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Information on Equine Artificial Insemination. 

1. The collection of equine semen starts with a clean penis.


Hygiene in horse artifical insemination is a necessity in order to obtain high fertility rates. The stallion penis harbours bacteria and other pathogens, and all kinds  of debris that can reduce the quality of the collected semen and reduce fertility rates.

Therefore, the start of the semen collection should be to clean the penis just befor the semen collection. The use of clean water is recommended in order not to disturb the natural population of bacteria.

The images below give an illustration.


The famous stallion Totilas is being brought for semen collection.

The penis is carefully cleaned by Bart Kools, while Totilas is being hold by his assistant.

The semen is collected in a special area and therafter immediately brought to the laboratory for dilution in Hippex extender.

2. A note on the ingredients of Hippex. 


In order to preserve equine sperm cells for fresh use or cooled transport in a liquid medium for several days, it is necessary to dilute the semen in a medium that contains proper ingredients that protect the sperm, especially the sperm cell wall.  The prime cell damaging events are oxidation of cell wall lipids (see Sieme et al. (2015) Reprod Domest Anim. 50 Suppl 3:20-6) and the removal of cell wall components by certain seminal proteins, called BSP proteins (see review by Plante et al. (2016) Cell Tissue Res 363:105-127). It is also important that the extender contains molecules that protect the sperm cells from a temperature-shock, since the diluted semen is lowered in temperature from around 30 °C to 4 °C to reduce cell metabolism.

The damaging oxidation of cell wall components can be prevented or stopped by antioxidants and by liposomes, which stabilize the cell wall. Hippex contains Lipex, a mixture of liposomes that consist of lipids similar to those of the sperm cell wall. The mechanism of action of these liposomes is unknown, but the product Lipex has proven to be very effective as antioxidant and cell wall stabilizing factor.

The function of the seminal BSP proteins is to bind to the sperm cell wall and to remove phospholipids and cholesterol in order to prepare the sperm cell for fusion with the egg cell.  This process can be delayed (to preserve the cells) by removing the BSP proteins using centrifugation. But then one also removes the more than 1000 proteins that fulfill functions in motility, modulation of the immune response (which is very important with respect to inflammation conditions such as endometritis), and fusion of the sperm cell and egg cell.

An alternative to centrifugation is the addition to the extender of certain proteins that bind to the BSP proteins, which prevents proper functioning of the BSP proteins in the storage medium. Hippex contains a balanced mixture of these protective proteins, collectively called Pronexcell. For proper protection, the Pronexcell proteins must have a higher concentration than the BSP proteins. Therefore, the volume of Hippex extender to be added to semen must be at least 2.5 times the size as that of the semen. In this way, Hippex can effectively be applied without centrifugation of the semen for 80-90% of the stallions.

Both practices (centrifugation and non-centrifugation) result in acceptable pregnancy rates. Therefore, it seems evident that non-centrifugation is the preferred method, because it saves time and money.

3. The importance of the degree of semen dilution. 


The degree of dilution of the equine semen determines the concentration final concentration of the protective ingredients. The following experiment illustrates this. A semen sample was split in 2 parts and diluted at different rate. 


This experiment showes a moderate difference between the 2 different degrees of dilution. In other cases the effect can be considerable.

Therefore, to ensure the preservation of equine sperm cells in Hippex extender for more than 2 days, it is recommended to dilute the semen with at least 3 volumes of Hippex extender.

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