Influence of allicin administration on reproductive efficiency, immunity and lipid peroxidation of rabbit does under high ambient temperature
Abstract
This study investigated the effect of daily oral administration with allicin levels (0, 5 and 10 mg/kg of female body weight), 30 days pre-insemination, on reproductive performance in vivo and in vitro, immunity, and oxidative stress of rabbit does under high ambient temperature. Niliparous NZW does (n = 105) were randomly divided into three groups (35 in each) treated with 0, 5 and 10 mg allicin dissolved in 2 ml distilled water, respectively, for 30 days pre-insemination. At the end of treatment (30 days), does were artificially inseminated with fresh diluted semen of 20 fertile NZW bucks. Reproductive performance and ovulatory response parameters were determined. Serum biochemicals, enzyme activity, immunoglobulins (IgG and IgM) and antioxidant status were determined on day 30 of treatment. Serum progesterone and prolactin were determined pre-insemination (30 days of treatment), on 15 days of pregnancy and 7 days post-partum. Results showed that both allicin levels increased live litter size, and bunny viability rat and litter size at birth and weaning. Allicin levels increased ovulation rate and improved embryo quality. Number of total follicles decreased only with 10 mg allicin. Progesterone increased pre-insemination, 15 days of pregnancy and 7 days post-partum progesterone by allicin levels. Prolactin pre-insemination and on day 7 post-partum increased with 10 mg allicin. Serum total proteins, albumin, globulin, IgG and IgM increased, while glucose, aspartate and alanine aminotransaminases, and thiobarbituric acid reaction decreased by both allicin levels. In conclusion, the mechanism by which allicin administration 30 days pre-insemination to improve the reproductive performance of rabbit does is based on that allicin can play an important role, as a natural exogenous antioxidant, increasing immune response and reducing lipid peroxidation.
1 INTRODUCTION
There are a variety of physiological and pathological factors causing a decrease in reproductive activity of intensively farmed rabbits (Castellini, 2007). The exposure to metabolic, environmental or nutritional stressors induces oxidative stress which may alter several biological activities, cellular and intracellular levels (Ibrahim, Eweis, El-Beltagi, & Yasmin, 2012), gonado–pituitary hormone (LH and FSH) disturbance, increases lipid peroxides and decrease enzymatic antioxidant activity (Al-Masri, 2015). Moreover, exposing to high ambient temperature leads to reduced reproductive and productive performance of rabbit does, and consequently results in substantial economic loss (Attia, Abd El-Hamid, Bovera, & El-Sayed, 2009). Improving the performance of animals by different agent additions can be achieved under heat stress (Marai, Habeeb, & Gad, 2003). Natural antioxidant supplementation has the ability to prevent cellular damage in antioxidant defence system by counteracting the oxidants and other cellular protection (Mittler, Vanderauwera, Gollery, & van Breusegem, 2004), which play a vital role in enhancing the reproduction and health status of rabbit does (El-Ratel, Abdel-Khalek, El-Harairy, Sara, & Lamiaa, 2017).
The phytobiotics have attracted major attention in animal production due to their different biological activities, including antioxidant, anti-microbial (Vamsi Duvvu, Ananda Rao, Venkata Seshaiah, & Srinivas Kumar, 2018), immunomodulatory, anti-inflammatory (Hanieh et al., 2010), hypolipidemic, antitoxicity and hypocholesterolaemic properties (Al-Shuwaili, Ibrahim, & Naqi Al-Bayati, 2015), and inhibition of lipid peroxidation and promotion of the activities of antioxidant enzymes (Marsoul, Abbood, & Abbas, 2016). Among these phytobiotics, garlic (Allium sativum) is one of the most essential and useful herbs for various medicinal purposes (Adulugba, Goselle, Ajayi, & Tanko, 2017).
