Description:FIELD OF THE INVENTION
[001] The present invention relates to a sperm extender for maintaining the viability and sperm character that determines the fertility of spermatozoa (sperm) during storage of spermatozoa in liquid nitrogen (-196°C). Particularly, the sperm extender preserves the sperm and/or maintains the fertility potential in liquid nitrogen (-196°C) for artificial insemination. Further, the present invention also relates to goat semen extender for cryopreservation. The said semen extender provides better rates of fertility and applicability in an industry that focuses on goat breeding.

BACKGROUND OF THE INVENTION
[002] Goat husbandry is an important component of animal husbandry system not only in India but throughout the world especially in developing countries. Majority of goats are being reared by poor landless, marginal and small farmers. With limited access to scientific goat rearing techniques and limited technical inputs, majority of goats have been reared in traditional manner without any focus on breeding strategies. This practice is being followed since decades. Selling out the superior male for slaughter and breeding goat with inferior quality male has become a constant feature of this sector. Consequently, Indian goat sector is now featured with low producing animal with reduced growth rate, fecundity and milk yield. According to 2019 census (Department of Animal Husbandry, Dairying and Fisheries, GOI) the male population of goat has recorded a negative growth of 14.15% compared to previous census. In addition to it the population of pedigreed animal has reduced to less than 10% together with non-descript animal close to 70%. The average milk production of goats in India is less than 100 ml. So, there was an urgent need to preserve and propagate the true breed elite indigenous superior goat germplasm for better health and productivity.
[003] Artificial insemination is a widely accepted assisted reproductive technique that focus on wider dissemination of male germplasm (semen) with use of cryopreserved semen through production of multiple insemination doses from single ejaculate. Semen for its use as insemination dose is cryopreserved semen stored in liquid nitrogen at -196° C through processing. The exposure of sperm to variable range of temperature disturbs the structural and functional integrity of sperm and kill them during processing and hence semen requires ingredients that protect and support sperm survival during semen processing during cryopreservation. Since the sperm cell are very sensitive to their microenvironment, the constituent/ ingredients that support sperm survival should be in appropriate proportion that are in accordance with sperm requirements otherwise will lead to sperm death. Semen extender is a fluid medium artificially prepared with a formulation/composition that has balance proportion of different ingredient to support sperm survival. Naturally the composition of seminal plasma, sperm and also sperm behaviour vary from species to species, hence required as different proportionate combination of ingredient that supports sperm survival with respect to particular species. Formulating a goat semen extender has challenge associated that make the process different from other animal species and requires an alternate approach to address the problem. Goat semen has Phospholipase A2, BUS III and BUSgp 60 (enzyme of bulbourethral origin that is specific to goat in its activity) and other similar proteins in the seminal fluid that reacts with egg yolk or skimmed milk to form lethal compound that cause sperm death making the process of semen dilution and cryopreservation different and difficult in goats. Egg yolk or skimmed milk powder act as cryoprotectant in semen extender and their elimination from extender composition make the sperm prone to cryoinjuries during cryopreservation leading to sperm death. To overcome this problem as an general practice for goat semen cryopreservation, prior to processing, the goat semen was centrifuged to remove the seminal plasma and then diluted with semen extender for cryopreservation. But the process causes stress on sperm and removes the naturally present protective factors in seminal plasma leading to damages and capacitate changes that alters the fertilizing capacity causing sperm death, disturb motility pattern and cause capacitate changes affecting the fertilizing capacity of sperm leading to poor conception. Also, few of the research have been conducted to develop the goat semen extender with reduced egg yolk concentration, but have achieved limited success in improving post thaw semen quality but only at lower egg yolk concentration (100-200 million sperm per ml of diluted semen) compared to the ideal recommended concentration (400 million sperm per ml of diluted semen) and also accordingly other constituent in semen extender to successfully cryopreserve the goat semen, that has limit the use for this technology for its commercial wide scale use.

