Introduction

Recently, global warming and heat stress represent the main challenges that contribute to the significant reduction in rabbit semen quality, leading to a significant decrease in sexual behavior, hormonal imbalances, an increase in abnormal sperm; a drop in sperm production, and finally a decrease in fertilization capacity and fertility1, especially with the highly sensitively to heat stress due to lack of sweat glands, except the ear1. Moreover, many blood biochemicals dis-balances in male rabbit were occurred by exposure to thermal stress2. Sperm are rich in polyunsaturated fatty acids with low levels within the cytoplasm3. Heat stress caused a higher level of inflammatory responses in the blood, resulting in a decrease in immunological function and a potential for apoptosis spermatozoa4. In addition, thermal stress caused an increase in oxidative stress, which in turn increased tissue damage by accelerating lipid peroxidation2 and significantly weakening antioxidant defense5, with lipid peroxidation followed in cellular damage6.

Several methods have been used to prevent the negative impacts of heat stress in buck rabbits, including phytogenic, minerals, and vitamins7. Many microelements are successfully used to reduce the damaging effects or avoid harmful impacts of heat stress1. Zinc is one of many series components that control the oxidative stress of cells8. In addition, zinc is the second richest trace element after iron, which plays an important role in the male reproductive process because of their high activity at the molecular level9,10, and involved in various enzyme activities and hormone functions11. Zinc is an effective antioxidant owing key role to catalytic, coactive, or structural roles in different cofactor including more than 300 enzymes12 such as SOD; more than 2000 transcription factors13, involved in the biosynthesis of DNA and cell division14, gene expression15, and immune protection16. Overall, zinc acts as an antioxidant stress agent by inhibition of oxidation of macromolecules such as DNA and proteins, as well as the inhibition of inflammatory response, subsequently downregulation of ROS production17. Inorganic zinc bioavailability in animals is low and large amounts of zinc are excreted into faeces, resulting in environmental pollution and waste of zinc resources. Nano-zinc can be used at lower doses and indirectly prevents environmental contamination18. The nano-zinc (particle size is below 100 nm) is more effective, available, and absorbed easier19, while it bypasses through the stomach wall and into body cells more quickly than ordinary minerals with larger particle size20.

Therefore, the objective of the present study was to determine whether the use of various zinc sources, such as zinc methionine, nano zinc oxide and zinc sulphate, under heat stress conditions could improve the semen of the quality of the rabbit bucks, their hormonal status, and their antioxidant status.

Material and method

Location and ethics statement

The study was carried out at the Department of Animal and Fish Production, Faculty of Agriculture (Saba- Basha), Alexandria University, Alexandria, Egypt (31° 12′ 20.7108′′ N, 29° 55′ 28.2936′′ E). All experimental procedures and handling of buck rabbits were approved by the Alexandria University Institutional Animal Care and Use Committee (Approval Number: 03/19/03/18/1/15), during the summer season (July to September 2021). The experiment was carried out according to relevant guidelines and regulations. Our animal research reports follow the recommendations of the ARRIVE Guidelines.

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 m2 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 of21,22 for ZnO NPs and close to recommendation doses of23 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 of24.

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 by25. The mean values of THI were classified according to26.

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 to27, 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 to28. Sperm concentration (*106/mm3) 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 microscope29. 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 to30, urea31, creatinine32,33, SOD34, CAT35,36, TBARS37, and TAC38. 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 ZnSo4); 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.

Figure 1
figure 1

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

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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.

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Body weight and semen quality

The data in Table 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.

Table 1 Effect of the different zinc sources on body weight of heat stressed V-line buck rabbits.
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Table 2 Effect of the different zinc sources on semen quality of heat stressed V-line buck rabbits.
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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.

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.

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Biochemical and hormonal analysis

As described in Table 3, liver functions (serum concentrations of AST and ALT) were decreased significantly with Zn-Met supplementation compared to the control group, while 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 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.

Table 3 Effect of the different zinc sources on liver and kidney function blood biochemical of heat stressed V-line buck rabbits.
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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.

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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 stress39. 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 process40, synthesis proteins, immune system activation, bone development41, DNA synthesis, cell division, gene expression, and biological functions42. In addition to reducing the negative impact of both cold and heat stressors43. 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 Ca44. Nano-zinc oxide is smaller than 100 nm causes increasing absorption rate and availability40, and is more biologically effective45 by bypassing conventional physiological ways of nutrient distribution, and transport across tissue and cell membranes, as well as protecting compounds against destruction before reaching their targets19, and reaching target cells more quickly than ordinary minerals with larger particle size20. 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 farming46, thus reducing the potential risks for environmental pollution18,47.

Physiological parameters such as respiration rate and rectum temperature are considered direct indexes of climatic stress48 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 minerals49. 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 by50,51 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 fluid9. 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 status52. Similar results were found by45 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 parameters54. 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 tissue55 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 peroxidation56. Other semen characteristics such as pH and motility (mass and individual) were not differ among experimental groups. Our results agree with those of57 and58 who reported that supplemental organic and non-organic zinc improved quantitative and qualitative characteristics of semen reproductive efficiency59 and fertility by lower blood kidney parameters that decrease ROS production and increased progressive motility, acrosome reaction and capacitation60. Viable sperm percentage did not effect by all zinc treatments in the current results, this observation has a harmoniousness with previous studies of9,61. In general, zinc affects reproductive characteristics by stimulating and protecting the germinal epithelium of seminiferous tubules and promoting the production and secretion of testosterone62, as shown in the present findings, and therefore influences spermatogenesis63. 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 by65 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 formation66. Our results were in agreement with67,68 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 with69,70 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 with71 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 suppression72. 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 function73, or by inhibiting the oxidation of macromolecules such as DNA and proteins17 or by maintaining the activities of radical scavenging enzymes40. 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 fertility11. 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 of74 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 physiology76. 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 concentration61, which helps in building protein, and is necessary for sexual desire and erections77.

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 1st 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.