This article explains the lecture given by Dr. María Peñas, from Recombina Biotech, at the XXXI Biovet International Symposium in Tarragona, during the third session dedicated to pronutrients, which took place on May 28^th, 2018. Under the title “Evaluation of the ability of Alquernat Magacal to increase the absorption of calcium in enterocytes” the conference described the effects of this product, based on pronutrients, which improves intestinal absorption. The veterinarian of the team of Biovet S.A, Dr. Antón Tallero, has made the adaptation of the presentation.

INTRODUCTION

Alquernat Magacal is a product based on pronutrients, which are active molecules of botanical source that improve the quality of the eggshell and prevent bone loss in layers by increasing calcium absorption at the intestinal level.

Thus, Alquernat Magacal helps to maintain adequate calcium levels in laying and breeding hens. Avoiding calcium deficiency means it’s possible to prevent conditions that affect egg and bone quality, such as cage fatigue syndrome and bone fracture. In order to achieve this, Alquernat Magacal acts directly on the cells of the intestinal mucosa (enterocytes) by increasing intracellular calcium absorption.

To demonstrate its effectiveness, an in vitro study was developed in the Recombina laboratory at the end of 2016, in which the calcium absorption of the enterocytes is quantified after the addition of this product developed by Biovet S.A.

OBJECTIVE

To evaluate the effect of Alquernat Magacal on the absorption of calcium by measuring the concentration of intracellular calcium in enterocytes.

EXPERIMENTAL DESIGN

• Determination of calcium absorption

In order to determine the amount of calcium absorbed by the enterocytes, FLUO-4 was used, which is a permeable indicator of calcium that allows to quantify the concentration of cytosolic calcium by fluorescence.

When FLUO-4 passes through the cell membrane, it is metabolized by esterases, generating Fluo-4 / Ca²⁺, which cannot pass through the membrane again.

The cell line used in the trial was IPEC-J2, which is an untransformed line, comes from piglet intestinal tissue and is used in a model for studies with challenges.

The enterocytes were cultured until confluence (2-3 days, 80-90% cell growth) in special plates to evaluate the fluorescence.

• Cellular treatments

Five batches or treatments were established: three with different concentrations of Alquernat Magacal, a negative control and a positive control with carbachol, a molecule that increases intracellular calcium mobilization. Each treatment was distributed in 12 wells: 6 with 2 mM calcium phosphate and 5 with 1 mM EGTA.

Group 4 and 5 are used as control groups. Group 4 is taken as a negative control, without any type of treatment. Carbachol is added to the positive control group, which is a substance that increases the intracellular mobilization of calcium.

• Treatments with Fluo-4

After incubation with Alquernat Magacal, the cells were washed and Fluo-4 was added.

The wells were incubated in a dark environment for 1 hour and the fluorescence was determined, which will indicate the concentration of calcium absorbed by the enterocyte.

 

RESULTS

In the upper graph we can observe the fluorescence levels that were determined after the incubation with Alquernat Magacal. The blue bars indicate the level of fluorescence due to the presence of calcium absorbed in the medium. The pink color EGTA expresses the level of cytosolic calcium in cells where calcium has not been added to the medium. That is, it represents basal calcium.

There is a relationship between the concentration of Alquernat Magacal and level of fluorescence. This is concentration of cytosolic calcium.

In the batch or treatment 3, where the concentration is higher, there is a greater difference between calcium absorbed from the medium and basal calcium and therefore a greater absorption capacity.

CONCLUSIONS

This test shows that Alquernat Magacal increases the intracellular calcium concentration by increasing the absorption in enterocytes.

A greater efficacy (significant calcium absorption increase) is observed using a 1:100 dilution of the active principles.

This means that Alquernat Magacal helps to obtain the right amount of calcium, important in multiple functions, including the development of the eggshell and bone.