Collection of the Green Coconut Fruit
The immature coconut fruits were harvested between November, 2011 and April, 2013 from Chief William kudofoke coconut farm, Ajara -Topa, Badagry, Lagos. The average weight of the fruit was 1.55kg. The fruit was authenticated at the federal institute of forestry research in Ibadan with plant’s ascension No FHI 109665 by Dr (Mrs) ugbogu, A. O.
Extraction of the Green Coconut Water
The unripe coconut fruits were washed and dehusk. The extraction of the liquid endosperm of the coconut fruit (coconut water) was done through the germinal pore, poured directly into an airtight bottle, preserved in the refrigerator at 4°c within three weeks. There was avoidance of metal contact with the coconut water (Bustamante, 2002) and cautions were taken in preventing particles from entering the water during the process of extraction (FAO, 2007).
Treatment Protocol
A total of fifty- five animals with established approximately 4days cyclicity were randomly divided into four major experimental groups 1 to 4.
Group 1: Negative control group received distilled water
Group 2: Induction and Withdrawal group
- 0.2mg/100g of MCH for 28 days
- 0.2mg/100g of MCH for 28 days - withdrawal for 8 days
- 0.2mg/100g of MCH for 28 days - withdrawal for 16 days
- 0.2mg/100g of MCH for 28 days - withdrawal for 28 days
Group 3: Post- treatment group
- 0.2mg/100g of MCH for 28 days -5ml/110g of ICW for 8 days
- 0.2mg/100g of MCH for 28 days - 5ml/110g of ICW for 16 days
- 0.2mg/100g of MCH for 28 days - 5ml/110g of ICW for 28 days
Group 4: Positive control group
- 5ml/100g of ICW for 8 days
- 5ml/100g of ICW for 16 days
- 5ml/100g of ICW for 28 days
Each experimental rat was served with volumes of the extract according to their weights using simple percentile faction. The administration was done by the use of an oropharyngeal canula.
Preparation of Histological Slides
The animals were sacrificed on the proestrous phase of estrous cycle. The ovaries were carefully dissected out and trimmed of fat. The ovaries were weighed and then fixed in 10% formal saline. The fixed tissues were transferred into ascending grades of alcohol. On day 1, the tissues were placed in 70% alcohol for 7 hours, then transferred into 90% alcohol and left in the latter overnight. On day 2, the tissues were passed through three changes of absolute alcohol for one hour each and finally cleared in Xylene. The tissues were then infiltrated in molten paraffin wax at 58°C. Three changes of molten paraffin wax at one hour intervals were made, after which the tissues were embedded in wax and blocked out. Serial sections of 5 µm thick cut by the rotary microtome were obtained from a solid block of tissue and floated in water bath. The sections were picked with clean slides onto which egg albumin had been coated and dried on the hot plate at 52oC. The mounted sections were dewaxed in xylene and then hydrated in descending grades of alcohol (90%, 70% and 50%). The sections were then stained with Haematoxylin for 10 minutes after which rinsed and differentiated in 1% acid alcohol for 10 seconds before rinsing in water. The sections were then counter stained in Eosin for one minute, rinsed in water and dehydrated in 50%, 70%, 90% and absolute alcohol. The sections were finally cleared in xylene and a drop of mountant was placed on the section and covered with cover slip (Drury and Wallington, 1960).
Hyperprolactinemia is an extreme common endocrine cause of infertility wherein women are more predisposed than men. It is essentially associated with chronic anovulation which is probably the major cause of female infertility (Rebar, 1997). This female reproductive dysfunction affects about one-third of infertile women worldwide (Corenblum et al., 1993). In northern Nigeria, there was 33.4% observation of hyperprolactinaemia in infertile women as it also incident in other causes of infertility such as polycystic ovarian syndrome, luteal phase defect and hypogonadism(Emokpae et al., 2011). Hyperprolactinaemia impairs ovarian structural and functional development thereby causing anovulation. Hyperprolactinemia accounts for approximately 80% of hypogonadotropic anovulation (Molitch, 2010) and occur in about 10 to 20 percent of amenorrhea non-pregnant women (Pouneh and Lisa, 2010). The decrease in the secretion of FSH and LH seen during hyperprolactinaemia may inhibit follicular maturation preventing the production of a mature egg (Nawroth, 2005) It also affects ovarian steroidogenesis as high prolactin tends to reduce granulosacellmitoses and hence inhibit follicular estradiol production in the body (Colao et al., 1998). There is accumulating evidence that PRL also exerts a direct inhibitory effect on gonadotropin actions in the ovary (Kelly et al.,1993).
