WO2016128858A1

WO2016128858A1 - Extender for cryopreservation of bovine sperm

Info

Publication number: WO2016128858A1
Application number: PCT/IB2016/050510
Authority: WO
Inventors: Armin TOWHIDI; Ghassem Amoabediny; Seyyed Mojtaba MOUSAVI; Mahdi ZHANDI
Original assignee: Towhidi Armin; Ghassem Amoabediny; Mousavi Seyyed Mojtaba; Zhandi Mahdi
Priority date: 2015-02-11
Filing date: 2016-02-01
Publication date: 2016-08-18

Abstract

An extender for cryopreservation of bovine sperm and a method of producing the extender are provided. The extender comprises soy lecithin nanoparticles, wherein an average diameter of the soy lecithin nanoparticles is less than 100 nm. The extender may better protect the sperm during the cryopreservation process compared to known extenders for cryopreservation of bovine sperm.

Description

Extender for cryopreservation of bovine sperm

The invention relates to an extender for cryopreservation of bovine sperm. The invention further relates to a method of producing an extender for cryopreservation of bovine sperm.

Cryopreservation of bovine spermatozoa may have applications in artificial insemination, assisted reproductive technologies, conservation of valuable genetics, and improvement of genetic progress gene transmission in breeding herds of dairy industry, etc. Cryopreservation often involves freezing–thawing process. Due to cold shock, however, freezing–thawing may impair sperm motility and reduce survival and fertilizing ability of frozen sperm samples. An extender may be a media that may be used to better protect the sperm during the cryopreservation process. Egg yolk is a known extender for sperm cryopreservation. However, because of some known problems associated with using egg yolk as an extender, e.g., egg yolk may contain harmful substances for sperm and may transfer pathogens, it may be advantageous to use other sources, e.g., non-animal origin and pathogen-free sources to produce extenders to improve cryopreservation of bovine sperm.

Akhter et al. (Reprod Domest Anim. 2012 Oct;47(5):815-9) describes an extender comprising soya-lecithin as a cryoprotectant of buffalo semen. Akhter et al. aims to show effects of using soya-lecithin on in vitro quality and in vivo fertility of buffalo semen. It is said that semen from three buffalo bulls was frozen in tris-citric extender containing 5.0%, 10% or 15% soya-lecithin or 20% egg yolk. Sperm motility, plasma membrane integrity and viability were assessed post-dilution, pre-freezing and post-thaw. In post-dilution and pre-freezing, the values for motility, plasma membrane integrity and viability remained higher in extenders containing 10% soya-lecithin and control compared with extender containing 5% and 15% soya-lecithin. However, motility, plasma membrane integrity and viability were higher in extender containing 10% soya-lecithin compared with control and extenders containing 5% and 15% soya-lecithin. Semen from two buffalo bulls was frozen in tris-citric extender containing either 10% soya-lecithin or 20% egg yolk. Higher (p < 0.05) fertility rate was recorded in buffaloes inseminated with semen containing 10% soya-lecithin (56%) compared with 20% egg yolk (41.5%). It is said that the results suggest that 10% soya-lecithin in extender improves the freezability and fertility of buffalo bull spermatozoa and can be used as an alternate to egg yolk in cryopreservation of buffalo semen.

It would be advantageous to produce an extender for improving efficiency of cryopreservation of bovine sperm.

To better address this concern, a first aspect of the invention provides an extender for cryopreservation of bovine sperm, the extender comprising soy lecithin nanoparticles, wherein an average diameter of the soy lecithin nanoparticles is less than 100 nm.

The invention is based on an insight that reducing size of lecithin particles in an extender for cryopreservation of bovine sperm such that an average diameter of the soy lecithin nanoparticles is less than 100 nm may advantageously improve quality of bovine sperm after freezing-thawing process in a cryopreservation of bovine sperm. When the extender as claimed is used in a cryopreservation process, characteristics such as mobility, vital characteristics, in vitro fertility, etc, of the sperm may be advantageously improved after freezing-thawing. As such, the extender may better protect the sperm during a cryopreservation process compared to known extenders for cryopreservation of bovine sperm. Moreover, the extender as claimed may be more uniform and more transparency compared to know extenders for cryopreservation of bovine sperm which may advantageously facilitate more accurate evaluation of sperm after freezing-thawing process.

