Introduction
Significant numbers of treatments to increase semen quality in males remain ambiguous. Traditional medicinal herbs are used extensively in different regions of the world and may be an alternative source of medicine for increasing reproductive performance and semen quality or therapy of infertility due to their low adverse effects compared with chemical drug use (Rabeh, Reference Rabeh2016; El-Zaher et al., Reference El-Zaher, Eid, Shaaban, Ahmed-Farid, Abd El Tawab and Khattab2021).
Alpinia galanga rhizome (AGR) is an important member of the Zingiberaceae family (Saboo et al., Reference Saboo, Chavan, Tapadiya and Khadabadi2014). It is a source of natural antioxidants and flavonoids such as galangin, alpinin, kampferide, and 3-dioxy-4-methoxy and vitamins A, B, and C (Mayachiew and Devahastin, Reference Mayachiew and Devahastin2008). Consuming it in the diet can raise sperm quality and thus increase the fertility rate (Ghasemzadeh et al., Reference Ghasemzadeh, Jaafar and Rahmat2010). AGR may enhance male fertility by increasing sperm quality and gene expression (Fedder et al., Reference Fedder, Jakobsen, Giversen, Christensen, Parner and Fedder2014). Also, AGR extract has spermatogenic activity in adult males due to its chemical compounds and can be helpful in producing drugs to improve buck fertility.
Zinc is a micromineral that has a role in a variety of animal metabolic processes. It is involved in ∼200 proteins and enzymes that are crucial for male fertility and it was established that it is required for correct growth (Kumar et al., Reference Kumar, Verma, Singh, Varshney and Dass2006).
In male rabbits, Zn supplementation improves the physical characteristics of semen, such as ejaculate volume, sperm count, motility, seminal plasma antioxidants, and fertility rate (El-Speiy and El-Hanoun, Reference El-Speiy and El-Hanoun2013; Amen and Muhammad, Reference Amen and Muhammad2016) and increases the secretion of the main sex hormones [follicle-stimulating hormone (FSH) and interstitial cell-stimulating hormone]. As a result, male rabbit reproduction is improved (Ogbu and Ezeokoli, Reference Ogbu and Ezeokoli2016). The objective of the current study was to investigate the synergistic effects of Alpinia galanga rhizomes (AGR) with or without Zn on the reproductive performance and characteristics of California rabbit bucks.
Materials and methods
Housing and management
The current work was carried out at El-Sabahia Poultry Research Station, which belongs to the Animal Production Research Institute, Agriculture Research Center, Giza, Egypt. Rabbits were housed in 60 × 55 × 40 cm wire galvanized cages in a naturally ventilated building. In the cages, pellet feeders and automatic drinkers were used; fresh water and feed were allowed ad libitum. An experimental diet, as mentioned in Table 1, was made and pelleted to meet the nutrient requirements of the rabbits (NRC, 1977).
Table 1. Composition and chemical analysis of the basal experimental diet
* Each 3 kg of premix contains: Vit. A: 12,000,000 IU; Vit. D3: 3,000,000 IU; Vit. E: 10.0 mg; Vit. K3: 3.0 mg; Vit. B1: 200 mg: Vit. B2: 5.0 mg Vit. B6: 3.0 mg: Vit. B12: 15.0 mg; biotin: 50.0 mg; folic acid: 1.0 mg; nicotinic acid: 35.0 mg: pantothenic acid: 10.0 mg; Mn: 80 g; Cu: 8.8 g; Zn: 70 g; Fe: 35 g; I: 1 g; Co: 0.15 g and Se: 0.3 g. **DE (kcal/kg DM) = 2118 + 12.18 x (% CP) – 9.37 x (% ADF) – 3.83 x (% hemicellulose) + 47.18 x (% fat) + 20.35 x (%n onstructural carbohydrate) – 26.3 x (% ash).
Compliance with ethical standards
All experimental procedures were reviewed and approved by the Animal Production Research Institute, Agriculture Research Center, Giza, Egypt.
