The BioTranZ® Express by ZDL

Sperm viability in human semen specimens cryostored
at 5°C using the Bio-Tranz container system for
semen transport

(From Middle East Fertility Society Journal, Vol. 3, No. 3, 1998)

Panayiotis M. Zavos
Juan R. Correa
William Clark
Panayota N. Zarmakoupis-Zavos

ABSTRACT

  • Objective: To asses a protocol designed for transport of unprocessed human semen specimens from the production site to distant laboratories.
  • Design: The viability of semen specimens stored from the time of collection to the time at which the specimens were to be processed and used (24 hr after collection) was evaluated using the Bio-Tranz™ technology. Specimens were assessed for percentage and grade of motility, and for the sperm membrane functional integrity as measured by the hypoosmotic swelling (HOS) test. The semen specimen was split into two aliquots (Aliquot 1 and 2) and transferred to 15.0 mL conical centrifuge tubes. Aliquot-1 was
    used without further processing and Aliquot 2 was mixed 1:1 (v/v) with TYB media. Aliquot 1 was maintained at 21°C. Aliquot 2 slowly cooled to 5°C by placing the tube into the middle compartment of the Bio-Tranz™ container.
  • Setting: Andrology Institute of Lexington, Lexington, Kentucky.
  • Patients: Semen specimens (n=30) were collected by each participant at intercourse via the use of the MFP TM and delivered to the Andrology Institute of Lexington for processing.
  • Main Outcome Measure(s): Viable cryostorage of semen specimens during transport at 5°C for 24 hr for andrological evaluation or use in assisted reproductive technologies.
  • Result(s): Significant differences (P<0.05) in all sperm parameters assessed were noticed between the unprocessed and TYB-prepared specimens after storage for 24 hr. Sperm characteristics were improved when preparing the specimens using TYB (Time 0; P<0.05). Sperm characteristics between the unprocessed (Time 0) and TYB-prepared specimens (24 hr) were not different (P>0.05).
  • Conclusion(s): Collection and preparation of human semen for transport at SoC is possible and that the Bio-Tranz ™ container maintains adequate sperm viability after 24 hours of cryostorage. The use of the Bio-Tranz™ container is convenient for patients that request semen processing services or for other clinical purposes at distant locations.
  • Key words: semen, spermatozoa, cryostorage, viability, transport

Transport of unprocessed human semen specimens from the production site to distant laboratories for
andrological evaluation and other clinical uses requires the development of proper protocols and devices for the maintenance of sperm viability and fertilizability during and after transport 1. Spermatozoa recovered following storage at 5°C in TEST-Yolk buffer (TYB), show enhanced survivability and fertilizing capacity than spermatozoa frozen at subzero (-196°C) temperatures following short-term incubation between 24 and 96 hr (1-7). It has also been documented that, the treatment of human spermatozoa with TYB can enhance their ability to penetrate zona-free hamster oocytes (ZFHO) and bind to the zona pellucida as measured by the sperm penetration assay (SPA) or hemizona (HZA) assays (1, 5, 8-11). Preincubation of spermatozoa in TYB have been shown to increase the percentage of human oocytes fertilized via in vitro fertilization via in vitro fertilization (IVF) procedures (1, 5, 11, 12). It seems that a higher proportion of the sperm population undergo capacitation during TYB incubation, which results in synchronization of the acrosome reaction following sperm washing and preparation for use in the various assisted reproductive technologies (5, 6, 11). Subsequently, the increased percentage of acrosome-reacted spermatozoa that is seen after incubation in TYB may indicate that a larger percentage of the sperm population acquired the ability to penetrate the ovum and account for the higher rates of fertilization observed (1, 5, 6, 11).

The Bio-Tranz™ container, which was designed to cool and maintain semen specimens at 5°C during transport, consists of a properly insulated Polystyrene container, TYB, a nonspermicidal condom-shaped semen collection device and test tubes. The viability of semen specimens stored from the time of collection to the time at which the specimens were to be processed and used (24 hr after collection), was assessed using the Bio-Tranz" technology. 