[004] Thus, there is a long felt need for a goat semen extender that contain all the ingredient in a very précised proportion/ concentration required for maintaining sperm character and fertilizing ability with successful sperm survival and minimum sperm loss during cryopreservation and do not require centrifugation prior to semen dilution with extender. Further, there is urgent need for a semen extender that is useful for cryopreservation and insemination of goat semen for breed conservation and improvement in country and worldwide.

OBJECT OF THE INVENTION
[005] One of the objects of the present invention is to provide goat semen extender.

[006] Another object of the present invention is to provide goat semen extender capable of prolonging the fertility of spermatozoa for cryopreservation or artificial insemination.

[007] A further object of the present invention is to provide a goat semen extender comprising buffering agent; energy source; cryoprotective agents; antibiotic; and low density lipoprotein/phospholipid source.

[008] Still another object of the present invention provides a goat semen extender that maintains the sperm character after freeze thawing and provides better fertility rates.

[009] Yet another object of the present invention is to provide a goat semen extender that is efficient with better fertility rates and is easy to prepare.

[010] Yet further object of the invention is to provide a semen extender to improve the post thaw motility and viability of cryopreserved goat sperm.

SUMMARY OF THE INVENTION
[011] In view of the foregoing disadvantages inherent in the known types of semen extenders now present in the prior art, the present invention provides a highly efficient goat semen extender capable of prolonging the fertility of spermatozoa for cryopreservation or artificial insemination. As such, the general purpose of the present invention, which will be described subsequently in greater detail, is to provide a goat semen extender to improve the post thaw motility and viability of cryopreserved goat sperm and which has all the advantages over the prior art and none of its disadvantages.

[012] The basic aspect of the present invention is to provide a goat semen extender comprising buffering agent; energy source; cryoprotective agents; antibiotic; and low density lipoprotein/phospholipid source in a balance proportion as need for goat sperm to conserve the characters of frozen semen to an extent comparable to fresh semen.

[013] In another aspect, the present invention provides the goat semen extender, wherein the buffering agent is 309-310 mM tirs base [tris (hydroxymethyl) aminomethane] and 176-177 mM citric acid.

[014] A further aspect of the present invention is to provide the goat semen extender, wherein the energy source is 34-35 mM glucose.

[015] In a still further aspect, the present invention provides the goat semen extender, wherein the cryoprotective agent is 4 – 6% (v/v) glycerol

[016] In a further aspect, the present invention provides the goat semen extender, wherein the antibiotic is 800 - 900 G/L streptomycin and 8,00,000 – 9,00,000 IU/L penicillin G sodium salt.

[017] In an another aspect, the present invention provides the goat semen extender, wherein the low density lipoprotein/phospholipid source is 13-14% (v/v) egg yolk.

[018] In yet another aspect, the present invention provides the goat semen extender, wherein the pH of the semen extender is 6.48 to 6.58.

[019] A further aspect of the present invention provides the goat semen extender, wherein the osmolarity of the semen extender is 1410 – 1420 mosm.

[020] A yet further aspect of the present invention provides the goat semen extender, wherein the egg yolk is hen egg yolk

[021] In this respect, before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. These together with other objects of the invention, along with the various features of novelty which characterize the invention, are pointed out with particularity in the disclosure. For a better understanding of the invention, its operating advantages and the specific objects attained by its uses, reference should be had to the accompanying drawings and descriptive matter in which there are illustrated preferred embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION
[022] In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described herein below, with reference to the accompanying examples and drawings. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially. It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof. The scope of the present invention is defined by the appended claims and their equivalents.

[023] In the following description, for the purpose of explanation, numerous specific details are set forth in order to provide a thorough understanding of the arrangement of the system according to an embodiment herein. It will be apparent, however, to one skilled in the art, that the present embodiment can be practiced without these specific details.

[024] The goat semen extender according to the embodiment of the present invention is specifically described below.

[025] The present invention provides a goat semen extender for diluting freshly collected goat semen. The said goat semen extender is utilized to prepare multiple doses from single ejaculate to inseminate large population of female goats either as fresh diluted (for instant use in synchronized herd) or to cryopreserve diluted semen for long term use as insemination dose.