Materials plant and chemicals
Dried Alpinia galanga was purchased from a local market. Zinc sulfate (ZnSO4, molar mass: 161.47 g/mol, water soluble), was purchased from the El-Gomhoria Company for Chemical, Drugs and Medical Instruments, Alex, Egypt.
Experimental design
The study was conducted in two stages. First stage: in total, 36 California rabbit bucks (aged 5 months) with an initial body weight of 2980 ± 30.33 g were assigned randomly into six groups. Experimental treatments were as follows:
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Group 1: basal diet and served as control (C)
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Group 2: basal diet + 1 g AGR/kg DM (AGR1)
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Group 3: basal diet + 2 g AGR/kg DM (AGR2)
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Group 4: basal diet + 200 mg zinc sulfate/litre water (Zn)
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Group 5: basal diet + 1 g AGR/kg DM + 200 mg ZnSO4/litre water (AGR1 + Zn)
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Group 6: basal diet + 2 kg AGR/kg DM + 200 mg ZnSO4/litre water (AGR2 + Zn)
AGR supplements (dry galangal powder) were added to feeds as a 1 or 2 kg/ton feed for groups 2 and 5 and groups 3 and 6, respectively, based on 50 or 100 mg/kg body weight doses of the buck/day/2 months (basically spermatogenesis time); dose suggested by Sarieh et al. (Reference Sarieh, Javad, Farzaneh, Mohammad and Mohammad2014).
Second stage: The previous bucks were used for fertilizer doses to study the impact of supplementation on fertility and reproductive traits of the different treatments. These animals represented the progeny of pooled semen and artificial insemination, eight does in each group (López and Alvariño, Reference López and Alvariño2000). Forty-eight mature untreated does (aged 6 months, nulliparous) with an initial body weight of 3050 ± 20.7 g were divided randomly into six treatments with eight individual replicates each. Only receptive females (red colour of vulva lips) were artificially inseminated with ∼30–40 million spermatozoa, in approximately three sequential equal parts. Does were given a 0.8 mg (0.2 ml) injection of gonadotropin-releasing hormone analogue (Buserelin, Suprefact®, Hoechst-Roussel, Germany; Receptal) at the time of insemination for the control and other supplements (Boussin, Reference Boussin1989). Does were artificially inseminated with semen as described previously by Adams (Reference Adams1981).
Feeds and diets analysis
Diet samples were analyzed for DM (method 930.15), ash (method 942.05), nitrogen (method 102 954.01), and ether extract (EE; method 920.39) (AOAC, 2000) using official methods.
Galangal active components analysis
Alpinia galanga rhizomes were subjected to the analysis of total bioactive components, as shown in Table 2. Folin-Ciocalteu reagent was used to determine total phenols (as gallic acid) in dry AGR 106 according to the method described by Kaur and Kapoor (Reference Kaur and Kapoor2002). Carotenoids as β-carotene were determined according to Nagata and Yamashita (Reference Nagata and Yamashita1992). At the same time, total flavonoids as (quercetin equivalent) content were determined by Miliauskas et al. (Reference Miliauskas, Venskutonis and van Beek2004). Total tannins (as tannic acid) were determined according to AOAC (2000) method. Saponins were determined by the method of Hiai et al. (Reference Hiai, Oura and Nakajima1976).
Table 2. Total concentrations of bioactive components identified in Alpinia galanga (AGR) on dry matter bases
The AGR sample was analyzed using mass spectrometry and gas chromatography (GCMS-QP 2010 system) (Shimadzu, Japan) was utilized to determine the components of the AGR extract using GC-MS. The sample was injected through an Rtx-5MS column at a rate of 0.9 ml min−1 at 260°C using helium as a carrier (30 m 0.25 mm, 0.25 m thick). The oven temperature was set to 61°C and the split injection mode was set at 50:1. At a 70 eV ionization potential, the ion source temperature was 230°C, whereas the interface temperature was 250°C. Based on their rate, the extract contents were identified using the NIST11 library (Gaithersburg, USA).
