Biosafety in embryos and semen cryopreservation, storage, management and transport.

This chapter summarizes pertinent procedures, data and opinions on the potential hazards of disease transmission through liquid nitrogen (LN)-cryopreserved and banked germplasm and tissues for somatic cell nuclear transfer (SCNT) The importance of applying internationally adopted sanitary washing procedures to germplasm as a crucial step towards their successful microbial-free cryopreservation and storage is emphasised. Special attention is given to the survival of pathogens in LN, variety of vitrification methods, sterility of LN, risks associated with the use of straws and cryovials, and LN Dewars including dry shippers. It was experimentally demonstrated that cross-contamination between LN and embryos may occur, when infectious agents are present in LN and if embryos are not protected by use of a sealed container. It is important, therefore, to prevent direct contact of germplasm and reproductive tissues with LN during cryopreservation and their storage as a mandatory measure for reducing the risk of contamination. This includes the usage of hermetically sealed high quality shatter proof freezing containers and/or the application of a secondary enclosure such as "double bagging or straw in straw". A periodic disinfection of cryo-Dewars should be considered as an additional precaution to diminish the potential for inadvertent cross-contamination. It would be advisable to use separate LN Dewars to quarantine embryos derived from infected donors of valuable genotypes or from unknown health status, extinction-threatened species.

Prevention of bovine herpesvirus-1 transmission by the transfer of embryos disinfected with recombinant bovine trypsin.

This study deals with the potential for the introduction of infectious agents through the use of animal-derived products. The efficacy of a recombinant bovine trypsin (RBTr) as a replacement for porcine pancreatic trypsin and a disinfectant for bovine herpesvirus-1 (BHV-1)-infected embryos was investigated according to the sanitary guidelines of the International Embryo Transfer Society. Treatment of in vivo and in vitro fertilized embryos contaminated with BHV-1 (10(5) TCID50/mL) in the presence of RBTr (525 U/mL) for 120 s, effectively removed the infectious virus compared with untreated and washed embryos (P < 0.05). Transfer of in vivo fertilized and disinfected embryos to BHV-1 seronegative recipients (n = 24) resulted in 14 pregnancies and 11 calves born free of BHV-1. In contrast, transfer of unwashed or undisinfected embryos to four recipients resulted in seroconversion and no pregnancies at term. It was concluded that the use of RBTr could be considered as an alternative method of rendering embryos free of BHV-1 and thus reduce the potential risk of disease transmission to embryo recipients and offspring.

Embryos produced from fertilization with bovine viral diarrhea virus (BVDV)-infected semen and the risk of disease transmission to embryo transfer (ET) recipients and offspring.

Bovine diarrhea virus (BVDV) causes a variety of economically important enteric and infertility problems in cattle. For that reason, several countries have eradicated the disease, and some others have schemes in progress to achieve freedom. Although there is a considerable amount of information about the risk of BVDV transmission through contaminated semen used for artificial insemination (AI), there is no evidence to indicate whether the resulting embryos, when used for embryo transfer, can lead to the transmission of BVDV to recipients or offspring. For this experiment, semen from a bull persistently infected with BVDV (10(5) 50% tissue culture infective doses/mL NY strain) was used for insemination (two times at estrus) of BVDV-seronegative, superovulated cows (N = 35). Embryos were collected 7 days after insemination and subsequently were washed according to the International Embryo Transfer Society recommendations or left unwashed. Out of 302 collected oocytes and embryos, 173 (57%) were fertilized and the remaining 129 (43%) had degenerated. Infectious BVDV was detected in 24% (17/71) of unwashed and 10% (8/77) of washed embryos, and in all (N = 11) follicular fluid samples, oviductal epithelial cells, endometrium, and corpora lutea tissues as determined by the virus isolation test. After transfer of 39 washed embryos to 27 BVDV-seronegative recipients, 12 (44%) cows became pregnant and 17 calves free of BVDV and BVDV antibodies, including five sets of twins, were born. After embryo transfer, all pregnant and nonpregnant recipients remained free of BVDV and antibodies. In conclusion, results herein suggest that BVDV can be transmitted by AI resulting in the production of some proportion of contaminated embryos. However, it appears that such embryos, when washed according to International Embryo Transfer Society and the World Organization for Animal Health guidelines do not cause BVDV transmission to recipients or their offspring.

Nontransmission of porcine circovirus 2 (PCV2) by embryo transfer.

