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The Lepex – Novum rabbit semen dilution and ovulation medium.

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In 2015, I developed the rabbit semen preservation medium Lepex, which has been on the market ever since. Recently, I have gained new knowledge about the biochemical mechanisms related to rabbit reproduction, which led me to design the new extender Lepex-Novum (Novum from New Ovulation Medium). The rabbit semen extender Lepex-Novum is a groundbreaking solution that preserves sperm cells and induces ovulation. The ingredients present in Lepex-Novum are specifically designed to assist the protein beta nerve growth factor (NGF)  naturally present in buck semen to induce ovulation. By supporting different molecular mechanisms involved in the natural ovulation process, Lepex-Novum enables natural mating-induced ovulation. In the following paragraphs, I will delve into the crucial elements of the development of Lepex-Novum and provide intricate details about the molecular processes of reproduction in rabbits. The powders to prepare the Lepex-Novum medium are stored separately.

1. Rabbits.


European rabbits are small mammals belonging to the family Leporidae. They were hunted and eaten by hominids during the Stone Age in large numbers in the northwestern Mediterranean areas, from Spain to Italy. The Phoenicians, who traded all over the Mediterranean area from Cyprus to the Iberian Peninsula, probably started the spread of the rabbit. The Romans discovered the rabbit as a food source, and the trade in the Roman Empire further spread the rabbits from the Iberian Peninsula to other parts of the world. Domestication took till the 18th century. Today, rabbits are bred bigger and fatter to get more meat. They are introduced worldwide in many cultures and are a part of daily life as food, clothing, a companion, and a source of artistic inspiration. Rabbits are used as a research model in fundamental science, and in applied science, rabbits are used among others for the production of antibodies that can be used in humans or to study human disease.

2. Rabbit meat production.

The rabbit is a good meat source to fulfill the demand for animal protein. With an optimal breeding program, a rabbit doe (female) can produce 60 young rabbits per year, representing over 100 kg of rabbit meat. Moreover, rabbits have a high reproductive rate compared to other livestock. They become sexually mature earlier, have a relatively short gestation period,  and can be rebred immediately after kindling. Artificial insemination (AI) of rabbit females appeared on European farms in the late 1980s. It was used earlier to obtain the animals' genetic improvement and solve sanitary problems. Significant improvements concerning both productive and economic aspects were later achieved. The technique of artificial insemination of rabbits continues to evolve.

France, Italy, and Spain historically produce a lot of rabbit meat. It is, therefore not surprising that a great deal of scientific research has taken place in those countries in particular and is still being conducted at a high level. Much of the study was published in a particular journal of the World Rabbit Science Association.

Below is an introductory text to a special issue of MDI Animals written by Spanish scientists who were guest editors.

Dear Colleagues,

In the coming decades, the demand for livestock products will increase with the dramatic rise in world population rises in addition to consumers becoming more conscious and demanding high-quality products. Rabbit husbandry, particularly important in the Mediterranean area and the semi-arid zones of Africa, Asia, or America, represents a valuable alternative source of good animal protein of high quality (low fat and low cholesterol). As rabbits are being chosen, worldwide, as a livestock species due to their reproductive and feeding advantages, modern rabbit breeding will face some important future challenges that must be resolved in the next few years to ensure it remains a competitive and sustainable sector. Different strategies should be used to maintain a balance between rabbit welfare, customer demands, and breeder requirements. An improvement in breeding systems is needed, and may involve avoiding hormones and antibiotics; enhancing the efficiency of reproductive procedures such as artificial insemination systems or freezing of semen and embryos, among others; extending the productive lifespan of does; and adjusting nutritional systems to optimize the physiological status of animals. This Special Issue aimed to publish high-quality manuscripts, including original research studies and reviews, that mainly address any of these topics in increasing rabbit productivity and sustainability.


Dr. Rosa María García-García

Dr. Maria Arias Alvarez

Guest Editors

3. Artificial insemination in rabbits.

The commercial use of AI in rabbit farming is relatively recent. The large-scale use of AI in rabbit farming started in the late 1980s. Thanks to the AI of rabbits, it is possible to increase the volume of high-quality products obtained with low labor costs, as opposed to using natural mating. Creating quality conditions for keeping a small number of males is easier. Conception rates after AI with fresh semen are equivalent to or even better than those obtained by natural mating.

Further advantages are: one can control offspring quality, increase the number of individuals with valuable, functional characteristics, prolonged fertility even during unfavorable times of the year, all year round cycle based production, and more efficient breeding programs.

AI was used earlier, before large-scale applications, to obtain the animals' genetic improvement and solve sanitary problems. However, significant improvements in both productive and economic aspects were later achieved, such as accelerating the desirable traits of high genetic value supported by semen preservation.