Spermatozoa characteristics
Semen was collected twice weekly from each buck by artificial vagina using a female teaser rabbit at the end of the trial, with a 3–4-day delay between each ejaculation. All ejaculates (an average of 120 samples for each treatment over the semen collection period) were kept at 37°C in a water bath until they were evaluated, which took no more than 15 min. Using a graduated tube, the volume of ejaculate (ml) was determined. Using a haemocytometer, the concentration of spermatozoa (number of sperm per ml) was determined (Smith and Mayer, Reference Smith and Mayer1955). A drop of sperm was examined under a low-power microscope with a hot stage at 37°C to determine the percentage of sperm with advanced motility. To measure mass motility rate, two drops of fresh semen were placed on a warmed slide and covered with a coverslip (20 × 20 mm). Mass sperm motility from at least three fields was examined at 37ºC under a phase microscope at ×40 magnification and assessed from 0 to 100%. A weak eosin solution was used at a rate of 1:99 before counting the cells, for evaluation of sperm concentration (×106/ml) using a haemocytometer slide. Total sperm output was calculated by multiplying semen ejaculate volume by semen concentration. The assessment of live and abnormal spermatozoa was performed using an eosin–nigrosine blue staining mixture. The percentage of live spermatozoa was determined using stains that penetrated cells with damaged membranes. The total number of motile sperm was calculated by multiplying the percentage of motile sperm by the total sperm outputs.
Libido (reaction time)
The reaction time (in seconds) from the time the doe was introduced to the buck until the buck began to mount and ejaculate during the first copulation was used to evaluate libido (sexual desire) (Ogbuewu et al., Reference Ogbuewu, Enumaibe, Kadurumba, Iwuji, Ogundu, Etuk, Opara, Okoli and Iloeje2013).
Blood collection
At the end of the experiment, blood samples (from bucks) were collected from 8:00 a.m. to 9:00 a.m. from the marginal ear veins under vacuum into clean tubes containing heparin for each treatment group before accessing feed and water. Blood plasma was obtained by centrifuging the blood at 3500 rpm for 20 min and stored at –20°C for later analysis.
Hormones assay
Plasma concentrations of oestrogen (E217β), progesterone (P4), and free testosterone (FT) levels were determined according to Wilke and Utley (Reference Wilke and Utley1987). Plasma FSH and LH concentrations were determined using a modified heterologous radioimmunoassay according to Bolt and Rollins (Reference Bolt and Rollins1983).
Determination of serum antioxidant and lipid profile
Biochemical analyses of plasma total antioxidant capacity (TAC) and malonaldehyde (MDA) were determined according to Ippoushi et al. (Reference Ippoushi, Ito, Horie and Azuma2005). For triglycerides (TG) (Fossati and Prencipe, Reference Fossati and Prencipe1982), total cholesterol was determined as described by Stein (Reference Stein and Tiez1986), high-density lipoprotein (HDL) was analyzed according to El Harchaoui et al. (Reference El Harchaoui, Arsenault, Franssen, Després, Hovingh, Stroes, Otvos, Wareham, Kastelein, Khaw and Boekholdt2009), low-density lipoprotein (LDL-c) was calculated using the formula: LDL-c {mg/dl = total cholesterol [HDL-c + (TG/5)]}, which was explained by Friedewald et al. (Reference Friedewald, Levy and Fredrickson1972).
Reproductive traits
Criteria were examined for all groups including fertility rate, litter size and weight at birth.
Statistical analysis
All data were subjected to analysis of variance as described in SAS (programme) (SAS, 2002 ). The significant means of differences among the groups were separated by Duncan’s multiple range test (Duncan, Reference Duncan1955) according to the following model:
$${\rm Y_{ij}= \mu + Tr_i} + e_{ij}$$
where Yij = observations, µ = overall mean, Tri = effect of ith treatment (i: 1–6), eij = experimental error.