MATERIALS AND METHODS

Semen Collection and Assessment

Thirty normozoospermic (World Health Organization standards) (13); donors participated in this study. Each donor produced a semen specimen after 3 to 4 days of abstinence via intercourse using a semen collection device provided with the Bio-Tranz™ (14, 15). The Bio-Tranz™ technology (ZDL, Inc., Lexington, KY, USA) consists of a Polystyrene (Styrofoam) container which contains a cold pak, TYB, the Hygiene™ seminal collection kit, and a Styrofoam separator that divides the Bio-Tranz™ container into two compartments (Fig. 1). The Hygiene™ semen collection kit consists of a nonspermicidal condom-shaped collection device (Male Factor Pak™), funnel and two 15.0 mL conical centrifuge tubes. Semen specimens were assessed for percentage and grade of motility, and for the sperm membrane functional status as measured by the hypoosmotic swelling (HOS) test (16). Grade of motility was measured using a scale of 0 to 4 to describe the progressive motility as described by Zavos et a1. (17).

Experimental Design

Semen specimens were split into two aliquots (Aliquot 1 and 2) and transferred to 15.0 mL conical centrifuge tubes following initial assessment. Aliquot 1 was used as control without further processing and Aliquot 2 was mixed 1:1 (v/v) with TYB media. Aliquot 1 was maintained at 21°C (ambient room temperature). Aliquot 2 was slowly cooled to 5°C (approximately 0.1°C/min) by placing the tube containing the diluted seminal specimen into the middle compartment of the Bio-Tranz™ container. A separator cover (thin piece of Styrofoam with two holes) was placed over the test tube. The specimen preparation procedure was completed by placing a frozen cold pak on top of the separator and then closing the Bio-Tranz™ container prior to transport. The semen-TYB mixture was cooled via conduction as it was placed closed to the frozen cold pak (-20°c). The low temperature of the frozen cold pak coupled with distance of the semen-TYB mixture in reference to frozen cold pak allowed the rate of cooling of the semen-TYB mixture (approximately 0.1°C/min). Semen specimens (Aliquot 1 and 2) were assessed after 24 hr of storage as previously described.

Semen Re-Warming

The semen-TYB mixture was removed from the Bio-Tranz™ container and placed in water bath (37°C) for 15-20 minutes as previously described (2, 3). The semen-TYB mixture was gently agitated periodically during the incubation period in order to keep the re-warmed spermatozoa in suspension.

Table 1: Sperm viability of Semen specimens diluted and cyrostored in TEST-yolk buffer (TYB) at 5C during 24 hours of cyrostorage
  Sperm characteristics assessed
Semen Treatments Motility (%) Grade (0 to 4) HOS * (%)
Fresh, 0 hours 55.0 ± 7.5 3.4 ± 0.3 64.0 ± 11.0
Fresh, 24 hours 17.7 ± 6.1 1.6 ± 0.5 23.0 ± 11.6
TYB, 0 hours 61.8 ± 7.6 3.5 ± 0.2 68.7 ± 10.8
TYB, 24 hours 52.8 ± 9.0 3.3 ± 0.2 58.7 ± 11.4

Statistics

The results were reported as means±SD. The data was analyzed by ANOVA procedures using the SAS Statistical Package (18). A level of P<0.05 was considered statistically significant. The statistical model included the effects of patients, temperature and sperm characteristics assessed.

RESULTS

The results obtained are summarized in Table 1. Significant differences (P<0.05) in all sperm parameters assessed were noticed between the unprocessed and TYB-prepared specimens after storage for 24 hr. Sperm characteristics were also improved when preparing the specimens using TYB after collection (Time 0; P<0.05). Sperm characteristics between the unprocessed (Time 0) and TYB-prepared specimens (24 hr) were not significantly different (P>0.05). The percentage and grade of motility, as well as, the integrity of the sperm membrane declined by 68, 53 and 65% after 24 hr in specimens stored at 21°C, respectively. Similarly, in specimens stored at 5°C using TYB, those sperm characteristics declined by 15, 6 and 15% after 24 hr of cryostorage.