Allicin (diallylthiosulphinate) is a defence molecule from garlic (Allium sativum L.) with a broad range of biological activities with chemical structure as C6H10OS2. Allicin is produced upon tissue damage from the non-proteinogenic amino acid alliin (S-allylcysteine sulphoxide) in a reaction that is catalysed by the enzyme alliinase (Borlinghaus, Albrecht, Gruhlke, Nwachukwu, & Slusarenko, 2014). This compound is capable of binding the free-radicals, as an agent against stress (El-Katcha, Soltan, Sharaf, & Hasen, 2016). The hypoglycaemic and hypolipidaemic effects of allicin were proved in broilers (Singh et al., 2017) and pigs (Omojola, Fagbuaro, & Ayeni, 2009), while impact of allicin on reducing enzyme activity of kidney and liver and improving immune response was reported in broiler chicken (El-Katcha et al., 2016). Recently, allicin, as a strong natural antioxidant, is used to increase the endogenous antioxidant enzymes activity and reduce inflammation and oxidative stress of male rabbits (Alam et al., 2018). Also, allicin can prevent apoptosis of rats by activating extracellular signal-regulated kinase (Abdel-Daim, Kilany, Khalifa, & Ahmed, 2017). Generally, phytochemicals enhance immune responses, reduce blood lipid profile and lipid accumulation (Abdelnour et al., 2019; Kata et al., 2018) and regulate the enzyme activity of glycolytic and gluconeogenic processes in the liver rats (Chetna et al., 2019).
Nevertheless, those limited observations do not give further support to the hypothesis that allicin may have impact on the reproduction and general health status of rabbit does kept under heat stress conditions.
Therefore, the present study was conducted to evaluate the efficacy of daily oral administration with two levels of allicin (5 and 10 mg/kg of female body weight) for 30 days pre-mating on the reproductive performance in vivo and in vitro, blood metabolites, oxidative stress, immunity and liver function of rabbit does under high ambient temperature.
2 MATERIALS AND METHODS
The current study was conducted at The Experimental Rabbitary Farm, and Laboratory of Physiology and Biotechnology, Animal Production Department, Faculty of Agriculture, Mansoura University, Egypt, during the period from June to September 2018.
2.1 Animals and experimental design
Niliparous NZW rabbit does (n = 105; 3.20 ± 0.25 kg body weight), handled according to the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the Protection of Animals Used for Scientific Purposes, were used in this study. Rabbit does were housed individually in wire galvanized cages (40 × 50 × 35 cm) accommodated with feeders for pelleted rations and automatic fresh water drinkers in a naturally ventilated building and lighted rabbitry (14.50 ± 0.30 hr daylight length). Does were kept under the same hygienic control. The experiment was done during summer season with average maximum and minimum values of temperate and relative humidity as mentioned in Table 1.
| Item | Minimum | Maximum |
|---|---|---|
| Ambient temperature (°C) | 22.15 ± 0.80 | 36.711 ± 1.40 |
| Relative humidity (%) | 42.60 ± 6.20 | 79.20 ± 8.50 |
Does were fed a commercial basal diet (CBD) containing 18.30% CP, 12.5% CF, 3.01% EE and 2,700 kcal DE/kg diet. Rabbits had ad libitum access to water and basal ration.
Does were further randomly divided into three experimental groups (n = 35 does in each). Does in the 1st group were orally treated with distilled water (2 ml) without any supplementation (control group). Those in the 2nd and 3rd groups were orally treated with 5 and 10 mg of allicin (Anhui Ruisen Biological Technology) per kg body weight dissolved in 2 ml distilled water, respectively, for 30 days pre-insemination.
At the end of treatment period (30 days), rabbit does (n = 105) were artificially inseminated with pooled semen (0.5 ml straw) collected from fertile NZW bucks (n = 20) diluted at a rate of 1:5 with glucose yolk citrate diluent and 50 μg/ml gentamycin. Artificially inseminated does were given an intramuscular injection with 0.25 ml/doe GnRH (Receptal, intervet equivalent B.V.) to induce ovulation.
