4°C and 66.9 ± 5.7%, respectively, were stressful to Japanese quails. In addition, Mehaisen et al. (Reference Mehaisen, Ibrahim, Desoky, Safaa, El-Sayed and Abass2017) demonstrated that AT values exceeding 30°C may be stressful for Japanese quails (Mehaisen et al., Reference Mehaisen, Ibrahim, Desoky, Safaa, El-Sayed and Abass2017). The high AT and RH which prevailed during the dry season, when the present study was conducted, have detrimental to the health, welfare, and reproductive performance of Japanese quails.
5.2. Heat stress and indicators of stress
The result of the present study demonstrates that supplementations with AA and/or BET in diets of male Japanese quails improved serum antioxidant enzyme activities when reared during the thermally stressful dry season. The findings of the present study agrees with those of Alirezaei et al. (Reference Alirezaei, Gheisari, Ranjbar and Hajibemani2012) who demonstrated that BET improved the antioxidant status of meat of broiler chicken subjected to heat stress conditions. Khan et al. (Reference Khan, Naz, Nikousefat, Selvaggi, Laudadio and Tufarelli2012) also showed that AA plays a role in enhancing antioxidant defenses in poultry birds. The findings of the current study suggest that AA and BET supplementation would enhance antioxidant defenses and improve thermotolerance in Japanese quails during the dry season.
5.3. Heat stress and testosterone levels
The results of the current study show testosterone synthesis in testes may have been too minimal to be detected at 21 days old male Japanese quails. The inability to detect testosterone at 21 days of age could be due to heat stress–induced delay in sexual maturity. The developmental phases of quails are classified as chick (0–21 days [or 0–3 weeks]); grower (28–35 days [or 4–5 weeks]); and adult (above 35 days [or 5 weeks]; Puspamitra et al., Reference Puspamitra, Mohanty and Mallik2018). The results of the present study are in agreement with Min et al., Reference Min, Sun, Niu and Liu2016, who showed stress results in decline in testosterone levels in serum, but that AA and vitamin E ameliorated this decline in rooster breeders. Parisi and Guerriero (Reference Parisi and Guerriero2019) showed that sex steroids, such as testosterone, are susceptible to oxidative stress due to thermal stress. The susceptibility of testosterone to the deleterious effects of free radicals requires antioxidants to ameliorate these effects (Sahin et al., Reference Sahin, Orhan, Tuzcu, Juturu and Sahin2017). The increased serum testosterone observed in the present study in birds fed dietary AA, both alone and it combination with BET, may be attributed to the antioxidant potentials of AA, acting in synergy with the methyl donor properties of betaine, when incorporated in the diets of male Japanese quails. These mechanisms of action of AA and BET could be beneficial to improve gonadal activities, libido, and fertility in male Japanese quails when exposed to the thermally stressful dry season.
5.4. Stress and sexual traits
The findings of the current study indicate that the size of cloacal gland increased in birds of the treated groups. The results of the present study agree with those of Biswas et al. (Reference Biswas, Mohan, Mandal and Lal2017) who demonstrated that selenium, an antioxidant, incorporated in diets of Japanese quails increased the size of cloacal gland and production of cloacal foam. Biswas et al. (Reference Biswas, Mohan, Sastry and Tyagi2007) also showed vitamin E improved cloacal gland size. Cloacal gland is a peculiar anatomical feature of Coturnix quails which is an indication of testicular size and function. Therefore, the assessment of cloacal gland size is referred to as a noninvasive tool to evaluate testicular activity and maturity in Japanese quails (Dominchin et al., Reference Dominchin, Busso, Kembro, Marin and Guzmán2016, Reference Dominchin, Busso, Kembro, Marin and Guzmán2017). Momoh et al., (Reference Momoh, Gambo and Dim2014) showed that the age of sexual maturity in Japanese quails reared under hot–humid condition is 5–6 weeks old. The present study did not evaluate the age of sexual maturity. This is one limitation of the present study. However, the result of the present study suggests that the bigger cloacal gland observed in AA and/or BET fed quails when compared with control group could be due to antioxidant effects of AA and BET on testosterone activity.
Similarly, the results of the present study show that testicular development increased with increase in age, especially, in those fed diets enriched with AA, alone and its combination with BET. The findings of the present study agree with those of Tsutsui et al. (Reference Tsutsui, Ubuka, Son, Bentley and Kriegsfeld2015), who demonstrated that rosemary oil exerted antioxidant protection against oxidative damage in the testes of heat-stressed Japanese quails. Heat stress–induced lipid peroxidation may result in developmental and functional damages in testes (Türk et al., Reference Türk, Çeribaşı, Şimşek, Çeribaşı, Güvenç, Kaya and Yaman2016). It is suggested that improved testicular development in male quails fed diets enriched with AA and/or BET in the present study could improve fertility during the dry season. The results of the current study and its potential benefits to reproductive responses in the birds corroborate with those of Egbuniwe et al., Reference Egbuniwe, Uchendu and Obidike2020, who demonstrated that diets enriched with BET and AA promote reproductive potentials as well as, sex and stress hormones (Egbuniwe et al., Reference Egbuniwe, Uchendu and Obidike2021) in Japanese quails during the dry season.
The results of this study may be useful to quail breeders who could use AA and BET to ameliorate heat stress effects on male sexual traits and optimize reproductive performance of male quails reared during the dry season.
6. Conclusion
From the results of the current study, it is demonstrated that heat stress conditions prevailing during the dry season in the zone adversely impact on some sexual traits of male Japanese quails. However, dietary enrichments with AA and/or BET could be utilized to ameliorate the negative impacts of dry season–induced heat stress in male Japanese quails. In conclusion, dietary AA and BET improve steroidal responses and some sexual traits in male Japanese quails during the dry season.
Acknowledgment
The authors thank Drs. Oguejiofor, Akogwu, and Obetta for their assistance during the period of the current study.
Funding statement
None to declare.
Conflict of interest
The authors declare no conflict of interest.
Data availability statement
The data that support the findings of this study will be openly made available in University of Nigeria, Nsukka Repository at https://oer.unn.edu.ng/reports?rdr=1.
Open access
Comments
Comments to the Author: It is understood that a correction was made by the author in this research article. It is recommended to rewrite the title, associating it with "heat stress". In the "Materials and Methods" section, it is recommended to write the method a little bit abbreviated. Also, due to the low number of results in this article, it is recommended to be published as "short communication".