Two experiments were conducted to determine the association of porcine circovirus type 2 (PCV2) with embryos and the risk of viral transmission by embryo transfer. In the first experiment, 240 embryos from uninfected donors were exposed to PCV2a 10(4)TCID50/mL in vitro before transfer to seronegative recipients; in the second experiment, 384 embryos recovered from infected donors, 10 days after donor inoculation with PCV2, were transferred to seronegative recipients. In total, 1120 embryos and/or ova were collected from 37 viral-free donors (experiment 1) and 1019 from 59 PCV2-infected donors (experiment 2) (P < 0.01). The washing and/or disinfection procedure recommended by the International Embryo Transfer Society was applied to embryos in both experiments. Transfer of embryos experimentally exposed in vitro to high titers of virus caused seroconversion of recipients (58%; N = 7/12) and their piglets (81%; N = 13/16). Postmortem, PCV2 DNA was detected in various organs of embryo transfer recipients and their embryo transfer-derived piglets. In contrast, the transfer of embryos recovered from infectious PCV2 donors did not result in the seroconversion of embryo recipients (N = 24) or their embryo transfer-derived piglets (N = 76). Neither PCV2 DNA nor infectious virus was detected in the tissues of either recipients or embryo transfer-derived piglets collected postmortem in the second experiment. The results obtained in this study indicate that the transmission of PCV2 from infected donors by embryo transfer is unlikely if the sanitary recommendations of the International Embryo Transfer Society are followed. In practical terms, this means that embryo transfer can be successfully used for the intentional elimination of PCV2 and to create virus-free offspring for the safe exchange of swine genetic materials.

A review of the risk of contamination of semen and embryos during cryopreservation and measures to limit cross-contamination during banking to prevent disease transmission in ET practices.

This review summarizes pertinent data and opinions regarding the potential hazard of disease transmission through cryopreserved and banked embryos in liquid nitrogen (LN). Special attention is given to the survival of pathogens in LN, new vitrification methods, sterility of LN, risks associated with the use of straws and cryovials, and LN dewars including dry shippers. It was experimentally demonstrated that cross-contamination between LN and embryos may occur, when infectious agents are present in LN and embryos are not protected by a sealed container. It is important, therefore, to prevent direct contact of embryos with LN during cryopreservation and their banking. This includes the usage of hermetically sealed, high-quality, shatter-proof freezing containers and/or the application of a secondary enclosure such as "double bagging or straw in straw." A periodic disinfection of cryo-dewars should be considered as an additional precaution to diminish the potential for inadvertent cross-contamination. It might be advisable to use separate LN dewars to quarantine embryos derived from infected donors of valuable genotype or from unknown health status, extinction-threatened species. Nevertheless, in summary, it has been concluded that over 25 yr with no direct evidence of disease transmission by transferred cryopreserved human and animal embryos, that the present cryopreservation technology is sanitary sound, with the stipulation that biocontainment measures recommended by the International Embryo Transfer Society (IETS) and the World Organization for Animal Health - Office International des Epizooties (OIE), are strictly followed.

Efficacy of the International Embryo Transfer Society (IETS) washing procedure for rendering oocytes matured in vitro free of bovine viral diarrhea virus (BVDV).

To ensure the freedom of embryos from pathogenic agents prior to embryo transfer (ET), a specific sanitary washing procedure has been recommended by the International Embryo Transfer Society (IETS). In the present study, the efficacy of removing the bovine viral diarrhea virus (BVDV) from cumulus-free matured oocytes at the stage of extruded first polar body (N = 240) was evaluated, using the IETS-recommended 10 sequential wash procedure, after exposure in vitro to BVDV type 2 (strain PA-131, 1 × 10(5.2) TCID(50)/mL for 1 h). In general, the percentage of contaminated oocytes was reduced (P < 0.03) after the first two washes. Nevertheless, after 10 washes, approximately 20% of oocytes still remained infectious or contaminated with virus, as detected by the virus isolation test (VI) and quantitative reverse transcription PCR (qRT-PCR) of viral RNA (on average 13 copies/oocyte). Similarly, a higher percentage of positive washing fluid samples were detected in the first three washes (50-100%). The six subsequent washes had lower but variable proportions of fluid samples contaminated with infectious virus. We concluded that the standard washing procedure may not render all oocytes free from the infectious virus adhered to the zona pellucida (ZP), and application of an additional method of oocyte disinfection was warranted to ensure nontransmission of BVDV to recipients by embryos derived from infected oocytes.