2.2 Ovulatory response (in vitro study)
Ten conceived does from each group were taken, transported to laboratory and slaughtered 60–64 hr post-insemination, for recording the ovulatory response parameters. Immediately after slaughtering, ovaries were excised, submerged in a flacon plastic tissue culture dishes (60 × 15 mm) containing saline solution at 38.5°C. Number of visible follicles with ≥2 mm in diameter (LF), haemorrhagic follicles (HF) and corpora lutea (CLs) on the ovarian surface were recorded for each dose. Total follicles (TF) and ovulation rate (OR) were calculated as the following: TF = Number of LF + HF and OR (%) = (Number of CLs/TF) × 100.
Embryos were recovered by flushing from reproductive tract of slaughtered does (all at morulae stage) using phosphate buffer saline containing 10% foetal calf serum and 50 μg gentamycin/ml. After embryo searching by stereoscopic microscope, embryos were counted, then embryo recovery rate (ERR) was calculated (ERR = [number of embryos/number of CLs] × 100). Number of embryos at morula stage was recorded (n = 459) and morphologically evaluated for good quality (n = 416) and poor quality (n = 43) embryos under stereoscopic microscope, morphology of mucin coat, zona pellucidae, blastomeres and refractive cytoplasm after washing for three times in PBS.
2.3 Reproductive performance of does (in vivo study)
The rest number of does in each group (n = 25) was left to continue post-insemination, and pregnancy was diagnosed by abdominal palpation on day 10–12 post-insemination. Number of proved pregnant does was recorded, and pregnancy rate (PR) was calculated (PR = [Number of pregnant does/number of mated does] × 100). Two days pre-expected parturition date, doe cages were provided with nest boxes. After parturition, kindling rate (KR) was calculated (KR = [Number of kindled does/number of pregnant does] × 100). At birth, born (TB) and number born alive (NBA), after 12 hr of kindling per doe were recorded, then viability rate (VRB) was calculated (VRB = [NBA/TB] × 100). Also, number weaned (NW) and viability rate were calculated at weaning. Moreover, individual body weight and litter weight at birth and at weaning were recorded.
2.4 Blood biochemical, immunity and antioxidant status
After the termination of treatment period (pre-insemination), blood samples were collected, from the ear vein of seven does in each group into sterile tubes. The whole blood was allowed to coagulate at room temperature by keeping it in slant position (45° angle) for 30 min, and then centrifuged at 3,500 rpm for 15 min. The clear blood serum was taken and kept in deep freezer until assay. Serum total proteins (TP), albumin (AL) and glucose were measured calorimetrically by using commercial kits (Diamond Diagnostics) according to the procedure outlined by the manufacturer. However, globulin (GL) was calculated by subtracting AL from TP concentration. Immunoglobulins (IgG and IgM) (ab187400) were assayed in blood serum by applying ELISA method by using ELISA kits. Activity of aspartate (AST) and alanine (ALT) aminotransaminases, activity of acid (ACP) and alkaline (ALP) phosphatases, reduced glutathione (GSH), superoxide dismutase (SOD) and thiobarbituric acid reaction (TBARS) were determined in blood serum by using commercially available kits (Diamond Diagnostics) according to the procedure outlined by the manufacturer.
2.5 Assay of serum progesterone and prolactin
Progesterone (P4) and prolactin (PRL) concentrations were determined in blood serum samples taken from the seven does in each group pre-insemination (taken at the end of treatment period), and from the same does that proven pregnant on day 15 of pregnancy (mid-pregnancy) and 7 days post-partum. The P4 (EIA 1561) and PRL (E-EL-RB1223) concentrations were assayed by applying ELISA method using ELISA kits.
