Counteract severe heat stress by including different forms of zinc in the rabbit bucks’ diet

The harmful influences of global warming on rabbit reproduction and industry attract global attention. Zinc (Zn) is an important trace element with a wide list of functions in the male reproductive system. The aim of this study was to estimate the effects of different forms of zinc supplementation, as organic (Zn methionine), nano (nano Zn oxide) as indirect way to minimized it impact on environment, and inorganic (Zn sulphate) on physiological parameters, semen quality, anti-oxidative status, hormonal profiles of male rabbits subjected to server heat stress. Thirty-six V-line bucks (6–7 months old, 2842.29 ± 34.46 g weight) were randomly distributed to 4 groups, bucks in 1st group (control group, Con) fed basal diet without Zn supplementation, the 2nd, 3rd and 4th groups fed basial diet with 30 mg/kg of zinc methionine (Zn-Met), nano zinc oxide (ZnO-NPs), and zinc sulphate (Zn-S), respectively for a period of 10 weeks suffered from severe heat stress of environmental ambient temperature (over 32 of temperature humidity index, THI). Semen samples were collected and evaluated for volume, pH, motility, concentration, viability, initial fructose, and seminal plasma antioxidant concentration. Liver, kidney function, hormonal and antioxidant profile were measured in blood serum. The results revealed that, compared to control, all forms of Zn supplementation used significantly improved kidney function (creatinine), serum antioxidant (SOD and CAT), physiological parameters, especially on 1st month of the experiment, and seminal plasma antioxidant (SOD and CAT) of heat stressed bucks. Likewise, semen quality in terms of sperm concentration, sperm viability, and initial fructose enhanced significantly (P ≤ 0.05) by ZnO-NPs supplementation. Zinc methionine supplementation significantly improved liver function and decreased seminal plasma TBARs. Treatments with Zn-Met and ZnO-NPs increased seminal TAC and blood testosterone levels with reduced blood cortisol levels compared to other groups. Severe heat stress could be counteract by inclusion Zn with studied forms Zn-Met and ZnO-NPs at recommended dose (30 mg/kg diet) to improve semen quality and antioxidant status.

Zinc source, animal, and diets.Thirty-six fertile V-line male rabbits, 6-7 months old with body weight (2842.29 ± 34.46 g), were used.Live body weights of rabbit bucks were recorded at the beginning of the experiment and at the end.Body weight change was calculated.The bucks were individually caged in wire batteries with a normal daylight (16-17 h).All rabbits were adapted in the experimental rabbit shed for 2 weeks.The rabbits were randomly distributed into four groups (n = 9 bucks per group) as follows: the first group (control group) fed a base diet without Zn supplementation, the second, third and fourth groups fed a base diet with 30 mg/kg of zinc methionine (Zn-Met, Mintrix ® Zn was purchased from United BioMed for feed additives, Cairo, Egypt), nano zinc oxide (ZnO-NP, size of < 100 nm; surface area m 2 g −1 , purity > 97 obtained by Sigma-Aldrich, Saint Louis, USA) and zinc sulphate (ZnS provided by El-Gomhoria Co. for Chemicals and Pharmaceuticals, Alexandria, Egypt), respectively according to recommendation doses of 21,22 for ZnO NPs and close to recommendation doses of 23 for Zn-Met and ZnS.The experiment was carried out according to relevant guidelines and regulations.The experiment lasted 10 weeks.Feed and fresh clean water were provided ad libitum.The basic diet was formulated as (16% yellow corn grain, 7% barley, 19% wheat bran, 20% soybean, 24% clover hay, 10% wheat straw, 2% beet molasses, 0.3% premix, 0.2% calcium carbonate, 0.8% dicalcium phosphate, 0.5% salt, 0.15% l-lysine, 0.05% methionine DL) and the calculated chemical composition was (88.71% dry matter, 17.21% crude protein, 14.81% crude fiber, 2.47% fat, 8.25% ash) according to the recommendation of 24 .
Microenvironment data.Throughout the experimental period (10 weeks), ambient temperature (°C) and relative humidity (%) were recorded daily at midday using a Mercury-room thermometer and hygrometer, respectively.the weekly temperature-humidity index (THI) was determined by the equation defined by 25 .The mean values of THI were classified according to 26 .