[026] According to an embodiment of the present invention, the goat semen extender comprises 309-310 mM tris base [tris (hydroxymethyl) aminomethane), 176-177 mM citric acid, 4 – 6% (v/v) glycerol, 34-35 mM glucose, 13-14% (v/v) egg yolk, 87,000 IU penicillin and 88 mg streptomycin.

[027] The pH of the said goat semen extender is 6.48 to 6.58 and osmolarity is 1410 to 1420 mosm.

[028] According to an embodiment of the present invention, the egg yolk is hen egg yolk.

[029] According to another embodiment of the present invention, the said semen extender maintains sperm characters during freeze-thawing process, maintains the fertilizing capacity, exhibits higher % live sperm in frozen-thaw semen that result in better conception.

[030] According to yet another embodiment of the present invention, the said goat semen extender does not require centrifugation and seminal plasma removal prior to dilution. It has lower hen egg yolk level in goat semen diluter that with minimum lethal interactive losses provide sufficient cryoprotection for sperm and maintains higher number of viable sperm in post thaw cryopreserved goat semen.

EXAMPLES:
[031] Embodiments of the present invention will now be discussed in more detail with reference to following examples which are provided for exemplification only and which should not be considered limiting on the scope of the invention in any way.

Example 1

[032] According to an embodiment of the present invention, the goat semen extender was prepared comprising:

S No. Ingredient Volume/Weight
1. Water 82.30 ml
2. Tris 3.78 gm
3. Citric acid 2.144 gm
4. Glycerol 4.526 ml
5. Glucose 0.619 gm
6. Yolk 13.17 ml
7. Pencilling 87,000 IU
8. Streptomycin 88 mg
9. pH 6.5±0.06
10. Osmolarity 1418 mosm

The above prepared semen extender was used to dilute the goat semen and with the diluted semen artificial insemination was done in atleast 1,000 female goats. The results indicated that the diluted semen showed that 50-60% goats were successfully inseminated.

Example 2
[033] According to another embodiment of the present invention, the goat semen was diluted using the goat semen extender as above, froze at -196°C and maintained in LN2 for 7 to 150 days (time). It was transported to the place where the insemination was to be done. The frozen diluted semen was thawed and artificial insemination was done for at least 900 goats. The results showed that the 50-60% goats were successfully inseminated indicating towards the efficacy of goat semen extender to maintain the properties of goat semen.

Example 3

[034] According to an embodiment of the present invention, the goat semen was diluted with the goat semen extender as prepared by the method described above and the said diluted goat semen was tested in the laboratory for motility etc. (Table 1-5). The results indicated that the motility of the sperms was maintained upto 70-80%.
Table 1

Table 2

Table 3

Table 4

Table 5

[035] According to another embodiment of the present invention, the goat semen diluted and cryopreserved using the semen extender of the present invention was compared with the goat semen cryopreserved and diluted using goat semen extender containing 20% egg yolk and other constituents. The results obtained clearly showed that the semen extender is highly efficient to maintain the viability and goat sperm character. (Table 6 and 7)

Table 1 - Centrifuged and diluted cryopreserved goat Semen

No. Viability Motile Progressive VAP VSL VCL STR LIN ALH BCF Wob
1 54.5 47.5 28.5 58.71 49.13 93.61 38.15 24.36 4.01 17.69 29.57
2 46.5 41.5 27.7 52.93 45.54 82.83 34.27 22.47 3.33 16.26 26.42
3 51.2 45.4 33.3 59.72 52.57 90.84 38.83 25.6 3.47 17.03 29.45
4 56.2 49.5 36 54.26 48.41 82.21 42.87 28.9 3.01 20.47 32.56
5 47.8 40.5 25.7 45.54 38.8 76.78 32.98 19.93 3.22 14.81 23.82
6 48.7 44.6 29.9 50.02 42.58 83.33 36.58 22.59 3.51 17.47 26.8
7 56.1 50.5 31.8 53.73 44.95 89.6 40.61 24.98 3.86 19.58 30.27
8 49.6 44.5 32 44.47 39.73 66.95 38.56 26.16 2.55 17.76 29.36
9 55.8 47.2 33.1 49.16 43.63 74.91 40 26.85 2.83 19.24 30.67
10 47.2 41.4 26.9 41.8 36.15 71.87 34.81 20.61 2.93 16.91 24.19