2.6 Statistical analysis
Data were statistically analysed by one-way analysis of variance using GLM procedures of SAS (SAS, 2002) to test the effect of allicin treatment as the following statistical model: Yij = µ + Ai + eij. Where: Yij = observed values of individual j, µ = overall mean, Ai = effect of treatment i (levels of allicin, 0, 5 and 10 mg/kg of doe body weight) and eij = random error. Duncan multiple range test (Duncan, 1955) was used for detecting the significant differences among the tested means at p < .05.
Pregnancy, rate of kindling, viability, ovulation, embryo recovery and embryo quality were statistically analysed using Chi-square test.
3 RESULTS
Reproductive performance (Table 2), including NBA and bunny viability rate at birth and at weaning as well as average bunny and litter weights at birth and at weaning, increased (p < .05) by inclusion of both allicin levels (5 and 10 mg per kg of doe body weight). Pregnancy rate and total litter size at birth tended to increase insignificantly (p > .05), while kindling rate was 100% in all groups.
| Item | Allicin levels | SEM | p-Value | ||
|---|---|---|---|---|---|
| Control | 5 mg/kg LBW | 10 mg/kg LBW | |||
| Reproductive traits | |||||
| Does mated (n) | 25 | 25 | 25 | — | — |
| Pregnancy rate, n (%) | 18 (72) | 21 (84) | 23 (92) | — | .2064 |
| Kindling rate, n (%) | 18 (100) | 21 (100) | 23(100) | — | — |
| Total born/doe (n) | 6.67 | 7.33 | 7.61 | 0.304 | .0971 |
| Born alive/doe (n) | 5.50b | 6.90a | 7.22a | 0.267 | .0001 |
| Total weaned/doe (n) | 4.56b | 6.43a | 6.87a | 0.293 | .0001 |
| Viability rate at birth (%) | 82.46b | 94.13a | 94.88a | 2.235 | .0001 |
| Viability rate at weaning (%) | 82.91b | 93.19a | 95.15a | 2.483 | .0015 |
| Bunny weight (g) | |||||
| Average bunny weight at birth | 45.78c | 51.81b | 54.09a | 0.530 | .0001 |
| Average bunny weight at weaning | 567.33c | 612.52b | 625.83a | 1.787 | .0001 |
| Litter weight at birth | 251.79b | 357.49a | 390.53a | 14.372 | .0001 |
| Litter weight at weaning | 2,587.02b | 3,938.50a | 4,299.45a | 179.189 | .0001 |
Note
- Mean values followed by different superscript letters in the same row are significantly different (p < .05).
The effect of allicin administration on ovulatory response is illustrated in Table 3. Number of follicles (large and bleeding), CLs and embryos per doe was not affected by allicin treatment; however, number of total follicles was significantly (p < .05) decreased only with 10 mg allicin. Oral administration of rabbit does with 5 and 10 mg allicin increased (p < .05) OR (Figure 1) and improved (p < .05) embryo quality, in term of increasing percentage of good quality and decreasing percentage of poor quality embryos compared with untreated does (Figure 2). The ERR was not affected by allicin treatment (Figure 3).
| Trait | Allicin levels | SEM | p-Value | ||
|---|---|---|---|---|---|
| Control | 5 mg/kg LBW | 10 mg/kg LBW | |||
| Does mated (n) | 10 | 10 | 10 | — | — |
| Large follicles/doe (n) | 19.40 | 17.90 | 17.30 | 0.9055 | .2576 |
| Bleeding follicles/doe (n) | 2.80 | 1.50 | 1.400 | 0.4492 | .0654 |
| Total follicles/doe (n) | 22.20a | 19.40ab | 18.70b | 0.9777 | .0415 |
| Corpora lutea/doe (n) | 15.10 | 15.60 | 16.20 | 0.7275 | .5705 |
| Total embryos/does (n) | 14.7 | 15.2 | 16.0 | 0.540 | .6130 |
Note
- Mean values followed by different superscript letters in the same row are significantly different (p < .05).