In a further aspect of the invention provides a method of producing an extender for cryopreservation of bovine sperm wherein the method comprises using soy lecithin nanoparticles in the extender, and wherein an average diameter of the soy lecithin nanoparticles is less than 100 nm.

It will be appreciated by those skilled in the art that two or more of the above-mentioned embodiments, implementations, methods, and/or aspects of the invention may be combined in any way deemed useful.

These and other aspects of the invention are apparent from and will be elucidated with reference to the embodiments described hereinafter. In the drawings,

Fig.1

shows an electron microscopy image of an example of a structure of soy lecithin nanoparticles in the extender for cryopreservation of bovine sperm.

Fig.2

shows a diagram of an example of size distribution of soy lecithin nanoparticle in the extender of Fig. 1.

Fig.3

shows a method of producing an extender for cryopreservation of bovine sperm, wherein the method comprises using soy lecithin nanoparticles in the extender.

Fig. 1 shows an electron microscopy image 10 of a non-limiting example of a structure of soy lecithin nanoparticles 15 in an extender for cryopreservation of bovine sperm wherein an average diameter of the soy lecithin nanoparticles 15 is less than 100 nm. An average diameter of the soy lecithin nanoparticles 15 may be, for example, between 50 nm and 60 nm, between 60 nm and 70 nm, between 70 nm and 80 nm, between 80 nm and 90 nm, between 90 nm and 95 nm, between 95 nm and 100 nm, etc.

Fig. 2 shows a diagram 20 of a non-limiting example of size distribution of soy lecithin nanoparticle in the extender of Fig. 1. Dynamic light scattering (DLS) method was used to determine the particle size. In this non-limiting example, lecithin contained in the extender was relented to nano-particles by using a sonication to an average diameter of 94.6 nm. In the diagram 20, size distribution by volume is shown. The first peak in the diagram 20 indicated that, in the provided non-limiting example, the size of %96.3 of nanoparticle were 76.7 nm. The second peak in the diagram 20 indicated that %4.7 of nanoparticle had size 4540 nm.

Optionally, an amount of the lecithin nanoparticles may be 1 %, 2%, 3%, 4%, etc, percentage of lecithin in the claimed extender.

In a non-limiting example, the herein claimed extender was generated and in a factorial design 4 × 2, the effect of lecithin restructuring to lecithin nanoparticles was evaluated (control and sonicated) as the first factor and the various levels of lecithin (1, 2, 3 and 4%) as the second factor on quality and in vitro fertility of sperm after freezing-thawing process. Bioxcell® commercial and egg yolk–based extender were used as control groups.

Semen samples were collected from six Holstein bovine, 2-4 years of age, by artificial vagina twice a week for 10 weeks, in total 112 ejaculates. Then semen samples were immediately transferred into a water bath (35 °C). Semen volume (by graduated tubes) and concentration (by calibrated photometer) (IMV, L’Aigle, France) were measured, respectively.

After primary evaluation, semen samples were pooled together. Then, pooled semen was divided on the number of treatments. Each part was diluted by one of extenders to a final concentration of 40 × 10⁶ spermatozoa/mL. Then, extended semen samples with different extender were loaded into 0.5 mL straws (IMV® Technologies, L’Aigle Cedex, France) and maintained for 4 hours in 4°C and then frozen by a computerized freezing machine (Digit Cool®, IMV® Technologies, L’Aigle Cedex, France) at a programmed rate (from 4°C to -10°C at 5°C/min; from -10°C to -100°C at 40°C/min; and from -100°C to -140°C at 20°C/min). Then, straws were plunged into the liquid nitrogen and stored until thawing. After freezing-thawing process, some parameters were evaluated including sperm motility (CASA), viability (Eosine- nigrosin), plasma membrane functionality (HOS) and apoptosis status (annexin staining). In an additional evaluation, in vitro fertility of sperm in 3% nanolecithin-based, Bioxcell and egg yolk extender was assessed by 6 replicates. Data were analyzed by GLM procedure using SAS 9.1 (SAS Institute, version 9.1, 2002, Cary, NC, USA).