The systematic use of AI in intensive rabbit production is one of the most effective approaches to distributing ejaculates from bucks of superior quality or genetic value. In short: AI offered large farms the opportunity to optimize the conditions for the highly economical production of rabbit meat.


4. Application of AI in rabbits requires the use of GnRH hormone.


Many mammals hold that ovulation is regulated by ovarian steroids released from mature ovaries. These steroids elicit the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. These mammals are called spontaneous ovulators.

Rabbits, however, are induced ovulators. This means that ovulation is induced by mating, which triggers GnRH secretion. Thus, when one uses AI instead of natural mating in large-scale rabbit farming, one must apply a gonadotropin-releasing hormone  (GnRH) to induce ovulation.

The application of GnRH can be done in several ways but is mainly done through intramuscular injection of the doe or intravaginal administration.

Examples of commercially available synthetic GnRH as veterinary medicines are Receptal from MSD Animal Health (part of US-based Merck & Co) and Dalmarelin from Fatro (based in Italy). Receptal contains 4.2 mcg of buserelin per ml, and Dalmarelin contains 25 mcg of lecirelin per ml

After intramuscular injection, the agonist is taken up in the blood vessels of the muscle tissue and distributed in the system. Immediate response….

Entry of the agonist in the blood causes an immediate release of luteinizing hormone from the anterior pituitary, resulting in ovulation.

About ten times more agonist is needed for intravaginal administration since the GnRH molecules need to pass the vaginal wall to reach blood vessels. Next to the wall passage, the vaginal environment is hostile to external protein.

The Hormone GnRH


Aa hormone is a regulatory substance produced in an organism and transported in tissue fluids such as blood to stimulate specific cells or tissues into action. A gonadotropin hormone is a hormone that acts on or stimulates the gonads,  i.e., an organ that produces sperm cells (testis) or egg cells ( ovary).

Gonadotropin-releasing hormone (GnRH) is a releasing hormone responsible for the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the anterior pituitary. GnRH is a tropic peptide hormone synthesized and released from GnRH neurons within the hypothalamus.

GnRH is a small protein or peptide consisting of 10 amino acids.

The identity of GnRH was clarified by the 1977 Nobel Laureates Roger Guillemin and Andrew V. Schally. The half-life of GnRH is only 2-4 minutes since the molecule is rapidly degraded by proteolytic enzymes, named peptidases. The short half-life was why GnRH analogs have been synthesized to increase their potency and duration of action for applications as human medicines.

For administration to mammals, synthetic GnRH is used. These molecules are called GnRH agonists or analogs. Synthetic GnRH is used to induce ovulation in rabbits. These molecules are called GnRH agonists or analogs. The analogs differ slightly from the natural (human) GnRH decapeptide but with specific modifications. These modifications are single or double substitutions of specific amino acids (in amino acid position 6). But also changes at position 9 (alkylation) or deletion of position ten occur. These modifications may inhibit rapid degradation in body fluids while maintaining function. Agonists with two amino acid substitutions are leuprorelin, buserelin, goserelin, and deslorelin. The agent's nafarelin and triptorelin are agonists with single substitutions at position 6.

5. Public resistance to the use of hormones.

Many people are not happy with the use of hormones in animal production. When it comes to AI in rabbits, the female is given an injection of the GnRH hormone, which can cause stress and spread diseases. Plus, the injection process generates a lot of plastic waste. To avoid this, the hormone can be added to the sperm used for insemination. Unfortunately, this turns the extender into a veterinary medicine, which can be costly for rabbit companies to register.

However, NGF has recently been discovered as a natural way to induce ovulation and improve animal welfare in rabbits. By adding NGF to the semen extender, ready-to-use seminal doses can be prepared without the need for injections or registration as veterinary medicine.

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6. NGF-induced ovulation as an alternative.


Until a few years ago, it was thought that ovulation in rabbits could only be induced by copulation. The notion was that the primary mechanism involved in releasing GnRH among induced ovulators consists of the activation of noradrenergic neurons in response to genital somatosensory signals generated by receiving an intromission from a male during mating. These noradrenergic neurons would then promote the release of GnRH. However, recent research into the molecular basis of reproduction in rabbits Indicates that the protein beta Nerve Growth Factor (NGF) plays a vital role as an ovulation-inducing factor.

Nerve growth factor (NGF) is a protein and neurotrophic factor that regulates specific target neuron growth, maintenance, proliferation, and survival. But many years after its discovery, it became clear that NGF also plays a vital role in reproduction, particularly in ovulation-induced species

The effect of NGF on target cells is mediated by two receptors: tropomyosin receptor kinase A (TrKA), which selectively binds.
NGF, and the p75 neurotrophin receptor. These receptors are at several locations in the doe reproductive tract. 