Table 4 is illustrated the effects of allicin treatment on reproductive hormones concentration (serum P4 and PRL). Allicin treatment of both levels (5 and 10 mg per kg) increased (p < .05) serum P4 concentration at mating, mid-pregnancy period and 7-day post-partum as compared to the control with higher (p < .05) values for 5 than 10 mg allicin per kg. Concentration of PRL at mating and on day 7 of post-partum was higher (p < .05) only with 10 mg allicin than other groups; however, a significant increase in PRL concentration has been observed due to both allicin levels at mid-pregnancy (p < .05).
| Reproductive stage | Allicin levels | SEM | p-Value | ||
|---|---|---|---|---|---|
| Control | 5 mg/kg LBW | 10 mg/kg LBW | |||
| Progesterone (ng/ml) | |||||
| Pre-mating | 0.34c | 0.62b | 0.83a | 0.018 | .0001 |
| Mid-pregnancy | 3.12c | 6.13b | 7.20a | 0.073 | .0001 |
| Seven days post-partum | 0.24c | 0.50b | 0.57a | 0.015 | .0001 |
| Prolactin (ng/ml) | |||||
| Pre-mating | 13.8b | 13.9b | 19.9a | 0.251 | .0001 |
| Mid-pregnancy | 16.3b | 19.8a | 21.5a | 0.613 | .0029 |
| Seven days post-partum | 19.1b | 20.9b | 22.9a | 0.250 | .0079 |
Note
- Mean values followed by different superscript letters in the same row are significantly different (p < .05).
The effects of allicin administration on blood serum constituents of rabbit does are tabulated in Table 5. Oral treatment with allicin (10 mg/kg body weight) significantly (p < .05) increased protein metabolism in term of increasing concentration of serum TP, AL and GL; however, serum glucose concentration, and activity of AST, ALT, ACP and ALP were significantly (p < .05) decreased by 5 and 10 mg allicin per kg LBW compared with the control.
| Parameter | Allicin level | SEM | p-Value | ||
|---|---|---|---|---|---|
| Control | 5 mg/kg LBW | 10 mg/kg LBW | |||
| Biochemicals | |||||
| Total proteins (g/dl) | 5.87b | 5.92b | 6.43a | 0.033 | .0001 |
| Albumin (g/dl) | 2.94c | 3.05 b | 3.26a | 0.031 | .0001 |
| Globulin (g/dl) | 2.87b | 2.92b | 3.18a | 0.061 | .0265 |
| Glucose (mg/dl) | 114.67a | 95.66b | 88.67c | 1.633 | .0001 |
| Hepatic markers | |||||
| AST (IU/l) | 52.67a | 40.00b | 31.65c | 1.5159 | .0002 |
| ALT (IU/l) | 35.33a | 22.28b | 20.60b | 1.188 | .0002 |
| ACP (IU/l) | 33.34a | 20.33b | 19.67b | 1.000 | .0001 |
| ALP (IU/l) | 62.00a | 51.33b | 46.67b | 1.689 | .0018 |
Note
- Mean values followed by different superscript letters in the same row are significantly different (p < .05).
- Abbreviations: ACP, acid phosphatases; ALP, alkaline phosphatases; ALT, alanine aminotransaminases; AST, aspartate aminotransaminases.
Results presented in Table 6 demonstrate the effects of allicin administration on immune response and antioxidant status. Serum immunoglobulins (IgG and IgM) concentrations, as immunity markers, were increased (p < .05) with increasing allicin level. The GSH and SOD were not affected by allicin (p ≥ .05); however, a marked decrease (p < .05) in TBARS, as a lipid peroxidation marker, has been observed due to allicin administration. Allicin administration at a level of 10 gm per kg LBW showed the highest improvement of immunity and lipid peroxidation of does.