Physiological parameters.
The rectal temperature (RT) and the respiration rate (RR) were recorded twice (4th and 8th weeks of the experiment) at 12:00-2:00 PM.A clinical digital thermometer and stopwatch were used to measure RT by inserting the thermometer 2 cm into the rectum for 2 min, inclined toward the wall of the rectum.Visual counting of nasal movement per 1 min without disturbing the rabbits by using a hand counter to determine the RR.
Semen collection and evaluation.The semen samples of each rabbit buck were collected weekly in the morning (8 am) for 10 weeks using an artificial vagina by exposing a mature female rabbit to the bucks as a teaser during collecting.After collection, the ejaculate volume without gel was recorded in graduated collecting tubes to the nearest 0.01 ml (excluding the gel plug).Semen pH was directly measured by using pH paper strips (range: 0-14, resolution: 1.0 pH unit; Sigma-AldrichVR).Sperm mass activity was determined manually according to 27 , giving an arbitrary score of 0 to 3. The individual progressive motility was examined by diluted semen with normal saline (1:100) and transferred to a warm glass slide (37 °C) covered with a cover slip and examined under a light microscope (400× magnification) on a scale of 0 to 100% according to 28 .Sperm concentration (*10 6 /mm 3 ) was evaluated in duplicate using a Neubauer hemocytometer after dilution with distilled water (1:200).Sperm viability was determined using semen smears mixed with Eosin-Nigrosin stain and viable and non-viable spermatozoa were determined by counting 200 sperm under a light microscope 29 .The initial fructose concentration of the collected semen was determined by a spectrophotometer using commercial kits (Biodiagnostic, Egypt) according to the manufacturer's instructions.At the 10th week of the experiment, the seminal plasma of 4 bucks of each treatment was separated from the mass of the sperm cells by centrifugation at 700g for 15 min.The supernatant (seminal plasma) was collected and kept at − 20 °C until analysis.Seminal plasma SOD, CAT, TBARs, and TAC were verified by colorimetric techniques using commercial kits (purchased from Bio-diagnostic, Giza, Egypt), according to the manufacturers' instructions.
Blood biochemical.At the end of 10th week of the experiment period, blood samples were collected morning before adding feed, from the margin ear of bucks after disinfecting with a sanitizer solution in sterilized tube.Then the blood samples were centrifuged at 700g for 10 min to obtain serum.The serum obtained was transferred to sterile Eppendorf ® tubes and stored at − 20 °C.Serum samples were analyzed for AST and ALT according to 30 , urea 31 , creatinine 32,33 , SOD 34 , CAT 35,36 , TBARS 37 , and TAC 38 .These analyses were performed using commercial kits (acquired from Bio-diagnostic, Giza, Egypt).
The cortisol (MBS2005085) and testosterone (MBS704954) enzyme-linked immunosorbent assay (ELISA) MyBioSource kits (sunny Southern California, San Diego, USA) were employed in quantifying the concentrations of cortisol and testosterone, respectively, following the manufacturer's protocols.

Statistical analyses.
The experimental data were subjected to a one-way analysis of variance analyzed (ANOVA) via the GLM procedure using Statistical Analysis Software (IBM SPSS Statistics for Windows, Version 20, Chicago, IL, USA).Each buck rabbit is an experimental unit that provides an independent measurement.All percentage data were arc-sine transformed prior to the approximate normal distribution.The Tukey test was used to compare the differences between the means when significant F values were observed at the levels of p < 0.05, according to the following statistical model: One-way model: YiK = μ + Xi + eik where: YiK = the response variable; μ = the overall mean; Xi = the fixed effect of treatment (control, 30 mg Zn MET, 30 mg Naon ZnO, and 30 mg ZnSo 4 ); eik = the residual error.Figures were fitted by the SigmaPlot (software 14.0 Systal software Inc).

Ethics approval and consent to participate. All experimental procedures and handling of buck rabbits
were approved by the Alexandria University Institutional Animal Care and Use Committee.