Effect of cryopreservation by slow cooling and vitrification on viral contamination of IVF embryos experimentally exposed to bovine viral diarrhea virus and bovine herpesvirus-1.

The objective was to determine the effect of cryopreservation by conventional slow controlled cooling (0.5 degrees C/min) and by vitrification on the presence of bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1) infectivity associated with frozen-thawed Day 7 bovine embryos. In this study, Day 7 embryos generated by in vitro fertilization (IVF) were exposed in vitro for 1.5h to BVDV (N=393) and BHV-1 (N=242) and subsequently tested before and after cryopreservation for the presence of infectivity. Exposure of embryos to viral agents resulted in 72% of them infected prior to cryopreservation. Stepwise exposure of embryos to cryoprotectants, as well as their removal, substantially reduced the proportion of contaminated embryos (46% vs. 72%, P<0.05). Overall, both freezing methods reduced the percentage of infectious embryos compared with that of embryos similarly exposed to viruses but not cryopreserved (31% vs. 72%, respectively; P<0.001). The percentage of embryos with infectious viruses was not significantly higher after vitrification than after slow cooling (38% vs. 22%). In addition, after cryopreservation, a higher percentage (P<0.002) of embryos exposed to BHV-1 (42%) remained infectious than did embryos exposed to BVDV (24%). In conclusion, cryopreservation reduced the proportion of infected embryos but did not render all of them free from infectious pathogens.

Risk of contamination of germplasm during cryopreservation and cryobanking in IVF units.

Cryopreservation of sperm, embryos and, more recently, oocytes plays an important and increasing role in assisted reproduction, due to improvements of old, and introduction of new technologies. In parallel, concerns are increasing about the technical and biological safety of these procedures. However, published data regarding the confirmed or theoretical hazards of these procedures are sparse and sometimes contradictory. The purpose of this review will summarize data and opinions about one of the most disputed risks, the potential hazard of contamination and disease transmission through cryopreservation. Special attention is concentrated on the weak points of the technology including open vitrification systems, sterilization of liquid nitrogen and safety of commonly used storage tanks including straws and cryovials. Suggestions are also made for practical measures to avoid these dangers while preserving the benefits and perspectives of new cryopreservation technologies.

Transmission of bovine viral diarrhea virus (BVDV) via in vitro-fertilized embryos to recipients, but not to their offspring.

The objective was to assess the potential of Day-7, IVP zona pellucida-intact blastocysts to transmit bovine viral diarrhea virus (BVDV) to embryo recipients. Embryos were exposed (1h) to two non-cytopathic (NCP) biotypes, either NY-1 (type 1) or two concentrations of PA-131 (type 2), washed 10 times, and transferred into recipients (two embryos/recipient) free of BVDV and its antibody. Six (30.0%) of the 20 pregnancies were lost after 30 d following transfer of the embryos exposed to the type 1 strain; none of the recipients or their 18 full term offspring seroconverted. Conversely, following exposure to the type 2 strain, 16 (51.6%) of the 31 pregnancies were lost >30 d after embryo transfer. Furthermore, 18 (51.4%) of 35 recipients receiving embryos exposed to type 2 seroconverted; 11 of those were pregnant at 30 d, but only 2 went to full term and gave birth to noninfected (seronegative) calves. Virus isolation tests were performed on single, virus-exposed, washed embryos (not transferred); 3 of 12 (25%) and 17 of 61 (28%) exposed to type 1 and type 2, respectively, were positive for live BVDV. Embryos exposed to type 2 virus had from 0 to 34 viral copies. In conclusion, a large proportion of recipients that received embryos exposed to BVDV, especially those exposed to a high concentration of type 2 virus, became infected after ET, and their pregnancies failed. However, term pregnancies resulted in calves free of both virus and antibody. Therefore, additional disinfection procedures are recommended prior to transferring potentially infected IVP embryos.

Disinfection procedures for controlling microorganisms in the semen and embryos of humans and farm animals.