Through interaction with the receptors, the protein NGF induces ovulation but also affects the doe's reproductive tract during and after ovulation.  Further, it also has a role in the biochemical environment during early embryo development. 

NFG may act as an ovulatory induction factor by two types of mechanisms in the female reproductive tract. First, NGF may stimulate uterine and cervical sensory neurons, which trigger neurons in the hypothalamus. Second, NGF can cause the further synthesis of NGF in the reproductive tract, which, together with that deriving from semen, is carried by the blood and target the brain with the ultimate release of GnRH. 

The most compelling evidence for the critical role of NFG is that synthetic NGF (a recombinant protein identical to the NGF found in rabbits) added to diluted semen induces ovulation and conception by the intravaginal route. Ovulation is generated in a dose-dependent matter. There were healthy offspring with no difference compared to GnRH.

The following picture emerges. About 1 mL of ejaculate containing about 300 million sperm cells suspended in seminal plasma containing NGF is deposited during mating. Through the action of NGF, a series of several molecular and cellular functions is initiated.  When spermatozoa reach the oviduct, within minutes in rabbits, only a small number of the sperm cells attach to the oviductal epithelium.  Ovulation is induced about ten h after coitus, and fertilization occurs 2–3 h after ovulation. All these events result from endocrine and neuronal interplay connected to mating, with NFG playing a pivotal role. 

NGF presents an alternative to the use of GnRH. Moreover, it is expected that NGF will play an essential role in the future of AI in many other animals. 

7. Matrix in Lepex improves the functioning of NGF-induced ovulation?


The rabbit semen extender Lepex contains Matrix, a polymer. As written above, this extender was developed in 2015 and used at the Peter Rutjens  Rabbit AI Station. In recent experiments that were aimed at the use of specific antibacterial peptides, a good performance was noted without the use of intramuscular injection of a GnRH-analogue. This made us suppose that Matrix, in one way or another, improved the functioning of NGF-induced ovulation.

The molecular mechanism of Matrix in extender application is several-fold; first, the sperm cells staple less; second, there is less backflow of diluted semen after insemination; and third, and most importantly, Matrix acts as a mucoadhesive. At that point, we supposed that the unexpected positive outcome of our experiments was caused by the improvement of NGF-mediated ovulation in rabbits, with a prime role of Matrix.

Matrix, a new role in the application of NGF

In 2012 and 2013, I investigated different polymers to apply in preservation media for swine semen to prevent the precipitation of the cells in the dilution by the presence in the medium of the three-dimensional network of the polymer. This would then be analogous to the action of the polymeric carrageenans in chocolate milk, which prevents the precipitation of the cocoa particles.

I started with a collection of several dozen polymers. After selection by in vitro research, namely sperm motility analysis during storage using the computer-assisted semen analysis system CEROS II from Hamilton Thorne, I came up with three potential candidates. After further selection using insemination tests, a single polymer with a specific range of molecular weights remained. This polymer was called Matrix.

Matrix was extensively tested in swine. The first experiments indicated that single insemination was sufficient for sows to become pregnant, in contrast to the current practice of two insemination. Matrix in the dilution medium for swine semen was tested over more than two years on various farms in the Netherlands. The conclusion was that with only one insemination, the medium with Matrix increased the farrowing rate by 2-4% and increased litter size by 0.9 -1.7 piglets per litter.

Unfortunately, large companies producing and selling semen diluents and consumables for AI were not interested in Matrix because it would always significantly reduce their turnover. After all, only one insemination was needed once instead of twice. 

After researching the use of Matrix in swine semen diluents, I turned my attention to developing a rabbit semen extender. In 2015, I successfully created Lepex. The image below demonstrates the motility of rabbit sperm diluted in Lepex during storage at 17°C.

motility rabbit.jpg

The motility of the various rabbit sperm samples was determined using the CEROS II computer-assisted sperm analysis system from Hamilton Thorne. This CASA system enabled me to analyze sperm based on tale settings to avoid counting all the particles in rabbit semen.


Matrix showed a positive effect on the storage capacity of Lepex. This and other experiments showed that Lepex has a  better storage capacity than other commercial rabbit semen extenders.

It was expected that Matrix would also have a positive effect on the reproductive performance of rabbits. After several exploratory experiments, the optimal percentage of Matrix was determined in a large-scale field trial.

The image below shows the results. At optimal Matrix concentration, the pregnancy rate is 4-5 % higher, and the litter size born alive is almost one higher compared to an extender without Matrix.

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Porex, Porexcell, and Pronexcell.