Green coconut water is an immature fruit. It is of special interest because the fruit at this stage of development possesses healing properties far beyond that any other fruit endosperm. The green coconut water is revered as a valuable source of medicine that literally comes in its own container. For thousands of years coconut products have held a respected and valuable place in local folk medicine. It common names include, fluid of life, dew from heaven, miracle water and nature’s sport. Coconut water has a host of yet scientifically unproven but traditional uses in cultures all over the world. From ancient times in Africa, reports support the position that about 85% of the world’s population rely on coconut fruit in traditional medicine. Coconut is use to treat a wide variety of health problems such as asthma, colds, cough, dropsy, dysentery, earache, fever, flu, gingivitis , gonorrhea, jaundice, lice, scabies, scurvy, skin infections, sore throat, typhoid and ulcers. It is use to conquer irregular or painful menstruation and also taken during pregnancy to give the unborn babies strength and vitality. It is also use to boost semen quality and induce libido. Also people suffering from loss of memory and many forms of memory defect can benefit from it (Sofowora, 1993). In the Indian and Caribbean folk medicine, it is believed to be highly beneficial in eliminating kidney stones and treating urinary tract infections (Corner, 1966). It was reported that GCW demonstrated estrogen-like properties when administered in several groups of postmenopausal rats. The rats demonstrated estrogen levels comparable to rats that still had their ovaries (Nisaudah et al., 2009). It has been suggested that GCW contains β-sitosterol in addition to other sterols, such as stigmasterol and fucosterol and α-spinasterol (plant sterols known to be involved in the synthesis of steroid hormones invivo) may be responsible for the strong estrogenic effect of GCW by facilitating the synthesis of endogenous estrogens. The β-Sitosterol is structurally related to animal cholesterol and can possibly act as a precursor of sex steroids (Punghmatharith, 1998). Cocos nucifera water has been shown to aid pregnancy as the number delivered at the end of the gestation period corresponded to the number of implantation sites counted on day 10 of pregnancy in mice. The water was also shown to promote diuresis with minimal loss of electrolytes. Cocos nuciferacan therefore be used in women with threatened or habitual abortion (Pragya, 2010; Kennedy et al., 2013). Women and men, even in their mid-60s, have reported to have increased libido after drinking the coconut juice of green coconuts. It also improves sexual vitality, boosts sperm count and enhances motility (Oettlé, 1993).
Accurate stereological estimations in ovarian follicles at various stages of development is an important indicator of the process of folliculogenesis in relation to the endocrine signals and paracrine/autocrine mechanisms that control the growth and maturation of the oocytes and their supporting follicular cells (West, 1990).Ovarian follicle is a three-dimensional structure with a central antrum surrounded by theca and granulosa cell. It secretes hormone that controls the reproductive cycle and has the ability to release an egg at ovulation. Volume density (VD) evaluations of the ovarianfollicle is used to measure development and information on the physiological status of the ovaries as different cell has its own function. This study therefore was carried out to investigate the effect of GCW on the VD of follicular components of the ovary of hyperprolactin rats.
MATERIAL AND METHODS
The stereological evaluation of volume density of the antrum, granulosa cell and theca cell were carried out only on mature follicles according to the method described by Gundersen. A restricted artificial boundary is marked on the follicle limited to boundary of the follicle. A counting grid with set of regularly spaced points is placed on the field. All points hitting profiles (disregarding their relationship to the frame) are counted.
A point is considered a hit if a profile (including its boundary) covers the upper right corner in the cross where the two lines cross each other. The volume density is calculated as proportion of volume by simple percentile fraction; VD (profile counted/total specimen profile x100) (Gundersen et al., 1988).
STATISTICAL ANALYSIS
Results were expressed as mean ± standard deviation. Analysis was carried out using analysis of variance (ANOVA) with Scheffe’s post hoc test. The level of significance was considered at p< 0.05