The Spermatogenic and Ovogenic effects of chronically administered Jaggery to rats

 The Spermatogenic and Ovogenic effects of chronically administered Jaggery to rats

Sperm count

 All the animal groups male and female administered jaggery  showed an increase in the number of sperms in both the testes and epididymides in a dose dependent manner compared with the control group. In particular, the epididymides from groups II and III showed a significant increase in the number of sperm compared with the control group and ova in ovaries in female rats

The weight of each of the organs was calculated as a weight percentage to the body weight.

BIOASSAY

Materials and methods

Study Design

Validated methods [8], specified by the United States

Pharmacopeia (USP), were used to determine the bioactivities of each product individually and subsequent mixtures of the two. The bioassays were performed at

Qualtech Laboratories Inc., Ocean, New Jersey. Ovarian

weights (FSH bioassay) or seminal vesicle weights (LH

bioassay) were obtained after treating prepubertal female

or male rats with Bravelle® (75 IU FSH; ≤2% LH activity),

Menopur® (75 IU FSH; 75 IU LH activity), or one of three

mixtures of Bravelle® and Menopur®: 150:75 IU (1 vial

Bravelle®:1 vial Menopur®); 300:75 IU (3 vials Bravelle®: 1

vial Menopur®); and 300:225 IU (1 vial of Bravelle®:3 vials

of Menopur®). 

These ratios represent a low combination

of FSH: LH activity (150:75 IU) as well as combinations

with a high FSH and low LH activity (300:75 IU) and high

FSH and high LH activity (300:225 IU). The FSH and LH

bioactivities were compared to a menotropin Reference

Standard traceable to the National Institute for Biological

Standards and Control (NIBSC).

Test Animals

In-bred Wistar female rats (n = 296) 21 days of age were

used for the FSH bioassay and Sprague Dawley male rats

(n = 248) 21 days of age were used for the LH bioassay.

The rats, certified to be free from murine viruses, were purchased from Hilltop Lab Animals Inc. (Scottdale, Pennsylvania). Upon arrival, the animals were weighed, their

health was assessed and they were randomized into

groups of eight. The animals were housed in polypropylene solid bottom cages with stainless steel wire lids containing shredded Aspen shavings. The animal room was

maintained at 18–19°C with a relative humidity of

approximately 50% and 12:12 hour, light: dark cycle. Rats

were given Purina rodent diet and tap water ad libitum.

For both the FSH and LH assays, one group of rats (N = 8)

was assigned to each of the low, middle, and high concentration groups for each gonadotropin, gonadotropin mixture and Reference Standard. Each assay was done in

duplicate, Therefore, for the FSH assay, 48 rats each were

assigned to six groups: Bravelle®, Menopur®, the three

ratios of Bravelle® plus Menopur®, and the Reference

Standard. For the LH assay, 48 rats each were assigned to

five groups: Menopur®, the three ratios of Bravelle® plus

Menopur®, and the Reference Standard. In addition, there

was a control group in each assay that had eight rats

treated with diluent alone.

Reference Standards for the FSH and LH assays

The Reference Standard diluent used for both gonadotropins was prepared by dissolving 10.75 g disodium hydrogen phosphate, 7.6 g sodium chloride and 1.0 g bovine

serum albumin in 1.0 liter of distilled water. The pH of the

solution was adjusted to 7.2 ± 0.2 with 1 N sodium

hydroxide.

As per the USP, for the FSH assay 70,000 units of hCG

were added to the Reference Standard diluent. A menotropin Reference Standard (Ferring, lot-DPH12250397,

122.2 FSH IU/mg, traceable to NIBSC) was reconstituted

and diluted in the Reference Standard diluent to the final

concentrations of 1.9, 3.8 and 7.6 IU/0.6 mL.

For the LH assay, a menotropin Reference Standard (Ferring, lot-DPH12250397, 99.3 LH IU/mg, traceable to

NIBSC) was reconstituted and diluted in the Reference

Standard diluent to the final concentrations of 7, 14, and

28 IU/0.8 mL.

The Reference Standard concentrations were established

in a geometric progression for the low, middle and high

doses respectively, based on previous dose response studies. The lowest concentration in this three-dose range produces a definite response in some of the rats as compared

to the control group and the highest concentration produces a submaximal to maximal response.

For both the FSH and LH bioassay, 29 vials of Menopur®

(lot-FHA004ULB) were each individually reconstituted

with 0.5 mL of 0.9% saline and pooled (diluent A).

Another 10 vials of Menopur® (lot-FHA004ULB) were

individually reconstituted with 1.0 mL of diluent A and

pooled (diluent B). For the FSH assay, two vials of Bravelle® (lot-FMA001) were each reconstituted with 1.0 mL of

diluent B and pooled. For the LH assay, five vials of Bravelle® (lot-FMA001) were each individually reconstituted

with 1.0 mL of diluent B and pooled.

For all mixtures, reconstitution and pooling were done

with commercially available BD plastic syringes. The solutions were further diluted to the same three concentrations as the Reference Standard with

 Reference Standard

diluent: 1.9; 3.8; and 7.6 IU/0.6 mL for the FSH assay and

7; 14; and 28 IU/0.8 mL for the LH assay.

Control Solution

The Reference Standard diluent was used as the control

solution for both assays.

FSH assay

Each female rat was injected subcutaneously with 0.2 mL

of the assigned dose at approximately the same time of

day for three consecutive days. Twenty-four hours after the

last injection, rats were sacrificed in a carbon dioxide

chamber. Left and right ovaries were carefully dissected

from each rat, freed of fat or fibrous tissue, dried by gentle

blotting on absorbent paper and weighed on an analytical

balance. For each rat, the left and right ovarian weights

were recorded.

LH assay

Each male rat was injected with 0.2 mL of the assigned

dose at approximately the same time of day for four consecutive days. Twenty-four hours after the last injection,

rats were sacrificed in a carbon dioxide chamber. Seminal

vesicles were carefully dissected from each rat, freed of fat

or fibrous tissue, dried by gentle blotting on absorbent

paper and weighed on an analytical balance.

THIS ABOVE METHOD OF BIOASSAY DONE WITH JAGGERY 

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