|Year : 2018 | Volume
| Issue : 4 | Page : 155-160
Effects of Lepidium sativum supplementation on growth and gonadotropins secretion in ovariectomized, estrogen-implanted rabbits
Oluwatosin V Imade1, Wuraola A Erinfolami2, Rasheed A Ajadi3, Monsuru O Abioja2, Samson A Rahman4, Olusiji F Smith2, Oladele S Gazal5
1 Department of Animal Physiology, College of Animal Science & Livestock Production, Federal University of Agriculture, Abeokuta, PMB 2240, Ogun State, Nigeria; Laboratory for Reproductive Research and Endocrine Analyses, Department of Biological Sciences, College of Science & Engineering, St. Cloud State University, St. Cloud, Minnesota 56301, USA
2 Department of Animal Physiology, College of Animal Science & Livestock Production, Federal University of Agriculture, Abeokuta, PMB 2240, Ogun State, Nigeria
3 Department of Veterinary Medicine & Surgery, College of Veterinary Medicine, Federal University of Agriculture, Abeokuta, PMB 2240, Ogun State, Nigeria
4 Department of Physiology & Pharmacology, College of Veterinary Medicine, Federal University of Agriculture, Abeokuta, PMB 2240, Ogun State, Nigeria
5 Laboratory for Reproductive Research and Endocrine Analyses, Department of Biological Sciences, College of Science & Engineering, St. Cloud State University, St. Cloud, Minnesota 56301, USA
|Date of Submission||19-Apr-2018|
|Date of Decision||30-Apr-2018|
|Date of Acceptance||22-May-2018|
|Date of Web Publication||24-Jul-2018|
Oluwatosin V Imade
Laboratory for Reproductive Research and Endocrine Analyses, Department of Biological Sciences, St. Cloud State University, 720 4th Avenue S., WSB-227, St. Cloud, MN 56301
Source of Support: None, Conflict of Interest: None
Objective: To test the effects of dietary supplementation of Lepidium sativum (LS) seed powder on growth performance and gonadotropins secretion in ovariectomized, estradiol- implanted rabbits. Methods: Ovariectomized, estradiol-implanted Chinchilla rabbits were assigned into four experimental groups: LS seed powder was included into normal rabbit chow at 0% (control), 5% (low), 7% (mid) and 10% (high) w/w. Experimental feed and water were given ad-libitum for 3 weeks. Weekly body weights and daily feed intake of rabbits were recorded. Twenty-one days post-feeding, blood samples were collected at 15-minute interval for 3 h (Period I) after which 2.5 μg gonadotropin-releasing hormone (GnRH) was injected intravenously and the sampling continued for another hour (Period II). Plasma was harvested and analyzed for luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by radioimmunoassay. Results: Feed intake was significantly (P<0.05) increased in LS-supplemented rabbits. However, the increase in feed intake did not result in significant body weight gain. LS seed supplementation significantly (P<0.001) increased mean plasma LH dose-dependently from the low- to the mid-LS level and then decreased LH at the high-LS level. LS supplementation increased (P<0.001) plasma FSH secretion. Injection of GnRH had no effect on plasma LH, however significantly (P<0.05) decreased overall plasma FSH secretion. Conclusions: LS seed supplementation stimulates feed intake and gonadotropins secretion in rabbits. Gonadotropins effect may be mediated through LS seeds phytosterols through the activation of estrogen receptors thereby producing agonistic effects resulting in LH and FSH secretion. The differential responses of gonadotropins to GnRH in LS-supplemented rabbits suggest differential regulation of the synthesis and secretion of these gonadotropins.