Semen and embryos generated by assisted reproductive techniques (ARTs) may be contaminated with numerous microorganisms. Contamination may arise from systemic or local reproductive tract infections in donors or the inadvertent introduction of microorganisms during ARTs, and may lead to disease transmission. This review describes sanitary procedures which have been investigated to ascertain whether they are effective in rendering semen and embryos free of pathogenic microorganisms, including internationally adopted washing procedures, which can be supplemented by antibiotics and enzymatic treatments. Other methods include treatment with antibodies or ozone, photoinactivation, acidification, and the use of novel antiviral compounds. In conclusion, despite the wide range of antimicrobial procedures available, none can be recommended as a universal disinfection method for rendering semen and embryos free from all potentially pathogenic microorganisms. However, some procedures are unsuitable, as they can compromise the viability of semen or embryos. In humans, washing by the gradient centrifugation method appears to be effective for reducing the microbial population in semen and is harmless to the spermatozoa. A useful procedure for embryos involving multiple washes in sterile medium has much to commend it for the prevention of disease transmission; furthermore, it is recommended by the International Embryo Transfer Society (IETS).

Risk of transmission of Mycobacterium avium ssp. paratuberculosis by embryo transfer of in vivo and in vitro fertilized bovine embryos.

Over a 5-year interval, experiments were conducted to determine if Mycobacterium avium ssp. paratuberculosis (Map) is associated with in vivo and in vitro fertilized (IVF) embryos and whether it can be transmitted by embryo transfer. The present studies included: collection of embryos from five asymptomatic, naturally infected donors and transfer to uninfected recipients; collection of oocytes from two naturally infected donors with overt clinical signs; exposure of in vivo and IVF embryos to Map and transfer to uninfected recipients; and the inoculation (transfer) of "clean" IVF embryos to the uterine lumen of infected cows. The presence of Map was confirmed in the uterine horns of all asymptomatic, infected donors. None of the tested embryos, which were not used for embryo transfer, or unfertilized ova (two per batch), were positive for Map, as determined by culture (n = 19) or by PCR (n = 13). However, all in vivo fertilized embryos exposed to Map in vitro (and subsequently sequentially washed) tested positive for Map, by both culture (12 batches) and PCR (15 batches), whereas IVF embryos treated in the same manner tested positive on culture (51%, 18/35 batches) and by PCR (28%, 20/71 batches). Transferring both in vivo embryos and IVF embryos potentially contaminated with Map into 28 recipients resulted in 13 pregnancies and eight calves born without evidence of disease transmission to either the recipients or the offspring over the following 5-year period. In samples collected from one of the clinically infected animals, two of seven (28%) cumulus oocyte complexes (COC) and follicular fluid tested positive by PCR and 10/10 cumulus oocyte complexes on culture for Map. From the second clinically infected cow, three of five batches of IVF embryos (n = 20) were positive on PCR and two of four batches containing unfertilized oocytes and embryos were positive on culture. Only 10% of embryos reached the morula and blastocyst stage 10 days after fertilization. In conclusion, Map is unlikely to be transmitted by embryo transfer when the embryos have been washed as recommended by the International Embryo Transfer Society.

Non-transmission of bacterial and viral microbes to embryos and semen stored in the vapour phase of liquid nitrogen in dry shippers.

Cryopreservation and storage of germplasm is an important factor in the prevention of disease transmission by embryo transfer and artificial insemination. Here we report the results of an investigation on transmission of selected bacterial and viral pathogens by the vapour phase of liquid nitrogen (VPLN) to embryos and semen in dewars designed for short-term storage and transportation of biological specimens. In this study transmission of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, BVDV, and BHV-1 was examined from: (1) contaminated dry shippers to germplasm; (2) between contaminated and non-contaminated cryopreserved germplasm; and (3) between stock culture of pathogenic agents and germplasm. Contaminated and non-contaminated samples of embryos and semen were stored in proximity in the vapour phase LN in open containers for 7 days prior to testing for the presence of microbes. The results showed that there was no cross-contamination from either the contaminated dewars to germplasm or between contaminated and non-contaminated samples of embryos and semen during 7 days storage under LN vapours. The outcome of our investigation indicates that VPLN is a safe means for short-term storage of embryos and semen in dry shipper dewars commonly used for transportation of bovine germplasm.

Experimental microbial contamination and disinfection of dry (vapour) shipper dewars designed for short-term storage and transportation of cryopreserved germplasm and other biological specimens.