Results and Discussion
Analysis of Alpinia galanga
Total phenols (12.34 mg/g DM) were found to be the most abundant component in Alpinia galanga, whereas carotenoids, total flavonoids, tannins, and saponins were found to be 0.57, 6.22, 2.16, and 0.27 mg/g DM, respectively, Also, high relative contents of thymol and carotol, and α-terpineol (25.25, 17.44, and 9.09% respectively) were found (Tables 2 and 3). According to the current findings, Alpinia galanga contains bioactive constituents as well as having antioxidant properties. These findings are in agreement with those of Abdullah et al. (Reference Abdullah, Subramanian, Ibrahim, Abdul Malek, Lee and Hong2015), Basri et al. (Reference Basri, Taha and Ahmad2017), and Rachkeeree et al. (Reference Rachkeeree, Kantadoung, Suksathan, Puangpradab, Page and Sommano2018), who discovered that Alpinia galanga was rich in bioactive components such as flavonoids, phenolic acids, and alkaloids, as well as flavones such as galangin, alpinin, and kaempferol.
Table 3. Chemical constituents identified by gas chromatography and mass spectrometry
Impact of Alpinia galanga and zinc on hormonal profile
Table 4 shows that the groups given AGR2 + Zn had considerably higher FSH, LH, FT, P4, and E217β concentrations than the control group, whereas FSH and E217β activity was statistically comparable across the AGR1 + Zn and AGR2 + Zn groups. When compared with the control group, the group treated with just Zn had improved FSH, LH, FT, and E217β activity. However, compared with the control group, the treated groups with different agents had significantly higher activity of FSH, LH, FT, and E217β hormones.
Table 4. Effect of Alpinia galanga and zinc supplementation on plasma hormonal profile of California rabbit bucks
Superscripts with different small letters (a–c) within the same row indicate significant difference at P < 0.05.
C: control. AGR1: Alpinia galanga powder in diet at 50 mg/kg body weight (BW); AGR2: Alpinia galanga powder in diet at 100 mg/kg BW, Zn: 200 mg/litre water Zn; AGR1 + Zn = Alpinia galanga powder in diet at 50 mg/kg BW + 200 mg/litre water Zn; AGR2 + Zn = Alpinia galanga powder in diet at 100 mg/kg BW + 200 mg/litre water Zn. E217β: oestrogen; FSH: follicle-stimulating hormone; FT: free testosterone; LH: luteinizing hormone; P4: progesterone.
The activity of medicinal plant extract directly activated the hypothalamic–pituitary–testicular axis, which regulates the gonads, and is hypothesized to be associated with increased serum levels of LH and FSH. Alpinia galanga is also a well known medical herb plant with antioxidant potential, according to Sharma et al. (Reference Sharma, Kumar, Sharma, Qayum, Singh, Dutt, Paul, Gupta, Verma, Satti and Vishwakarma2018). According to Mazaheri et al. (Reference Mazaheri, Shahdadi and Nazari Boron2014) and Sarieh et al. (Reference Sarieh, Javad, Farzaneh, Mohammad and Mohammad2014), rats given an alcoholic extract of Alpinia galanga had substantially higher serum testosterone levels, and also LH, and FSH levels. Conversely, Honmore et al. (Reference Honmore, Kandhare, Kadam, Khedkar, Sarkar, Bodhankar, Zanwar, Rojatkar and Natu2016) found that Alpinia galanga possessed various biological features such as galangin, saponins, and a flavonol, flavonoids that have a leading influence on the testes, in response to an increase in testosterone production concentration. Surprisingly, Negm and Ragheb (Reference Negm and Ragheb2019) found that supplementation with Alpinia galanga resulted in considerable favourable effects on testes, including an increase in serum total testosterone levels, FSH, and LH concentrations. Also, according to Kolangi et al. (Reference Kolangi, Shafi, Memariani, Kamalinejad, Bioos, Jorsaraei, Bijani, Shirafkan and Mozaffarpur2019), Alpinia galanga, a traditional medication for men, can improve the reproductive profile hormones (testosterone and FSH).