Keywords: Ovariectomized rabbits, Lepidium sativum, Growth, LH, FSH
|How to cite this article:|
Imade OV, Erinfolami WA, Ajadi RA, Abioja MO, Rahman SA, Smith OF, Gazal OS. Effects of Lepidium sativum supplementation on growth and gonadotropins secretion in ovariectomized, estrogen-implanted rabbits. Asian Pac J Reprod 2018;7:155-60
|How to cite this URL:|
Imade OV, Erinfolami WA, Ajadi RA, Abioja MO, Rahman SA, Smith OF, Gazal OS. Effects of Lepidium sativum supplementation on growth and gonadotropins secretion in ovariectomized, estrogen-implanted rabbits. Asian Pac J Reprod [serial online] 2018 [cited 2021 Feb 28];7:155-60. Available from: http://www.apjr.net/text.asp?2018/7/4/155/237052
| 1. Introduction|| |
Traditional uses of medicinal plants and their associated properties have been documented by several authors. One of the medicinal plants that has been reported to have multi-system effects and also possess important biological activities on reproductive characteristics is Lepidium sativum (LS),,. LS, also known as garden cress, has been recognized to possess properties such as abortifacient, aphrodisiac, teratogenic antifertility, antiovulatory and lactogenic effects,. This plant reportedly increased sexual stamina and sexual retentivity in both human males and females.
Recently, there has been an increased interest in unravelling the mechanism of action of LS on reproduction. Available evidence indicates a divergent effect of LS on reproduction. Although proceptive and receptive activities of the seed were reported in female rat, mammogenic and lactogenic activities in female rat and in ewe, contraceptive effects have also been reported in female mice,,. Further, administration of tocopherol extract increased epididymal sperm concentration, sperm count, sperm motility, grade activity, sperm viability and decreased abnormal sperm morphology percent in rabbit,. Therefore, the effect of LS on reproduction may depend on the organism, the physiological state and the mode of administration of the seed or the extract.
Reproduction in all mammals is controlled by the hypothalamic-pituitary gonadal axis through which gonadotropin-releasing hormone (GnRH) from the hypothalamus regulates the synthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary. These gonadotropins in turn regulate gonadal gametogenesis and the secretion of gonadal hormones. To date, there is a dearth of information on the effects of LS on the synthesis and secretion of gonadotropins and specifically on its pulsatility. Therefore, this study was carried out to determine the effects of LS seed supplementation on growth performance and gonadotropins secretion in ovariectomized, estradiol-implanted (OVX+E2) rabbits.
| 2. Materials and methods|| |
2.1. Plant material and preparation
LS (garden cress) seeds were purchased from a local market in Gombe, Nigeria and confirmed in the Department of Forestry and Wildlife Management, Federal University of Agriculture, Abeokuta, Nigeria. The seeds were ground into fine powder using electric mill and kept in air-tight container until required for use.
2.2. Experimental animals
The research was carried out at the Veterinary Teaching Hospital, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria. Ethical approval (COLANIM/ANP; April 22, 2015) for this study was obtained from the Research Ethics Committee, Federal University of Agriculture, Abeokuta, Ogun State. Twenty primiparous Chinchilla rabbits [mean weight: (2.10 ± 0.15) kg] were used in this study. All rabbits were bilaterally ovariectomized under ketamine-xylazine and diazepam anesthesia and then received subcutaneous estradiol implants at the neck region. The animals were housed individually in a wooden cage where feed and fresh water were given ad-libitum. The house was naturally ventilated and maintained under clean environmental condition.
2.3. Experimental design and blood sampling
Twenty-one days post ovariectomy, the animals were randomly assigned to receive either normal rabbit chow (control; n = 5) or rabbit chow supplemented weight for weight with LS seed powder at either 5% (low; n = 5), 7% (mid; n = 5) or 10% (high; n = 5). All animals were fed the control or experimental diet. Daily feed intake and weekly live body weight were recorded. Blood samples were collected after 21 days of feeding through an indwelling marginal ear vein catheter every 15 min for 3 h (Period I) after which GnRH (2.5 μg/animal) was injected intravenously and then sampling continued for 60 min at the same frequency (Period II). After each sampling, the blood was replaced by an equal volume of heparinized saline solution (10 U heparin/mL). Blood samples collected were centrifuged at 3 000 rpm in refrigerated centrifuge (4 °C) to obtain plasma. Plasma was stored at -20 °C until hormonal assay was done.