Results
Microclimate and physiological parameters.Figure 1 shows the weekly ambient temperature, humidity, and THI during the experiment period.The weekly temperature humidity index under the experimental conditions ranged from 31.58 to 38.49.The recorded THI values clearly showed that the bucks suffered severe heat stress.Physiological parameters (RR and RT) during 1st and 2nd months of the experimental period are presented in Fig. 2. The result revealed that all sources of Zn supplementation in this study significantly succeeded in decreasing both RR and RT values of heat stressed bucks on the first month and RR only on the second month.From Fig. 2 it can be shown that the inclusion of zinc in heat-stressed buck rations still has an effect on RT but the effect is not significant.1 indicated that the initial and final body weight of heat stressed bucks did not differ in all experimental groups.While the change of body weight due to zinc treatments was significant compared to the control group Table 2 shows that the most semen parameters were significantly improved by zinc supplementation except semen pH, mass motility and individual motility..The highest semen volume was collected from the ZnS group.While both ZnO NPs and ZnS significantly recorded the highest sperm concentration compared to other treatments.All zinc treatments significantly enhanced the percentage of viability of the sperm.The seminal plasma initial fructose level (P < 0.05) was increased by inclusion zinc with different forms in heat stressed bucks' rations compared to the control group and this effect was significant with zinc Met and ZnO NPs only.

Body weight and semen quality. The data in Table
The antioxidant status of seminal plasma of heat stressed bucks such of SOD and CAT levels (Fig. 3) were significantly improved by zinc treatment with different sources.As well, the TBAR concentration in seminal plasma showed a decrease in all zinc supplementation groups compared to the control group and this decrease was significant with Zn Met.  3, blood antioxidant status, whereas, SOD, CAT and TAC were improved and TBARS was decreased by adding Zn from different sources to heat stressed bucks rations and this effect was significant with SOD and TAC.Furthermore, the results of our study in Fig. 4 showed that all zinc treatments decreased serum cortisol levels with elevated serum testosterone concentrations of heat stressed bucks.