Regarding the function of zinc on hormonal profiles, our findings agreed with those of Abdel-Wareth et al. (Reference Abdel-Wareth, Al-Kahtani, Alsyaad, Shalaby, Saadeldin, Alshammari, Mobashar, Suleiman, Ali, Taqi, El-Sayed, El-Sadek, Metwally and Ahmed2020), who found that supplementing nano-zinc oxide in the diet of male Californian rabbits increased testosterone levels in the serum. El-Speiy and El-Hanoun (Reference El-Speiy and El-Hanoun2013) also found that administering zinc sulfate to rabbits bucks enhanced testosterone, FSH, and LH hormones.
Impact of Alpinia galanga and zinc on lipid profile and antioxidant status
Table 5 shows that all groups using AGR with or without zinc had significantly lower concentrations of triglycerides, total cholesterol, low-density lipoprotein, and MDA, while HDL-c and TAC were significantly increased but were significantly decreased compared with the control group. These findings are consistent with those of Kaushik et al. (Reference Kaushik, Kaushik, Yadav and Pahwa2013), who discovered that the AGR extract reduced total cholesterol, blood triglycerides, and blood LDL while increasing HDL. Furthermore, Abdel-Azeem and Basyony (Reference Abdel-Azeem and Basyony2019) found that supplementing a diet with Alpinia galanga extract (AGRE) significantly reduced plasma total cholesterol, triglycerides, low-density lipoproteins, and total lipids, as well as TAC, glutathione S-transferase, superoxide dismutase (SOD), catalase, and glutathione peroxidase. Similarly, Negm and Ragheb (Reference Negm and Ragheb2019) found that supplementation with AGRE increased SOD levels while decreasing malondialdehyde (MDA) levels. Furthermore, there was a considerable drop in blood lipid profiles TG and TC, as well as a large increase in HDL-c. Our findings were consistent with Kumar and Alagawadi’s (Reference Kumar and Alagawadi2013) findings, which revealed that galangin enhanced liver function and serum lipid profile. According to Jantan et al. (Reference Jantan, Rafi and Jalil2005) the rhizomes of A. officinarum lowered serum TG and TC while increasing serum HDL levels in mice. In terms of the role of zinc, the findings of this study agreed with those of Boiko et al. (Reference Boiko, Honchar, Lesyk, Kovalchuk and Gutyj2020), who discovered that supplementing rabbit water with nano-zinc citrate reduced cholesterol, triglycerides, and lipid hydroperoxides while increasing glutathione reductase and catalase activity. Also, Mazani et al. (Reference Mazani, Argani, Rashtchizadeh, Ghorbanihaghjo, Hamdi, Estiar and Nezami2013) also found that Zn supplementation boosted overall antioxidant capacity, glutathione peroxidase, and SOD activity while decreasing MDA levels in the blood serum.
Table 5. Effect of Alpinia galanga and zinc supplementation on lipid profile and antioxidant status in plasma of California rabbit bucks
Superscripts with different small letters (a–c) within the same row indicate significant difference at P < 0.05.
C: control. AGR1: Alpinia galanga powder in diet as 50 mg/kg BW; AGR2: Alpinia galanga powder in diet as 100 mg/kg BW, Zn: 200 mg/litre water Zn; AGR1 + Zn = Alpinia galanga powder in diet as 50 mg/kg BW + 200 mg/litre water Zn; AGR2 + Zn = Alpinia galanga powder in diet as 100 mg/kg BW + 200 mg/litre water Zn. HDL-c: high-density lipoprotein; LDL-c: low-density lipoprotein; MDA: malondialdehyde; TAC: total antioxidant capacity; TC: total cholesterol; TG: triglyceride.
Impact of Alpinia galanga and Zn on reproductive traits
Table 6 displays the effects of AGR and Zn on semen characteristics. Results demonstrated that all supplemented agents significantly improved the ejaculate volume, advanced motility sperm concentration, cell integrity, fertility rate, and litter size at birth compared with the control group. Also, the diminishing reaction time, abnormal sperm and dead sperm were improved due to a significant decrease compared with the control group. Overall, group treatment with AGR1 + Zn and AGR2 + Zn illustrated highly significant improvement in the different treatments compared with the control group.