DISCUSSION

The use of cryopreserved semen for artificial insemination using donor semen (AID) is steadily increasing, although the cryopreservation (-196°C) techniques presently employed reduce significantly the fertilization potential of human spermatozoa (19). Spermatozoa cryostored at 5°C in TYB show improved survivability than spermatozoa frozen at subzero temperatures (2, 15). The use of cryopreserved spermatozoa provides the advantage of arranging patients and performance of assisted reproductive techniques (ART) such as intrauterine insemination (lUI), IVF, intra cytoplasmic sperm injection (ICSI) and others (3, 20, 21). A newly established technique has been introduced to optimize fertility rates and to decrease the difficulty in aligning patients or making semen available from patients that either travel on the day that the procedure is to be performed or they happen to be residing at another distant location from where the procedures are performed. This technique consists of collecting, diluting and cryostoring (5°C) human spermatozoa diluted with TYB for approximately 24 to 48 hr and up to 96 hr (3, 6, 7). The fertilization potential of spermatozoa incubated and cryostored in TYB can also be enhanced by selection of spermatozoa via various sperm preparation techniques employed in ART after the cryostorage period (3, 17, 22-24). It has also been documented that the treatment of human spermatozoa with TYB can enhance their ability to penetrate ZFHO (1). Preincubation of spermatozoa in TYB have been shown to increase the percentage of human oocytes fertilized via IVF procedures (12). It seems that following sperm incubation in TYB at 5°C, more spermatozoa undergo capacitation and synchronization of the acrosome reaction (5, 6). Subsequently, the increased proportion of capacitated spermatozoa that is noted after TYB incubation may indicate that a higher percentage of spermatozoa achieve the acrosome reaction stage and consequently the ability to penetrate the ovum, which could account for the higher fertilization rates observed (1,5,6,8-11). Most recently, it was also shown that incubation of spermatozoa in TYB followed by filtration using the Sperm Prep™ Sephadex filtration method can improve the recovery of antisperm antibody (ASA)-free spermatozoa by selectively masking and entrapping spermatozoa with ASA bound to its surface (25).

The development of methods for the transport of semen specimens processed and cryostored in TYB combines the advantages of maintaining sperm viability, and possibly the sperm fertilizing ability, while in transit. Following delivery to the andrological or ART laboratory, the specimens are then processed for clinical evaluation or used for various forms of ART including lUI. The employment of this technology also allows the performance of various inseminations obtained from the cryopreserved specimen, without the patient having to produce multiple specimens within a short period of time (24 to 96 hr). The results obtained in the current study show that the collection and preparation of human semen for transport at 5°C is possible and that the Bio-Tranz™ container maintains adequate sperm viability after 24 hr of cryostorage. The use of the Bio-Tranz™ container is convenient for patients from distant locations that request semen processing services or for other clinical purposes such as semen cryopreservation, evaluation and processing for use in the various forms of ART and also lUI procedures. The Bio-Tranz" is a rather unique product and will revolutionize the way liquid (non-frozen) semen is shipped and utilized for patients that require various clinical and laboratory procedures performed on their semen and they happen to be located at distant locations from the site where those procedures are performed.