Table 2 - Semen diluted and cryopreserved using semen extender of the present invention

No. Viability Motile Progressive VAP VSL VCL STR LIN ALH BCF Wob
1 75.4 Motile Progressive 111.63 96.89 171.93 91.59 54.65 6.23 38.59 63.52
2 79.1 66.2 42.6 72.25 60.92 116.03 58.05 37.53 4.85 27.87 44.58
3 73.5 71.2 46 77.77 67.6 128.02 54.28 33.49 4.97 25 38.81
4 73.2 64.2 47.5 73.67 63.03 123.79 54.34 33.19 4.95 26.22 38.97
5 81.5 65 46.7 98.86 80.03 157.39 58.67 37.56 6.56 28.34 46.76
6 79.2 74.7 43.8 82.8 67.78 131.22 56.93 36.62 5.77 27.12 44.75
7 76.2 71 43.8 81.6 64.39 142.7 49.93 28.95 6.25 23.31 37.15
8 76.3 65.3 35.9 83.29 68.55 136.4 56.54 35.45 6.09 26.33 43.27
9 69.3 70.6 44 76.36 63.68 119.69 49.01 31.89 5.07 22.9 38.7
10 76.1 61.3 37.2 83.39 71.1 129.4 56.72 37.31 5.26 27.09 44.05

*VCL: Curvilinear velocity (µm/sec)
VSl: Straight Line Velocity (µm/sec)
VAP: Average Path Velocity (µm/sec)
LIN: Linearity (%)
STR: Straightness (%)
WOB: Wobble (%)
BCF: No. of times the curvilinear trajectory crosses the average path trajectory/sec. (hz)
ALH: Amplitude of Lateral Head Displacement (µm)

[036] It is to be understood that the above description is intended to be illustrative, and not restrictive. For example, the above-discussed embodiments may be used in combination with each other. Many other embodiments will be apparent to those of skill in the art upon reviewing the above description. The benefits and advantages which may be provided by the present invention have been described above with regard to specific embodiments. These benefits and advantages, and any elements or limitations that may cause them to occur or to become more pronounced are not to be construed as critical, required, or essential features of any or all of the embodiments. While the present invention has been described with reference to particular embodiments, it should be understood that the embodiments are illustrative and that the scope of the invention is not limited to these embodiments. Many variations, modifications, equivalent replacements, additions and improvements to the embodiments described above are possible and fall within the scope of the present invention.
, Claims:We claim:

1. A goat semen extender comprising
buffering agent;
energy source;
cryoprotective agents;
antibiotic; and
low density lipoprotein/phospholipid source.

2. The goat semen extender as claimed in claim 1, wherein the buffering agent is 309-310 mM tirs base [tris (hydroxymethyl) aminomethane] and 176-177 mM citric acid.

3. The goat semen extender as claimed in claim 1, wherein the energy source is 34-35 mM glucose.

4. The goat semen extender as claimed in claim 1, wherein the cryoprotective agent is 4 – 6% (v/v) glycerol.

5. The goat semen extender as claimed in claim 1, wherein the antibiotic is 800 - 900 G/L streptomycin and 8,00,000 – 9,00,000 IU/L penicillin G sodium salt.

6. The goat semen extender as claimed in claim 1, wherein the low density lipoprotein/phospholipid source is 13-14% (v/v) egg yolk.

7. The goat semen extender as claimed in claim 1, wherein the pH of the semen extender is 6.48 to 6.58.

8. The goat semen extender as claimed in claim 1, wherein the osmolarity of the semen extender is 1410 – 1420 mosm.

9. The goat semen extender as claimed in claim 1, wherein the egg yolk is hen egg yolk.