Discussion
Temperature humidity index (THI) is used to evaluate the degree of heat stress by the combined both ambient temperature and relative humidity estimate the severity of heat stress.This index is widely used in hot and humid areas around the world to assess the impact of heat stress 39 .The weekly THI values clearly indicated that the buck rabbits suffered severe thermal stress.Zinc is an effective antioxidant and plays a vital physiological role for animal health, synthesis of more than 300 enzymes involved in metabolism process 40 , synthesis proteins, immune system activation, bone development 41 , DNA synthesis, cell division, gene expression, and biological functions 42 .In addition to reducing the negative impact of both cold and heat stressors 43 .Zinc methionine is organic forms of Zn are generally bound to methionine amino-acid complex, it may be better absorption and more available to animal by interaction with other elements, especially Cu, Fe, and Ca 44 .Nano-zinc oxide is smaller than 100 nm causes increasing absorption rate and availability 40 , and is more biologically effective 45 by www.nature.com/scientificreports/bypassing conventional physiological ways of nutrient distribution, and transport across tissue and cell membranes, as well as protecting compounds against destruction before reaching their targets 19 , and reaching target cells more quickly than ordinary minerals with larger particle size 20 .At the same time, using nano-Zn oxide is useful to avoid excessive supplementation with Zn in animal diets and lowest the accumulation of Zn in soils after intensive animal farming 46 , thus reducing the potential risks for environmental pollution 18,47 .Physiological parameters such as respiration rate and rectum temperature are considered direct indexes of climatic stress 48 and accordingly measure the degree of discomfort/comfort or adaptability of bucks.Our result revealed that all sources Zn supplementation significantly decrease RR and RT values of heat stressed bucks on 1st month and RR only on 2nd month.The positive impact of zinc on physiological parameter of heat stressed   buck may be due to that zinc reduced stress on the rabbits by enhancing the bioavailability of other minerals 49 .
The insignificant RT values in the 2nd month of all zinc treatments may be due to the development of metabolic mechanisms of all experimental bucks to adapt to heat stress to a tolerable level.The initial and final body weights of the heat-stressed bucks did not differ in all experimental groups.Although the body weight change of all zinc treatments was significantly greater compared to the control bucks, that cleared that all zinc treatments alleviated the negative effect of heat stress.
Zinc sulphate treatment significantly improved semen volume, sperm concentration and sperm viability; a similar improvement was obtained by 50,51 who concluded a significant increase in semen volume, total live sperm concentration and sperm mobility due to zinc sulphate dietary supplementation in heat-stressed rabbit males.Increased semen volume of Zn-s treatment may contribute to increased prostatic fluid 9 .Furthermore, nanozinc significantly increased each of sperm concentration, sperm viability, and initial fructose compared to the control group could be due to improvement in buck antioxidant status 52 .Similar results were found by 45 who reported that nanoparticles have biological effects that improve male fertility by enhancing sperm quality.Also, 53 observed that supplemented young rams' diet with 50 mg/kg or 100 mg/kg nano zinc practical improved epididymal semen quality.Increasing the percentage of sperm viability of nanozinc bucks may be due to improvement the functionality of sperm plasma membranes without any significant effect on motility parameters 54 .Furthermore, the improvement in sperm concentration of bucks fed a diet supplemented with nano-zinc could be due to increased antioxidant enzymes activity of testicular tissue 55 or seminal plasma in the current study that protects sperm against oxidative stress.In addition, Zn-Met significantly enhanced the viability sperm percentage and initial fructose in the present findings.
Additionally, the membrane of rabbit sperm cells is rich in polyunsaturated fatty acids that make sperm susceptible to lipid peroxidation 56 .Other semen characteristics such as pH and motility (mass and individual) Table 3.Effect of the different zinc sources on liver and kidney function blood biochemical of heat stressed V-line buck rabbits.Means in row with different superscript letters (a-b) are significantly different (p ≤ 0.05).Zn-Met = zinc methionine; ZnO-NPs = zinc oxide nanoparticales; Zn-S = zinc sulphate.www.nature.com/scientificreports/ were not differ among experimental groups.Our results agree with those of 57 and 58 who reported that supplemental organic and non-organic zinc improved quantitative and qualitative characteristics of semen reproductive efficiency 59 and fertility by lower blood kidney parameters that decrease ROS production and increased progressive motility, acrosome reaction and capacitation 60 .Viable sperm percentage did not effect by all zinc treatments in the current results, this observation has a harmoniousness with previous studies of 9,61 .In general, zinc affects reproductive characteristics by stimulating and protecting the germinal epithelium of seminiferous tubules and promoting the production and secretion of testosterone 62 , as shown in the present findings, and therefore influences spermatogenesis 63 .Besides, the positive result of all zinc treatments on sperm concentration and spermatogenesis may be due to the production of sperm necessitates extensive cell division by influencing mitotic and meiotic cell divisions, synthesis of DNA and RNA by improving the activity of DNA and RNA polymerase (Zn-containing enzymes) 64 .Increasing initial fructose in all zinc treated bucks as compared to the control bucks may be attributed to the effect of treatments on fructogenesis in seminal vesicles of heat stressed rabbits.Antioxidant status such as SOD and CAT levels of the seminal plasma of heat-stressed bucks in the present findings were significantly improved by different sources of zinc treatments.Although zinc methionine supplementation was more effective in reducing the seminal plasma TBAR concentration compared to other groups.These results agreed with those presented by 65 who reported that Zn supplementation had a beneficial effect on the anti-oxidative status of seminal plasma, which may provide better protection to spermatozoa from oxidative damage, and also improve the concentration of testosterone as is shown in the current study which may play a role in the production of good quality semen.
Liver functions were significantly improved as AST and ALT concentrations were reduced in Zn-Met group compared to the control group with no significant effect of other zinc treatments.The positive effect of Zn treatments on semen quality may be due to the revealed negative relationship between liver enzymes such as ALT and GGT and sperm concentration, total testosterone and LH due to the formation of pro-oxidant species formation 66 .Our results were in agreement with 67,68 who found that supplementation with ZnO-NP in rabbit diets significantly reduced serum levels of AST and ALT activities compared to the control group.All zinc treatments improved kidney function as reflected in the decrease in creatinine values.The current results were agreed with 69,70 that found the diets supplemented with 50 and 100 mg/kg of Zn-Met significantly decreased blood creatinine concentration compared to the control group.On the other hand, the result disagreed with 71 who reported that plasma creatinine concentration did not change in rabbit fed diet supplemented with Zn.
Heat stress decreases antioxidant status, which is one of the most important physiological changes in response to high ambient temperature in animals that increases oxidative stress and immune suppression 72 .Dietary supplementation of different Zn sources in the current study improved the serum antioxidant status (SOD and TAC levels) with no significant effect between ZnO NPs and control groups.The improvement in blood antioxidant status may be due to zinc being considered an effective antioxidant by having catalytic, coactive, or structural functions in different enzymes such as SOD, which controls many physiological processes such as metabolism and immune function 73 , or by inhibiting the oxidation of macromolecules such as DNA and proteins 17 or by maintaining the activities of radical scavenging enzymes 40 .The improvement in blood antioxidant status may be associated to the enhancing in seminal antioxidant status as mentioned earlier in our study, which illustrated that zinc prevents the oxidative damage to the sperm cells and thereby improve the fertility 11 .From Fig. 3 TBARs seminal plasma was affected by Zn supplementation, whereas, TBARs was decreased in Zn treatments compared to the control group but this effect was nonsignificant.Our results disagree with the findings of 74 who showed that plasma TBARs and plasma lipid peroxides concentration was reduced significantly by zinc supplementation in rabbit diets.
Animals that exposed to ecological stressors such as extremes of temperatures, neurons in the CNS are stimulated, that activated of the hypothalamic pituitary adrenal axis leading to stimulate the cortex of the adrenal gland to produce and release corticosteroids (cortisol) 75 .However, zinc had an effect on the severe heat stress by decreasing cortisol levels which observed in the current study, whereas all zinc treatments declined serum cortisol levels.Testosterone as a sex hormone has a positive impact on semen quality and reproductive physiology 76 .In the present findings, all zinc forms led to elevated serum testosterone concentration of heat stressed bucks, which reflected the improvement in semen characteristics which was in consistency with previous studies.Many studies supported our results on improving blood testosterone concentration 61 , which helps in building protein, and is necessary for sexual desire and erections 77 .

