|Title of Invention||
A SPERM PROCESSING KIT
|Abstract||The present invention provides a pesticidal composition for controlling insects or representatives of the order Acarina and microorganisms, which composition comprises: (A) an insecticidally effective amount of at least one neonicotinoid or phenylpyrazole insecticide, and (B) a fungicidally effective amount of at least three fungicides including: (B 1) at least one phenylamide (acylalanine type), (B2) at least one phenylpyrrole and (B3) at least one triazole.|
|Full Text||DESCRIPTION OF THE INVENTION:
This invention relates to sperm processing kit for assisted reproductive procedures.
In normal, coitus semen is deposited in vagina. As the sperm progresses through the cervical mucus, it is separated from seminal plasma, which is known to have an adverse effect on fertilizing potential of sperm. In addition, the passage of the sperm through the female reproductive tract results in ce^acitation and acrosome reaction, which are pre¬requisites for fratilization.
In assisted reproductive techniques sperm is either directly introduced in the uterus (lUI) or injected directiy into the egg (ICSI) or in the fallopian tubes (GIFT) or brought into direct contact with eggs in vitro (IVP). Therefore, it is essential to process the semen before it can be used in assisted reproductive procedures.
The success of any assisted reproductive procedure is to a large extent dependent on the quality of the processed semen, thus it is important to use those techniques of semen preparation, which yield an enriched population of progressively motile, and morphologically normal sperm with minimum damage to the sperm membranes.
A variety of semen processing technique have evolved. These include:
(i) Simple configuration;
(iii) Percoll density gradient centrifugation;
(iv) FicoU density gradient centrifugation;
(v) Glasswool column filtration; and
(vi) Sephadex column filtration
Each of these methods has their advantages and disadvantages. At present the most commonly used methods of semen preparations are Percoll density gradient or swim-up.
Swim-up techniques works very well with normal semen samples but are not very effcient in processing substandard semen samples-the yield of motile sperms even after long periods of incubation is very low.
For the past decade percoll density gradient centrifugation was accepted as the most effective method of semen preparation. However, recently there has been some concern that silica content of residual percoll in the sranen sample may induce inflammatory reaction in the body.
Although percoll centrifiigation technique has been used for many years in assisted reproductive medicine without any adverse effects, pharmacia biotech, the manufacturers of percoll have now taken active steps to prevent the usage of Percoll for the isolation of cells to be used for clinical or medical treatment. Research efforts are in progress to develop media, which can replace percoll media. A few new media, which can replace percoll media, have been reported (Ref).
Accordingly, it is an object of the present invention to provide sperm processing kit consisting of a sperm-separation media, a sperm wash solution, sterilized plasticware consisting of two pipette, two centrifuge tubes and a semen collection container.
The kit of the present invention has several advant^es, as given below:
1. It is easy to use, as it does not require an elaborate laboratory set-up to process the sample.
2. As each kit is only meant for a one-time use, there is no necessity for storage of opened media; any storage of opened media invariably results in contamination because of lack of training of laboratory personnel in aseptic methods and the unavailability of a sterile environment, especially in clinical set-ups of small-town physicians;
3. Sealed ampoules can be stored safely in the refrigerator for at least a year without any change in pH.
The following wUl illustrate the manufacture of the q>erm processing kit of the present invention.
1. Preparation of Sperm Separation Media:
The media is based on Arabinogalactam, a water-soluble polysaccharide polymer extracted from Larix-Accidentabis (i.e.) larch trees. (Reference No 1) because of its non-toxicity, low viscosity and water-solubility, it is used for separation and purification of a variety of cell types (Reference No. 2,3,4).
A stock solution of 30% w/w cellsep (a highly purified form of Arabinogalactan) is first prqiared by using Milli Q water. Ninety parts of this stock solution is mixed with 8 parts of lOX phosphate buffered saline and 2 parts of water to form an isotonic cellsep solution. The pH of the solution is adjusted between 7 and 8, with the addition of IN HCL or IN NaoH. The osmolality of the solution is adjusted between 250 and 400m Osm/L and the density between 1.08 and 1.12 g/ml.