Many years ago, I accidentally discovered the positive effect of whey isolates on the storage capacity of swine semen extenders. Adding 1 % of a particular brand of whey isolate was named Porex. Porex was tested in TRIXCell, the extender of IMV Technologies. TRIXcell+ was the extender with Porex. During the six years of insemination trials, 35 farms that participated had each year a higher farrowing rate and a higher number of piglets born alive when TRIXcell+ was used instead of BTS, the reference extender..  resulted in a higher farrowing rate and a higher litter size.

TRIXcell+ showed an average increase in the number of piglets born alive by 0.6. Earlier studies indicated that TRIXcell had a similar farrowing rate and litter size performance to BTS (Haugan et al., 2007).

The additive to the extender was named Porex. A further improvement of Porex was the addition of soy lecithin. With the addition, the name was changed to Porexcell. Both Porex and Porexcell have been used for many years in swine semen extenders.

Around 2014, it became apparent that Porexcell’s positive effect became less and even negatively impacted the preservation capacity of swine semen extenders. It turned out that it contained increasingly more pesticides over the years. This negative effect on motility during the storage of diluted sperm was not noticeable in stallion semen extenders or rabbit semen extenders. It appears that swine sperm cells are highly susceptible to pesticides.  Efforts were undertaken to develop a product with no pesticides; since I did not want the development to affect stallion and rabbit sperm cells, eventually, I changed to a protein mixture derived from milk but purer than whey protein. The new product was named Pronexcell. To this day on, Pronexcell is used in Hippex semen extenders for stallion sperm and in Lepex, the rabbit sperm extender.

The positive effects of these proteins add to the performance of Matrix.



8. Matrixcell.


The results of the initial experiments indicated that Lepex-Novum performs very well. Additional ingredients had to be added to reach a performance similar to using GnRH. As written above, the NGF protein interacts with two receptors, TrkA, and p75, that can be found throughout the female reproductive tract to induce ovulation. Multiple receptor activations are necessary, and the response is directly proportional to the number of NGF-receptor encounters per unit of time. Effective signal transduction requires proper concentrations and a suitable biochemical environment. A sperm extender has a very different biochemical milieu than vaginal mucus. The surface of the female reproductive tract is covered with mucus, a complex viscoelastic gel that acts as the first line of defense against harmful agents from the outside environment. Mucus plays an essential role in many biological functions. The mucus houses vaginal flora, critical in maintaining the vaginal mucosal barrier. To improve the mixing of diluted sperm with mucus and to enable NGF to interact with its receptors, Matrixcell is one of the critical ingredients of Lepex-Novum.


NGF delivery at the site of receptors in the female reproductive tract may be affected by mucus in numerous folds, cavities, and ridges in the epithelium, which increase the vagina's surface area and provide distensibility.  Moreover, leakage may reduce the amount of NGF available for interaction with receptors. With the application of AI, there are fewer vaginal fluid dynamics and natural biomechanical forces than with natural mating.

Thus,  the efficacy of NGF activity relies on adequate concentrations and retention on the surface of the walls of the reproductive tract. Mucoadhesive Matrix proved successful, prolonging NGF presence in the mucosa by forming physical and chemical bonds with the mucus. However, to improve NGF penetration into mucus, Matrixcell was used, which is a more pure and refined form of Matrix.

The mucus penetration encompasses different phases. The first establishes an intimate contact between the insemination medium and the mucus and involves spreading the medium. In the second phase, an interpenetration between Matrix polymers and mucus glycoproteins occurs by forming physical entanglements between the two macromolecular species. In particular, Matrixcell performs better than Matrix in this phase, as indicated by in vitro experiments. In the third phase, consolidation occurs, strengthening the Matrix polymer-mucin binding by creating Van der Waals forces and hydrogen bonds.



9. NGF half-life


The presence of peptidases in both diluted semen and the female reproductive tract affects the half-life of NGF in the insemination dose. Peptidases are found in rabbit seminal plasma, primarily coming from the epididymis and accessory sex glands, while microbes colonize the female reproductive tract and also secrete peptidases. Both the sperm and the vaginal fluid contain protein-degrading enzymes. 

Matrixcell, present in the extender, acts as a gel to extend the half-life of NGF. In addition, the half-life of NGF proteins was extended by adding a cocktail of inhibitors of protein-degrading enzymes, competing peptides, and proteins (a.o., Pronexcell), 

10. Concluding remarks


The new insights into the reproduction of rabbits led to the development of the new semen extender Lepex-Novum. The various unique ingredients of the Lepex-Novum medium support the molecular mechanisms that lead to ovulation and pregnancy in rabbit. The excellent performance of Lepex-Novum has been confirmed by several experiments at different locations in 3 countries.


Barex Biochemical Products

The art of semen preservation

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