The results of sperm motion evaluation showed that the herein claimed extender containing lecithin nanoparticles had higher level of total and progressive motility and the most sperm velocity characteristics compared to Bioxcell® commercial and egg yolk–based extender. Among optional 1 %, 2%, 3% and 4% percentage of lecithin in the claimed extender, the extender containing 3% lecithin nanoparticles, had the highest values in terms of sperm motility, VSL, VCL, VAP, ALH and BCF. The extender containing 3% lecithin nanoparticles had 68.36% total motility and 41.75% progressive motility.

Bovine sperm morphology test showed that extender containing 3 %lecithin nanoparticles had the highest percentage of normal sperm compared with the other groups, although no significant difference was observed.

The plasma membrane functionality in post-thaw spermatozoa did not differ between egg yolk-based extender and extender containing 3% lecithin nanoparticles.

In addition, the apoptotic test showed that the percentage of live spermatozoa was significantly higher in egg yolk-based extender and extenders containing 3 and 4 % soy lecithin nanoparticles. Although no significant difference was observed among them.

The results of in vitro fertility assessment indicated a rate of blastocyst production of 3% lecithin-based extender may be substantially higher than those in Bioxcell, but may not differ with egg yolk extender.

It is noted that reduction of lecithin size and providing phospholipid vesicles or liposomes, may be a cause of more dissolved lecithin in the extender and may have improved interaction of lecithin with sperm membranes.

It is noted that optionally, the extender further comprises: Tris, Citric acid, Fructose and distilled water. In an example, the extender may comprise: 3.025% (w/v) Tris, 1.7% (w/v) Citric acid, 1.25% (w/v) Fructose, and Glycerol 7% (v/v) in 100 cc distilled water.

It is noted that optionally, the extender may have has a pH of more than 6.5 and less than 7.0.In an example, the extender has a pH of 6.8. It is further noted that in an example, the extender may have an osmolarity of 300 mOsm/Kg.

Fig. 3 shows a method 30 of producing an extender for cryopreservation of bovine sperm, wherein the method comprises using 32 soy lecithin nanoparticles in the extender, and wherein an average diameter of the soy lecithin nanoparticles is less than 100 nm.

Optionally, the method 30 may comprise generating the soy lecithin nanoparticles using a sonication device. Generating the soy lecithin nanoparticles may optionally comprise 5 seconds work and 10 seconds rest of the sonication device. Sonication, which is know in the art per se, may be the act of applying sound energy to agitate particles in a sample, for various purposes. Ultrasonic frequencies (>20 kHz) may be used, leading to the process also being known as ultrasonication or ultra-sonication. Sonication may be applied using an ultrasonic bath or an ultrasonic probe, colloquially known as a sonicator.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb "comprise" and its conjugations does not exclude the presence of elements or stages other than those stated in a claim. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. In the device claim enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

Claims

An extender for cryopreservation of bovine sperm, the extender comprising soy lecithin nanoparticles (15), wherein an average diameter of the soy lecithin nanoparticles (15) is less than 100 nm.
The extender according to claim 1, wherein the average diameter of the soy lecithin nanoparticles is in a range of 70-100 nm.
The extender according to claim 1, wherein the average diameter of the soy lecithin nanoparticles is between 90-95 nm.
The extender according to any of preceding claims, wherein the extender comprises 3% (w/v) soy lecithin nanoparticles.
The extender according to any of preceding claims, wherein the soy lecithin nanoparticles have negative electric charges.
The extender according to any of preceding claims, wherein the extender further comprises: Tris, Citric acid, Fructose and distilled water.
The extender according to claim 6, wherein the extender comprises: 3.025% (w/v) Tris, 1.7% (w/v) Citric acid, 1.25% (w/v) Fructose, and Glycerol 7% (v/v) in 100 cc distilled water.
The extender according to any of preceding claims, wherein the extender has a pH of more than 6.5 and less than 7.0.
The extender according to any of preceding claims, wherein the extender has a pH of 6.8.
The extender according to any of preceding claims, wherein the extender has an osmolarity of 300 mOsm/Kg.
A method (30) of producing an extender for cryopreservation of bovine sperm, wherein the method (30) comprises using (32) soy lecithin nanoparticles in the extender, an average diameter of the soy lecithin nanoparticles being less than 100 nm.
The method (30) according to claim 11, wherein the method (30) comprises generating the soy lecithin nanoparticles using a sonication device.
The method (30) according to claim 12, wherein generating the soy lecithin nanoparticles comprises 5 seconds work and 10 seconds rest of the sonication device.