REFERENCES

  1. Bolanos JR, Overstreet JW, Katz DF. Human sperm penetration of zona-free hamster eggs after storage of the semen for 48 hours at 2°C to 5°C. Fertil Steril 1983;39:536-41.
  2. Zavos PM, Goodpasture JC, Zaneveld LJD, Cohen MR. Motility and enzyme activity of human spermatozoa stored. for 24 hours at +5°C and -196°C. Fertil Steril 1980; 34:607-9.
  3. Zavos PM, Correa JR, Sofikitis N, Kofinas GD, Zarmakoupis PN. A method of short-term cryostorage and selection of viable sperm for use in the various assisted reproductive techniques.
    Tohoku J Exp Moo 1995; 176: 75-81.
  4. Jaskey DG and Cohen MR. Twenty-four to ninety-six-hour storage of human spermatozoa in test-yolk buffer. Fertil Steril 1981; 35: 205-8.
  5. Falk RM, Silverberg KM, Fetterolf PM, Kirchner FK, Rogers BJ. Establishment of TEST-yolk buffer enhanced sperm penetration assay limits for fertile males. Fertil Steril 1990; 54: 121-6.
  6. Kofinas GD, Zavos PM. Selection of viable spermatozoa via sperm filtration following 24 h cryostorage at 5 °C in test-yolk buffer. Mol AndroI 1992; 4: 113-9.
  7. Kofinas GD, Zavos PM. Short term cryostorage technique for human spermatozoa: its possible application in an artificial insemination program. Infertility 1992; 15: 44-54.
  8. Lanzendorf SE, Holmgren WJ, Jeyendran RS. The effect of egg yolk medium on human sperm binding in the hemizona assay. Fertil Steril 1992;58: 547-550.
  9. Paulson RJ, Sauer MV, Francis MM, Macaso TM, Lobo RA. A prospective controlled evaluation of TEST-yolk buffer in the preparation of sperm for human in vitro fertilization in suspected cases of male infertility. Fertil SteriI1992;58: 551-555.
  10. Soffer Y, Golan A, Herman A, Pansky M, Caspi E, Ron-El R. Prediction of in vitro fertilization outcome by sperm penetration assay with TEST-yolk buffer preincubation. Fertil Steril 1992; 58: 556-562.
  11. Garnzu R, Yavetz H, Lichtenberg D, Paz G, Homonnai ZT, Yogev L. The effect ofegg yolk on the binding capacity of human spermatozoa to zona pellucida. Fertil Steril 1994; 62: 1221-5.
  12. Katayama KP, Stehlik E, Jeyendran RS. In vitro fertilization outcome: glass wool-filtered sperm versus
    swim-up sperm. Fertil Steril 1989; 52: 670-2.
  13. World Health Organization. Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 3rd ed. Cambridge University Press, Cambridge, 1992.
  14. Zavos PM. Seminal parameters of ejaculates collected from oligospermic and normospermic patients via masturbation and at intercourse with the use of a Silastic seminal fluid collection device. Fertil Steril 1985 Oct;44(4):517-20
  15. Zavos PM, Goodpasture Je. Clinical improvements of specific seminal deficiencies via intercourse with a seminal collection device versus masturbation. Fertil Steril 1989 Jan;51 (I): 190-3
  16. Jeyendran RN, Van der Ven, H.H., Perez-Pelaez, M., Crabo, B.G. & Zaneveld, L.J.D. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Ferti 1984; 70: 219-228.
  17. Zavos PM, Correa JR, Zarmakoupis PN. Epididymal spermatozoa: recovery and subsequent improvements of mouse epididymal spermatozoa via the SperrnPrep™ filtration method. Tohoku J Exp Med 1995; 175: 101-9.
  18. SAS Institute, Inc. (1989) SAS User's Guide: Statistics, Cary, Ne.
  19. Zavos PM. Principles of cryopreservation of human spermatozoa: state-of-the-art. Infertility 1990; 13: 239-246.
  20. Zavos PM, Zarmakoupis-Zavos PN, Correa JR, Abouabdalla M, Aslanis A. Variations in pregnancy rates following intrauterine insemination among infertility centers: can the inseminators make a difference? Middle East Fertil Soc J 1997; 2: 24-29.
  21. Zavos PM, Barnes FL, Correa JR, Zarmakoupis-Zavos PN, Tesarik J. Methods for the isolation and purification of post-ejaculate human round spcrmatids for possible use in intra cytoplasmic round spermatid injection. Middle East Fertil Soc J 1997; 2:147-150.
  22. Zavos PM, Correa JR, Sofikitis N, Toda T, Zarmakoupis-Zavos PN. The usefulness of pentoxifylline for the recovery of human spermatozoa in assisted reproduction technologies. Middle East Fertil Soc J 1996; I: 128-133.
  23. Correa JR and Zavos PM. Preparation of frozen-thawed bovine spermatozoa via various sperm selection techniques employed in assisted reproductive technologies. Theriogenology 1996; 46: 413-420.
  24. Correa JR, Zarmakoupis-Zavos PN, Zavos PM. Quantitative and qualitative characteristics of frozenthawed bovine spermatozoa recovered via a conventional and a standardized swim-up technique. Tohoku J Exp Med 1997; 181: 267-274.
  25. Zavos PM, Correa JR, Zarmakoupis-Zavos PN. Antisperm antibody treatment mode: levels of antisperm antibodies with Test-yolk buffer and filtration using the SperrnPrep™ method. Fertil Steril 1998; 69(3): 517-21.