| Parameter | Allicin levels | SEM | p-Value | ||
|---|---|---|---|---|---|
| Control | 5 mg/kg LBW | 10 mg/kg LBW | |||
| Immunity markers | |||||
| IgG (mg/dl) | 422.73c | 491.00b | 520.67a | 4.829 | .0001 |
| IgM (mg/dl) | 123.40c | 129.33b | 134.33a | 1.130 | .0158 |
| Antioxidant enzymes | |||||
| GSH (mg/dl) | 12.33 | 14.37 | 16.33 | 1.163 | .1276 |
| SOD (IU) | 5.52 | 5.92 | 6.21 | 0.292 | .3121 |
| Lipid peroxidase | |||||
| TBARS (nmol/ml) | 1.27a | 1.09b | 0.98c | 0.017 | .0001 |
Note
- Mean values followed by different superscript letters in the same row are significantly different (p < .05).
- Abbreviations: GSH, reduced glutathione; IgG, immunoglobulin G; IgM, immunoglobulin M; SOD, superoxide dismutase; TBARS, thiobarbituric acid reaction.
4 DISCUSSION
Under heat stress, cellular damage could be prevented by antioxidants, which increase the ability to in antioxidant defence system by counteracting the oxidants and other cellular protection (Mittler et al., 2004). Antioxidants from natural sources play a vital role in improving reproductive efficiency, immune response and health status of rabbit does (El-Ratel et al., 2017). Allicin as a natural antioxidant was reported to have many biological activities for good health status, including anti-microbial, anti-inflammation, immunomodulatory and hypoglycaemic (Indrasanti, Indradji, Hastuti, Aprilliyani, & Rosyadi, 2017). According to the obtained results, oral administration of rabbit does with both allicin levels improved their reproductive efficiency (Table 1). Similar results were reported on poultry fed diet supplemented with allicin (El-Katcha et al., 2016), female Albino rats treated with aqueous garlic extract (Raji, Fayemi, Ameen, & Jagun, 2012) and ewes fed garlic plant either as powder or oil diet (Nassar, El Shereef, & Abo Bakr, 2017). It is worthy noting that improving the reproductive performance of does was in parallel with the ovulatory response of allicin-treated groups. Does treated with allicin (10 mg/kg) resulted in more stimulation of ovulatory response in terms of decreasing number of total follicles and increasing number of CLs and embryo yield (Table 2), and subsequently increasing ovulation rate (Figure 1) as well as improving quality of embryos (Figure 2). The noticed tendency of increase in number of CLs (p ≥ .05) in allicin treatment groups may be attributed to the effect of allicin on increasing LH surge as compared to the control group, reflecting higher ovulation rate. The rabbit is a reflexively ovulating species in which sensory and neuro-endocrine stimuli act together to induce a LH pre-ovulatory surge (Dufy-Barbe, Franchimont, & Faure, 1973) and determine the ovulatory response. It was reported that garlic extract stimulates gonadotropins secretion through anterior-pituitary activation (Obochi, Malu, & Obi-Abang, 2009). Natural plant-derived antioxidants may exhibit beneficial effects on ovulation and ovary functions (Zhong & Zhou, 2010). Interestingly, improving embryo quality in treatment groups may be due to direct effect of allicin on ovarian tissues and activity, and/or indirect effect on heathy status and immunity of rabbit does.