2.4. Radioimmunoassay of LH
LH concentration was determined using techniques described by Bernard et al. The LH used for the standards and iodination reaction was obtained from Sigma Aldrich Chemicals, St. Louis, Missouri, USA. The antiserum AFP C5288113 (obtained from Dr. A. F. Parlow) was used at 1: 35 000 dilution as the first antibody and Goat Anti-Rabbit Immunoglobulin G (GARGG, EQUITECH-BIO INC, Kerrville, Texas, USA) was used as the second antibody.
Briefly, on day 1, standards, antibody (100 μL) and tracer 125I-LH (18 000-20 000 cpm/100 μL) were pipetted into 12 mm χ 75 mm glass tubes (VWR International, Pennsylvania, USA) containing 150 μL of blood plasma and incubated at 4 °C for 48 h. Thereafter, 100 μL of GARGG diluted with phosphate buffer saline-ethylene diamine tetraacetic acid and 500 μL 6% polyethylene glycol were added. Tubes were centrifuged at 3 000 rpm at 4 °C for 15 min. The supernatant was discarded. The tubes containing pellets were loaded and counted in a Packard Cobra Π Gamma counter (Perkin Elmer, Waltham, MA, USA). The minimum LH detected by assay was 0.4 ng/mL, and average intra- and interassay coefficients of variation were 5.1% and 7.4%, respectively.
2.5. Radioimmunoassay of FSH
Plasma FSH concentration was determined using a modified heterologous radioimmunoassay in duplicates of 50 μL volume in a 5-day assay. The FSH used for the standards and iodination reaction was obtained from Sigma Aldrich Chemicals, St. Louis, Missouri, USA. The iodinated FSH (NIDDK-oFSH-19-SIAFP, AFP 4117A) and the first antibody (NIDDK-anti-oFSH-1, AFP-C5288113) were obtained from Dr. A.F. Parlow, and Goat anti-rabbit Gamma Globulin (GARGG, EQUITECH-BIO INC, Kerrville, Texas, USA), the second antibody, were used in the assay.
Briefly, on day 1, 50 μL plasma and antibody (100 μL of 1:15 000 AFP-C5288113) were pipetted into 12 mm 75 mm glass tubes (VWR International, Pennsylvania, USA) and incubated for 24 h at 4 Then, 125I-oFSH (18 000-2 0000 cpm/100 μL) was added on day 2 and incubated at 4 Ό for another 24 h. Thereafter, 200 μL of the second antibody, GARGG was added and tubes were incubated for 48 h at the same temperature. Then, 3 mL of 0.01M phosphate-buffered saline was added and tubes were centrifuged at 3 600 rpm at 4 °C for 1 h. The assay detection limit was 0.125 ng/mL and intraassay coefficient of variation was 4.2%.
2.6. Statistical analysis
Data were statistically analyzed using the general liner model program of SAS. Multiple comparisons were done with Tukey’s test. The main effects of treatments on plasma LH, FSH concentrations, feed intake and body weight gain were determined. Differences were considered significant at P<0.05. All the data were reported in Mean ± standard error (Mean±SE).
| 3. Results|| |
3.1. Effects of LS supplementation on growth performance in OVX+E2 rabbits
Average daily feed intake was significantly higher (P<0.001) in LS-supplemented ovariectomized rabbits compared to normal rabbit chow-fed controls. However, changes in body weight between LS-supplemented and control rabbits were similar, although there was a trend toward an increase in the earlier group [Figure 1].
|Figure 1: Effects of LS seed supplementation on growth performance indices in OVX+E2 rabbits.|
Animals were fed either normal rabbit diet or LS seed supplemented diet for 21 days. Data are Mean ± SE; Bars with asterisks are significantly different; LS: Lepidium sativum.