2. Preparation of Sperm wasliing Media:
HEPES buffered HTF media, purchased from Irwine Sciraitific (USA) is fortified
with the following:
Human Serum Albumin 1%
Streptomycin 0.1 mg/ml
Penicillin 1001 jx/ml
pH is adjusted between 7 and 8
3. Stock isotonic sperm-separation solution is diluted with the required volumes of sperm wash solution to obtain 50 to 80% of isotonic sperm sq>aration solution;
4. All solutions are sterile filtered using 0.2pm filter under a laminar flow hood;
5. Glass ampoules are washed and sterilized in the oven;
6. Ampoules are filtraed with the required volumes of the two media and sealed;
7. All ampoules are labeledandpacked in the cardboard box.
Extensive expoiments in our laboratory have shown that separation media made with cellsep powder, which is purified Arabinogalactam, a water-soluble polymer extracted from the wood of larch tress (Ref No. 1) is highly useful in preparing ^erm for assisted reproductive technology. So far to our knowledge, there had been one more report on the success of this media, in sperm preparation for IVF and ICSI (Ref.). We have compared the use of this media vis a vis percoU media and find that it is as good as the traditionally used percoU media (ref).
Cellsq) is a natural polysaccharide, soluble in water and totally non-toxic, therefore liighly suitable for preparation of variety of cells for clinical use.
1. Purchased from Larex Inc. 2852 Palton Road, St. Paul, Minnesota 55113, U.S.A
2. Corash,LM.etal. Blood 49:71-87
3. Ostasiewicz et al. Anal of Biochem, 213:395,1993
4. Turner J. M. et al, presented at ASH meeting 1996.
1. A sperm processing kit consisting of:
(a) a sperm separation media based on Arabinogalactam having a pH between
, ,,e7 and 8 an osmolality between 250 to 400 Osm/L, and a^density between
1.08 and 1.12g/ml;
(b) a sperm wash solution consisting of HEPES buffered HTF media fortified with human serum albumin, penloxyfyline, streptomycin and penicillin with a pH between 7 and 8;
(c) a pair of sterilized pipettes and centrifuge tubes; and
(d) a semen collection container.
2. A sperm processing kit as claimed in claim 1, wherein the sperm separation media is obtained by preparing a stock solution of 30% w/w Cellsep using MiUi -Q water, mixing ninety parts of this stock solution, with 8 parts of lOX phosphate buffered saline and 2 parts of water to form an isotonic Cellsep solution, adjusting the pH of the solution between 7 and 8 by the addition of IN HCL or IN NaoH, the osmolality of the solution is adjusted between 250 and 400 mosm/L and the density between 1.08 and 1.12 g/ml.
3. A sperm processing kit as claimed in claim 1 or 2, wherein the sperm wash solution is prepared by fortifying HEPES buffered HTF media with the following:
Human Serum Albumin 1%
Streptomycin 0.1 mg/ml
Penicillin 100 l/ml
4. A sperm processing kit as claimed in claim 3, wherein the pH of the sperm
wash solution is adjusted between 7 and 8.
5. A sperm processing kit as claimed in any one of claims 1 to 4, wherein the
Arabinogalactan is "Cellsep" powder.
6. A sperm processing composition consisting of 50 to 80% of isotonic sperm
separation solution based on Arabinogalactan, the rest being sperm wash
solution consisting of HEPES buffered HTF media fortified with the
Human Serum Albumin 1%
Streptomycin 0.1 mg/ml
Penicillin 100 In/ml
7. A sperm processing composition as claimed in claim 6, wherein the
Arabinogalactam is "Cellsep" powder.
8. A process for preparing sperm processing composition as claimed in claim 6
or 7, which comprises the step of forming a stock isotonic sperm-separation
solution by diluting with the required volumes of sperm wash solution to
obtain a 50 to 80% of isotonic sperm-separation solution, sterilizing the said
solution followed by filtration under a laminar flow hood.
|Indian Patent Application Number||84/MAS/1999|
|PG Journal Number||8/2007|
|Date of Filing||22-Jan-1999|
|Name of Patentee||M/S. CRYO-GENIE INDIA PVT. LTD|
|Applicant Address||'SHREYAS', NO. 1872, 38TH 'A' CROSS, 11TH MAIN 4TH 'T'BLOCK, JAYANAGAR, BANGOLORE 560 041|
|PCT International Classification Number||A01N 001/02|
|PCT International Application Number||N/A|
|PCT International Filing date|