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BIOLOGICAL TRANSPORTER/SPERM SHIPPER INSTRUCTIONS FOR COLLECTION, SHIPPING, AND USE Read and understand all instructions before attempting to collect and ship your specimen. Although all materials in this kit are designed for providing an adequate environment to ship refrigerated human sperm, no guarantee of viability can be made as all specimens have different initial quality and may differ in their ability to remain viable with shipping and refrigeration. Most specimens that have initial quality of normal or above will respond favorably and retain their motility after use of this kit. Specimens of lower than normal quality or quantity may fail to provide adequate motility or viability after use of this kit. Immediately Upon Receipt of the BioTranz. Preparing for a shipment, you will need to abstain from ejaculation for 2 to 7 days prior to your specimen collection day if you wish to maximize both sperm count and motility. If you are shipping sperm for insemination use, you need to time your abstinence and the shipping of the specimen to their ovulation. Be sure to read the shipping instructions on the following page prior to arranging collection of the specimen. Getting ready to collect and ship a specimen. Remove the bottle of Sperm refrigeration media from the freezer and allow thawing by one of the following methods: 1 setting at room temperature for approximately 1 hour prior to specimen production, or 2 setting the sperm refrigeration media vial into the refrigerator on the day previous to specimen production, or 3-setting the sperm refrigeration media into a glass of warm water for fifteen minutes or until thawed, but still cool Set a glass of cold tap water (1/2 full) into the refrigerator at this time. There should be a 2-7 day period of sexual abstinence (no ejaculation) prior to semen collection. Use 1 of the 2 following methods of collection. The semen sample must be produced on the day that it is to be shipped. The closer to shipping time the better. Option-1) Intercourse with the collection condom The Male Factor Pak (MFP) condom is used in the same manner as an ordinary condom during intercourse. Have a clean pair of scissors available. Remove MFP condom from its packaging and place against top of erect penis. To eliminate trapping air, hold reservoir tip pinched closed while unrolling condom onto penis. Proceed through intercourse and obtain maximum sexual stimulation before ejaculation. After ejaculation, hold open end of condom to prevent slippage. Once erection has subsided, remove condom being careful not to spill any semen. Holding reservoir end of condom over the open specimen container, use scissors to make a cut in the tip. Carefully squeeze all possible semen from condom into the specimen container. After all semen has collected in the specimen container, discard condom. Skip to step “3” below or Option-2) Masturbation with the use of the specimen collection cup (orange or blue top) Remove the specimen collection cup from the kit. It is preferable that you not use a lubricant, but if you must, use only plain baby (mineral) oil, sperm friendly lubricant. Be careful not to contaminate the semen specimen with any lubricant or foreign material as this can effect your specimen. Proceed with masturbation until you have achieved maximum sexual stimulation and ejaculate into the collection cup, being careful to collect the entire ejaculate. Proceed to step “3” below. 3) Preparing the specimen for cooling and shipping At this point, allow the fresh semen to sit at room temperature for 15 to 30 minutes in the collection cup. This allows the semen to liquefy,(or become more watery). This step will allow the sperm to mix with the refrigeration media solution more evenly, and will allow for the best protection of the sperm. Remove the two snap top tubes from the black foam packaging tubes and label with your name, and current date. Transfer the semen mixture from the collection cup to one of the snap top tubes, by using one of the enclosed pipettes. If the volume of the semen is in excess of 5 cc, then split the semen evenly into the second snap top tube. (Remove and dry the sperm refrigeration media bottle from the glass of warm water, if used for thawing.) Open the sperm refrigeration media bottle carefully, and slowly add an amount of media equal to the volume of the semen in the snap top tube(s). If the total semen volume is 5cc or greater, add the entire volume of the refrigeration media. Mix gently 3-4 times by inverting the specimen tubes being sure that the specimen tube lid(s) are inserted fully and snapped on. Place closed tube/s with semen/media mixture into the glass of refrigerated water in the refrigerator for 15 minutes to allow for initial cooling of the mixture. Remove the specimen tube(s) from the cold water, and dry the outer sides of the tubes Double check each specimen tube lid for tightness before placing each tube into one of the black foam insulated carriers and placing a black rubber stopper into the open end as they were originally shipped. Place both black tubes with test tubes into zip lock baggy with towel absorbent and seal the zip lock. It is important to make sure and pack the specimens as specified in order to meet the “Laboratory Specimen” packaging regulations of the carriers. Specimens must be double packed (tube and baggy) and have absorbent to catch spills (paper towels). Place the frozen refrigerant packs from your refrigerator freezer into the Styrofoam cooler, and then the baggy with test tubes of semen next to them as shown on the enclosed photo (Figure 2). Place the lid on the Styrofoam container and seal it with strong shipping tape to secure the container lid to the cooler body. Make