Table 6. Effect of Alpinia galanga and zinc supplementation on semen quality and reproductive traits of California rabbit bucks
Superscripts with different small letters (a–c) within the same row indicate significant difference at P < 0.05.
C: control. AGR1: Alpinia galanga powder in diet as 50 mg/kg BW, AGR2: Alpinia galanga powder in diet as 100 mg/kg BW, Zn: 200 mg/litre water Zn, AGR1 + Zn= Alpinia galanga powder in diet as 50 mg/kg BW + 200 mg/litre water Zn, AGR2 + Zn = Alpinia galanga powder in diet as 100 mg/kg BW + 200 mg/litre water Zn. AdM: advanced motility sperm conc.: sperm concentration; EjV: ejaculate volume; LSB: litter size at birth; RT: reaction time; SAb: sperm abnormal.
The current results indicated that Alpinia galanga contains bioactive compounds and antioxidant activity. These results are compatible with Rachkeeree et al. (Reference Rachkeeree, Kantadoung, Suksathan, Puangpradab, Page and Sommano2018) who found that Alpinia galanga is rich in bioactive compounds such as flavonoids, phenolic acids, and alkaloids and comprises various flavones such as galangin, kaempferol and alpinin. Also, Nampoothiri et al. (Reference Nampoothiri, Esakkidurai and Pitchumani2015) showed that oral consumption of Alpinia galanga had increased sperm motility and sperm counts in male mice without any spermatotoxic effect. Mazaheri et al. (Reference Mazaheri, Shahdadi and Nazari Boron2014) mentioned that the application of Alpinia galanga has also significantly increased the sperm rate, viability, and motility in male rats. Moreover, Mazaheri et al. (Reference Mazaheri, Shahdadi and Nazari Boron2014) illustrated that Alpinia galanga might improve male fertility by promoting sperm quality, and increasing sperm concentration, viability, motility and testosterone hormones. Conversely, a previous study by Kolangi et al. (Reference Kolangi, Shafi, Memariani, Kamalinejad, Bioos, Jorsaraei, Bijani, Shirafkan and Mozaffarpur2019) observed that Alpinia galanga could impact the melioration of sperm quality and sperm concentration without causing harmful effects. Its power is attributed to its antioxidant and scavenging activity against the ROS via its phytochemical chiefly including galangin. Finally, Bebars et al. (Reference Bebars, El Habeby, Issa and El-Dien2021) found that the extract of Alpinia galanga increased sexual behaviour and mount latency and produced excellent preservation of testicular structure in male rats. Conversely, Akbar (Reference Akbar2020) found that consumed Alpinia galanga led to increased reproductive function in male albino rats in the form of an increase in the testosterone hormone level and augmented sperm count. These effects were attributed to the direct action of the extract on the testes and moreover found that it improved semen quality, sexual desire, and erection.
Regarding the role of zinc on reproductive traits, Baiomy et al. (Reference Baiomy, Hassanien and Emam2018) documented that supplementation with ZnO significantly improved sperm concentration and live spermatozoa. Moreover, fertility rate and litter size at birth were larger than that of NZW rabbit bucks. Also, Amen and Muhammad (Reference Amen and Muhammad2016) recommended that supplementation of Zn led to improved physical characteristics of semen, including ejaculate volume, sperm count, motility, seminal plasma antioxidants and fertility rate and augmented the secretion of the major sex hormones (FSH and LH) in male rabbits. Conversely, Chia et al. (Reference Chia, Ong, Chua, Ho and Tay2000) recorded that for zinc there was a strong relationship between zinc and spermatogenesis.
Conclusion
It is concluded that the application of Alpinia galanga and zinc significantly increased sperm percentage, motility and reproductive hormones (testosterone, FSH, LH, E217β, P4). This suggested that this plant may be favourable for improved sperm quality and fertility parameters.
Acknowledgements
The authors acknowledge the Animal Production Research Institute, Agriculture Research Center for providing the infrastructure and staff necessary for this study.
Authors’ contributions:
All authors contributed substantially towards the paper.
Conflict of interest
The authors declare that they have no conflict of interest.