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3.2. Effects of LS supplementation on LH and FSH secretion in 0VX+E2 rabbits
[Figure 2] showed the plasma LH levels in control and LS-supplemented ovariectomized rabbits obtained during a 4-hour frequent blood sampling period after 21 days of normal rabbit chow or LS seed powder supplementation.
|Figure 2: Plasma LH concentrations in OVX+E2 rabbits fed normal rabbit diet or LS seed-supplemented diet for 21 days.|
Blood samples were obtained at 15-interval for 3 h before 2.5 μg GnRH injection (i.v.) and sampling for 1 h at the same frequency; LH: luteinizing hormone; LS: Lepidium sativum.
Click here to view
[Figure 3] showed the mean plasma LH levels during the entire 4-hour window sampling period. The window sampling period consisted of a 3-hour baseline component followed by a 1-hour post-GnRH injection sampling component.
|Figure 3: Main effects of LS seed supplementation on plasma LH concentrations in OVX+E2 rabbits.|
Data showed Mean ± SE; Bars with asterisks are significantly different; LH: luteinizing hormone; LS: Lepidium sativum.
Click here to view
Corresponding values for plasma FSH were shown in [Figure 4] and [Figure 5] respectively. Both basal plasma LH and FSH levels were very high in the rabbits [Figure 2] and [Figure 4]. Pulsatile LH secretion tended to be increased by LS seed supplementation [Figure 2] and [Table 1] and overall, LS seed supplementation significantly (P<0.001) increased mean plasma LH concentration dose-dependently, increasing from low- to mid-LS supplementation and then decreasing at the highest level of LS supplementation. Basal pulsatile plasma FSH secretion was significantly increased (P<0.001) by LS supplementation and this increase was dose-dependent [Table 1].
|Figure 4: Plasma FSH concentration in OVX+E2 rabbits fed normal rabbit diet or LS seed supplemented diet for 21 days.|
Blood samples were obtained at 15-interval for 3 h before 2.5 μg GnRH injection (i.v.) and sampling for 1 h at the same frequency; FSH: folliclestimulating hormone; LS: Lepidium sativum.
Click here to view
|Figure 5: Main effects of LS supplementation on plasma FSH concentrations in OVX+E2 rabbits.|
Data showed mean ± SE; Bars with asterisks are significantly different; FSH: follicle-stimulating hormone; LS: Lepidium sativum.
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|Table 1: Effect of graded doses of LS seed supplementation and GnRH injection on LH and FSH secretion in OVX+E2 rabbits.|
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The study results showed that there was a tendency for LS seed supplementation to affect the basal temporal secretion of LH in PeriodI (before GnRH injection) and to significantly affect mean levels of LH during our 4-hour sampling period. With respect to FSH, baseline FSH concentrations were significantly affected by LS seed supplementation during Period I and this effect was dose- dependent, with the lowest LS supplementation having the most effect.
3.3. Effects of GnRH injection in OVX +E2 rabbits fed LS supplemented diet
[Table 1] presented the effects of GnRH injection on LH and FSH secretion in LS-supplemented OVX+E2 rabbits. GnRH administration had no effect on plasma LH secretion but significantly decreased (P<0.05) overall plasma FSH levels in comparison to pre-GnRH injection levels. Further, there was a tendency (P=0.053) for the plasma LH concentration to be dependent on the interaction between LS supplementation and GnRH stimulation.
| 4. Discussion|| |
Generally, administration and implantation of estrogen have been reported to reduce gonadotropins in ovariectomized rats,, rabbits and monkey by exerting a negative feedback effects on the LH and FSH secretion. In the current study, LS supplementation increased plasma LH and FSH secretions in OVX+E2 rabbits in a dose dependent manner. Stimulation of LH and FSH may be ascribed to phytosterol constituent of LS, which has estrogenic activity. The effect could be through temporary or permanent alteration of the feedback loop in the hypothalamus, pituitary and the gonad by mimicking the effects of endogenous estrogen which then trigger their specific receptors, thereby resulting in stimulation of FSH and LH secretion. Phytosterol has been reported to increase basal gonadotropins secretion in immature male and female rabbits. In addition, plasma estrogen levels in the OVX+E2 rabbits were very high which was undoubtedly due to estradiol implantation. This report is in agreement with the results from a previous study where the increases in LH, prolactin, estrogen and progesterone were observed in young adult virgin rats fed with LS-supplemented diet for only 14 days. However, reduction in LH and FSH levels at high level of LS supplementation may be as a result of adenohypophysial negative feedback of the estrogenic action of phytosterol, which induces a decrease or loss of progressive sensitivity or the affinity for estrogen receptors binding and then causes subsequent LH and FSH inhibition.