Improving the reproductive performance was associated with significant (p < .05) enhancing in P4 and PRL hormones profiles as affected by allicin administration (Table 3). Enhancement in P4 level may be due to tendency of increasing total litter size, while increasing PRL as a hormone of milk initiation resulted in significant (p < .05) increase in viability rate at weaning (Ragab, Vicente, Minguez, & Baselga, 2014), in allicin level-dependent manner from 0 up to 10 mg/kg. It is well known that P4 is essential for regulating the reproductive processes, such as ovulation, zygote implantation and pregnancy maintenance (Graham & Clarke, 1997). Level of P4 was observed to be the highest at mid-pregnancy as reported by Kleden, Soetanto, and Kuswanto. (2017). The observed higher P4 concentration during mid-pregnancy in does treated with allicin (10 mg/kg) than in the controls may be in relation with number and size of CLs on the ovarian surfaces and implantation of numerous foetuses onto uterus. The noticed increase in P4 level in G3 as affected by high allicin level may be attributed to increasing GnRH (LH-RH) release, leading to elevating LH level to maintain CLs. In this respect, allicin as antioxidant may improve gonado–pituitary hormone disturbance in rabbit does exposed to oxidative stress under heat stress condition (Al-Masri, 2015). Also, garlic extract was reported to stimulate gonadotropins secretion and hormones of the ovary through anterior-pituitary activation (cited from Behnaz, 2014), which might be one reason for the observed increase in level of P4. It is worthy noting that pre-mating PRL level in all groups has pronounced positive relationship with reproductive performance, being the best with the highest pre-mating PRL level. Pre-mating plasma prolactin concentrations were 15.5 and 16.3 ng/ml in non-ovulating and no mated rabbit does (Lamb et al., 1991). During pregnancy, circulating levels of PRL show a gradual rise in all groups to reach a maximum at mid-pregnancy. After delivery, prolactin levels fall but basal concentrations do not reach the non-pregnant range until one week post-partum. Similar trends were reported in rabbits by McNeilly (1965). Improving PRL profile in treatment groups is in agreement with Obochi et al. (2009), who reported that garlic extract stimulates hormones of the ovary through anterior-pituitary activation.
Serum biochemicals reference values might introduce valuable information about the physiological status and provide an indication of the health status in rabbits. The present results indicated that allicin administration (10 mg/kg body weight) significantly (p < .05) increased protein metabolism, while both allicin levels significantly decreased serum glucose and enzyme activity compared with the control (Table 4). Increasing TP concentration was associated with increase in the AL and GL concentrations as affected by containing garlic organo-sulphur compounds, which have a protective action on liver function (Ajayi, Adeniyi, & Babayemi, 2009). Similar results were reported by El-Katcha et al. (2016) on broiler chickens treated with allicin, or on rabbits (Alagawany, Elwy, & Fayez, 2016) and buffalo calves (Vamsi Duvvu et al., 2018) treated with garlic. The recorded reduction in glucose level in does treated with allicin is in accordance with findings on mice (Kumar & Reddy, 1999) and broilers (Singh et al., 2017). Allicin as a sulphur compound may be related to the reduction in glucose by competing with insulin which results in increased free insulin (Banerjee, Dinda, Manchanda, & Maulik, 2002). Increasing insulin secretion leads to an increase in production of the nitric oxide and significant decrease in the inflammatory cytokines, glycosylated haemoglobin, post-prandial blood glucose and fasting blood glucose level (Kumar et al., 2013). The reduction in enzyme activity reflected impact of both allicin administrations on liver function. Treatment with allicin showed marked reductions in serum activity of ALT and ALP in infected rabbits, confirming the antibacterial activity of allicin that reduced the damaging effects of bacteria on the liver (Abu El Hammed, Soufy, El-Shemy, Nasr, & Dessouky, 2016). In addition, aqueous garlic extract improved hepatic steatosis in rabbits (Arhan et al., 2009) and this effect was attributed to the organo-sulphur compounds (allicin) present in garlic. Allicin may lead to cell membrane stabilization and protection of the liver against harmful agents and free radical-mediated toxic damages to the hepatocytes through reduce the activity of enzymes (Alam et al., 2018).