Conclusions
Current data revealed a significant positive improvement in body weight change, blood kidney function (creatinine), serum antioxidant (SOD and CAT), physiological parameters, especially in the 1 st month of experiment, and seminal plasma antioxidant (SOD and CAT) of heat stressed bucks with the three forms of dietary Zn supplementation.Furthermore, semen quality in terms of sperm concentration, viability, and initial fructose improved significantly with ZnO-NPs supplementation.Zinc methionine supplementation significantly improved liver function and reduced seminal plasma TBARs.Both ZnO-NPs and Zn-Met improved seminal TAC and blood testosterone with reduced blood cortisol levels.Therefore, it concluded that the recommended forms of Zn in the dose of 30 mg/kg diet are zinc methionine and ZnO-NPs, rather than ZnS during the severe heat stress conditions to improve semen quality, and serum and seminal antioxidant status of buck rabbits.

Figure 1 .
Figure 1.Microclimate date of the experimental period (temperature, humidity, and temperature humidity index, THI).

Figure 2 .
Figure 2. Effect of the different zinc sources on physiological parameters, respiration rate (RR) and rectum temperature (RT), of heat stressed V-line buck rabbits during 1st half and 2nd half of the experiment.Zn-Met = zinc methionine; ZnO-NPs = zinc oxide nanoparticales; Zn-S = zinc sulphate.

Figure 3 .
Figure 3.Effect of the different zinc sources on seminal plasma antioxidant status of heat stressed V-line buck rabbits.Zn-Met = zinc methionine; ZnO-NPs = zinc oxide nanoparticales; Zn-S = zinc sulphate.

Figure 4 .
Figure 4. Effect of the different zinc sources on blood hormones of heat stressed V-line buck rabbits.Zn-Met = zinc methionine; ZnO-NPs = zinc oxide nanoparticales; Zn-S = zinc sulphate.

Table 3
, liver functions (serum concentrations of AST and ALT) were decreased significantly with Zn-Met supplementation compared to the control group, while Vol:.(1234567890)Scientific Reports | (2023) 13:12987 | https://doi.org/10.1038/s41598-023-39928-3www.nature.com/scientificreports/ the other Zn treatments did not differ than the control group.All zinc treatments improved kidney function as reflected in decreasing creatinine level.From Table

Table 1 .
Effect of the different zinc sources on body weight of heat stressed V-line buck rabbits.

Table 2 .
Effect of the different zinc sources on semen quality of heat stressed V-line buck rabbits.