In the OVX+E2 rabbits, pre-GnRH injection levels of LH and FSH were high and suppression of only FSH was observed following GnRH challenge. Although previous studies indicate importance of species differences in the sensitivity of the gonadotropins to GnRH, the dose of GnRH used in this study, much to our chagrin, did not stimulate further increase in the secretion of gonadotropins. This result indicates that LS-supplemented estrogen-primed pituitary did not respond to GnRH stimulation. The lack of pituitary response may be due to the high level of baseline LH level and therefore a depletion of releasable gonadotropins. Besides, the high level of estrogen observed with estrogen implantation may reduce the responsiveness of the gonadotropins to GnRH stimulation. In our hands, plasma estradiol-17 β level in implanted rabbits was similar to the level observed in pregnant rabbits. Thus, the sub-maximal gonadotropins response to GnRH in this study may be due to the high negative estrogen feedback or a diminished anterior pituitary responsiveness to GnRH. The level of estrogen measured in OVX+E2 New Zealand white rabbits is of many magnitudes lower than the value measured in this study with OVX+E2 Chinchilla breed. Thus, there may be a breed effect observed in this study. Most rabbit experiments have used the New Zealand breed of rabbit,. Further, the dose of GnRH injected may be subthreshold; the dose used was based on a previous study in which the Fauve de Bourgogue strain was used. In contrast, the Chinchilla breed was used in the present study.
The differential response of LH and FSH to GnRH stimulation in this study confirms that the synthesis and release of the gonadotropins are differentially regulated. This difference must relate to the differences in the GnRH-induced responses in LH- β and FSH- β genes. It has been suggested that this may reflect differences in the frequency of GnRH stimulation. Many studies have indicated that LH- β gene promoter activity is stimulated to a greater extent at higher GnRH pulse frequency,,. These higher frequencies of GnRH increase GnRH receptor number which reportedly preceded the differential regulation of LH- β and FSH- β genes.
Significant increase in feed intake in OVX+E2 rabbits may indicate the positive influence of LS on the appetite of the rabbits and nutrient digestion. The lack of difference in weight gain between controland LS-supplemented rabbits suggests that LS seed may contain anti-nutritional factors which hampered the bioavailability and utilization of the nutrients. It has been previously reported that whole LS seed flour contains tannins, phytic acids, oxalic acid and cyanogens which might obstruct the bioavailability of nutrients. This result is in agreement with previous findings,. Whereas weight decreasing effect of LS supplementation in rats was reported, other studies in poultry, have found positive effect of LS on weight gain. It will appear that the effect of LS on weight gain may be dependent on the animal model, the dosage, fractions and method of LS administrations.
In conclusion, these findings suggest that biological activities of LS and mechanism underlying its effects on reproduction may be mediated through multiple pathways which depend greatly on the amount of LS ingested. Increases in feed intake without observable increase in body gain deserve further investigation.
Conflict of interest statement
The authors declare that they have no conflict of interest.
We thank Musa Muideen, Ogungbenro Ifeoluwa, Olawale Abiola, Adigun Grace, Adepoju Esther, Dr. Bisola Adeniyi and Tijani Tope for their support during the animal work. The support and contributions of members of the Laboratory for Reproductive Research and Endocrine Analyses, St. Cloud State University (SCSU), St. Cloud, Minnesota, USA are also appreciated.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]