Additionally, the obtained results showed that both allicin levels improved serum immunoglobulins (IgG and IgM), as immunity markers, while decreased lipid peroxidation marker (TBARS), showing the highest improvement of immunity and lipid peroxidation of does with allicin at a level of 10 mg per kg BW (Table 5). In accordance with the present results, allicin as a vital compound in garlic exerted positive impact on immunity of young animals (Wang et al., 2011), broiler chicken (El-Katcha et al., 2016) and infected animals through increasing of immunoglobulins levels (Kamel & El-Shinnawy, 2015). In rabbits, allicin treatment increased serum IgG and IgM levels in blood (Alam et al., 2018). In supporting this finding, garlic as an herbal plant and natural antioxidant was reported to be rich in flavonoids, which improve immune functions (Acamovic & Brooker, 2005) and stimulate humoral immune response. Previous reports indicated that garlic and their contents could activate proliferation of lymphocytes, release of cytokine and phagocytosis (Wang et al., 2011). In rabbits, garlic was suggested to limit the growth and colonization of several pathogenic and non-pathogenic bacteria in gut, leading to better feed utilization (Nouzarian, Tabeidian, Toghyani, Ghalamkari, & Toghyani, 2011) and improved the immunity of growing rabbits. This effect could be attributed to that garlic inhibits certain thiol-containing enzyme in micro-organisms, and the thiosulphinate compounds in garlic stimulate the oxidation of intracellular bacterial thiol content (Lee & Gao, 2012). Also, dietary garlic enhanced T-cell proliferation and might have directly/indirectly enhanced B-cell proliferation and differentiation. Moreover, Salem and Salem (2016) stated that the positive effect of garlic administration may be attributed to various organo-sulphur compounds, particularly allicin, which has anti-microbial and immune-stimulant properties.
Concerning the oxidative stress, the results indicated that allicin treatment ameliorated the oxidative stress and reduced lipid peroxidation by reducing TBARS in blood serum of does through its anti-oxidative action (El-Sheakh, Ghoneim, Suddek, & Ammar, 2016). Similarly, feeding garlic-supplemented diet reduced lipid peroxidation compared with control rabbits. Although the SOD plays an important role in protecting cells from damage caused by ROS, no observed increase was found in SOD activity in association with reducing TBARS. This may be due to dietary supply of the appropriate nutrients (Ashour, Alagawany, Reda, & Abd El-Hack, 2014).
Allicin is an active ingredient of garlic and considered as an organic disulphide formed from alliin (Borlinghaus et al., 2014). It showed antioxidant and anti-inflammatory activities (Abdel-Daim et al., 2019) and has ability to attenuate the signalling pathways of ROS and increase the endogenous antioxidant enzymatic activity (Abushouk, Ismail, Salem, Afifi, & Abdel-Daim, 2017). Allicin has direct or indirect effect through upregulation of phase II detoxifying enzymes in a nuclear factor related-2 factor-dependent pathway (Abdel-Daim et al., 2019).
5 CONCLUSION
The mechanism by which allicin increased reproductive performance is based on that allicin can play an important role in improving ovulation rate and embryo quality and subsequently litter size of does. Also, allicin improved protein metabolism, liver function and immune response. Furthermore, allicin, as a natural exogenous antioxidant, has properties of protecting damage of tissues. Therefore, oral administration of rabbit does with allicin at a level of 10 mg/kg for 30 days prior to mating for improving reproductive efficiency and health status under heat stress condition.
CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest.
AUTHOR CONTRIBUTION
Substantial contributions to conception and design (Ibrahim Talat El-Ratel and Abdel-Khalek E. Abdel-Khalek); acquisition of data (Ibrahim Talat El-Ratel and Mohamed E. Hammad); analysis and interpretation of data (Ibrahim Talat El-Ratel and Sherif A. Gabr); statistical analyses (Mohamed E. Hammad and Hanan I. El-Morsy), drafting the manuscript (Ibrahim Talat El-Ratel, Abdel-Khalek E. Abdel-Khalek and Sherif A. Gabr); critically revising the manuscript for important intellectual content (Ibrahim Talat El-Ratel and Abdel-Khalek E. Abdel-Khalek); and final approval of the manuscript for publication (all authors).
ANIMAL WELFARE STATEMENT
The experimental procedures were conducted according to the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the Protection of Animals Used for Scientific Purposes.