sure that the lid of the cooler fits into the lip of the cooler box. Tape the circumference of the cooler, where the lid meets the box, to prevent cool air from escaping. Place the cooler back into its corrugated shipping box and seal the box securely with cellophane or other appropriate shipping tape. Apply the “Lab Pack” sticker to the outside of the shipping box. Be sure to complete the Federal Express air bill carefully and include the phone number of the recipient. Other major carriers such as DHL and UPS offer similar services. The service you choose to use is at your discretion. We are not responsible for the failure of any third party carrier to meet delivery times or guarantees in any way. As you will be shipping perishable product, the carrier will most likely not be able to offer any type of refund upon service failure Andrology Institute of America, ZDL Inc, Fertmart.com, and The International Institute for Gender Selection will not offer any type of product replacement. Shipping the specimen---plan ahead and make sure that everything is ready to minimize transit time and mistakes The specimen must be shipped on the same day that it is produced to retain viability. The specimen must be shipped via overnight service. Specimens experiencing greater than 12-24 hour transit time will most likely show additional decreased viability and motility. Call Fed Ex, or the carrier of your choice to have the package picked up, or you may deliver it to their office. Fed Ex will pick up express shipments at no additional charge if you are not located in an extended area, and the arrangement is made with the carrier prior to 12 PM. Please check with your carrier of choice prior to actually needing service to prevent problems after your specimen has been produced. If asked by the carrier if the packaging meets laboratory specimen shipping regulations say yes, they are double sealed and have an absorbent and meet all current regulations. . Most Carriers will not deliver on Sundays, nor to many outlying areas on Saturday, so plan accordingly. It is recommended that you drop off the lab pack in the afternoon (preferably within 2-4 hours after collection). Upon Receipt of Specimen: The specimen may be taken to a physician for warming, washing, and intrauterine insemination, or used directly for vaginal (only) insemination once warmed to body temperature if so instructed by your physician and you have no allergies to penicillin, gentamycin, or streptomycin antibiotics. Danger: May cause bodily harm, shock, or death if used contrary to your physicians’ instruction. Do not use this product if you are allergic to any antibiotics. Never attempt to place anything, including raw semen, or raw semen mixed with refrigeration extender media into the cervix or uterus. Semen and semen mixed with extender must first be washed by your health care professional prior to use for intracervical or intrauterine insemination. Tape the circumference of the cooler, where the lid meets the box, to prevent cool air from escaping. Place the cooler back into its corrugated shipping box and seal the box securely with cellophane or other appropriate shipping tape. Apply the “Lab Pack” sticker to the outside of the shipping box. Be sure to complete the Federal Express air bill carefully and include the phone number of the recipient. As you will be shipping perishable product, the carrier will most likely not be able to offer any type of refund upon service failure and Andrology Institute of America, ZDL Inc, Fertmart.com, and The International Institute for Gender Selection will not offer any type of product replacement. Shipping the specimen---plan ahead and make sure that everything is ready to minimize transit time and mistakes The specimen must be shipped on the same day that it is produced to retain viability. The specimen must be shipped via overnight service. Specimens experiencing greater than 12-24 hour transit time will most likely show additional decreased viability and motility. Call Fed Ex, or the carrier of your choice to have the package picked up, or you may deliver it to their office. Fed Ex will pick up express shipments at no additional charge if you are not located in an extended area, and the arrangement is made with the carrier prior to 12 PM. Please check with your carrier of choice prior to actually needing service to prevent problems after your specimen has been produced. If asked by the carrier if the packaging meets laboratory specimen shipping regulations say yes, they are double sealed and have an absorbent and meet all current regulations. You will find the phone number on the air bill provided for either Fed Ex. You may need to arrange for package pick up a day in advance depending upon your location. Please call the carrier in advance so that you know when and where you need to arrange for pick up. Most Carriers will not deliver on Sundays, so plan accordingly It is recommended that you drop off the lab pack in the afternoon (preferably within 2-4 hours after collection). Upon Receipt of Specimen: The specimen may be taken to a physician for warming, washing, and intrauterine insemination, or used directly for vaginal (only) insemination once warmed to body temperature if so instructed by your physician and you have no allergies to penicillin, gentamycin, or streptomycin antibiotics. Warning: May cause bodily harm if used contrary to your physicians’ instruction. Do not use this product if you are allergic to any antibiotics. Never attempt to place anything, including raw semen, or raw semen mixed with refrigeration extender into the cervix or uterus. Semen and semen mixed with extender must first be washed by your health care professional prior to use for intracervical or intrauterine insemination.