Cryopreservation, storage and transport of cryopreserved germplasm without the risk of disease transmission is of great concern to animal and human health authorities. Here we report on the efficacy of microbial decontamination of the liquid nitrogen (LN) dry (vapour) shippers used for short-term storage and transportation of germplasm and other biological specimens. Dry shippers containing either a hydrophobic or a non-hydrophobic LN absorbent were experimentally contaminated with high titers of cultures of Pseudomonas aeruginosa, Escherichia coli, Staphylococus aureus, bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1). Biocidals with broad spectrum antimicrobial activity and gas vapours of formalin and ethylene oxide were used for disinfection of the dewars. Among the biocidals used, treatment with sodium hypochlorite solution, the quaternary ammonium-based disinfectants and peracetic acid were the most effective and useful for dry shippers with a hydrophobic LN absorbent. None of the bacterial or viral microorganisms were detected in samples of semen and embryos stored in dry shippers following their disinfection with these biocides. An application of some other disinfectants, due to their foaming properties or to the permeability of the absorbent hydrophobic membrane (HM) was not effective or may have caused irreversible damage to the LN absorbent. Gas sterilization by ethylene oxide in contrast to formalin was fully effective for both types of dry shippers.

Observations on the fertilization and development of preimplantation bovine embryos in vitro in the presence of Tritrichomonas foetus.

Tritrichomonas foetus, a world-wide distributed parasitic protozoan is a cause of infertility and abortion. There is no documented information on the susceptibility of bovine embryos to the parasite. To determine the effect of T. foetus on fertilization and embryonic development of preimplantation bovine embryos, we added approximately 10(4)/ml or 10(6)/ml T. foetus (Belfast strain) to sperm cells and oocytes prior to in vitro fertilization (IVF) or to presumptive zygotes 24 h post-fertilization. Light and scanning electron microscopy (SEM) revealed that exposure of oocytes or embryos at any stage of development to T. foetus caused rapid adhesion of the trichomonads to the embryonic intact zona pellucida (ZP) and to trophoblastic cells of hatched blastocysts. Treatment of contaminated embryos with 0.25% trypsin for 3 min did not render them free from T. foetus. Motile parasites were not observed after 18 h incubation in IVF medium, or after 72 h in synthetic oviductal fluid (SOF) embryo culture medium. The percentages of cleaved zygotes, blastocysts and hatched embryos resulting from culture of experimental and uninfected control groups of embryos were not different (P > 0.05). Tritrichomonas foetus was not detected in embryonic cells of ZP-intact or hatched embryos when examined by transmission electron microscopy (TEM). In conclusion, T. foetus has no detrimental effect on the fertilization and development of IVF embryos and the potential risk of transmission of trichomonosis is unlikely, due to the limited survival of the parasite in IVF culture conditions.

Microbial contamination of embryos and semen during long term banking in liquid nitrogen.

We report on microbial contamination of embryos and semen cryopreserved in sealed plastic straws and stored for 6-35 years in liquid nitrogen. There were 32 bacterial and 1 fungal species identified from randomly drawn liquid nitrogen, frozen semen, and embryos samples stored in 8 commercial and 8 research facility liquid nitrogen (LN) tanks. The identified bacteria represented commensal or environmental microorganisms and some, such as Escherichia coli, were potential or opportunistic pathogens for humans and animals. Stenotrophomonas maltophilia was the most common contaminant identified from the samples and was further shown to significantly suppress fertilization and embryonic development in vitro. Analysis of the strains by pulsed field gel electrophoresis revealed restriction patterns with no relatedness indicating that there was no apparent cross-contamination of S. maltophilia strains between the germplasm and liquid nitrogen samples. In addition, no transmission of bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1) from infected semen and embryos straws to clean germplasm stored in the same LN tanks or LN was detected.

Adherence of bovine viral diarrhea virus to bovine oocytes and embryos with a hardened zona pellucida cultured in vitro.

The purpose of this study was to investigate the adherence of bovine viral diarrhea virus (BVDV) to bovine mature, or immature, cumulus-free oocytes and to in vitro fertilized embryos, maintained in vitro in a ligated bovine oviduct to allow for the hardening of the zona pellucida. Incubation of the oocytes and embryos in the oviduct for 5 h caused hardening of the zona pellucida as measured by resistance to pronase digestion (which increased from approximately 3 min to 7 h; P > 0.001). However, there was no difference between the number of infected oocytes and embryos (n = 965 in 193 samples) following experimental exposure to BVDV regardless of whether or not they were previously incubated in the oviduct (P > 0.05). It was concluded that the modification of the proteolytic resistance properties of the zona pellucida during in vitro oviductal incubation did not influence the adherence of BVDV to zona pellucida of oocytes or in vitro fertilized embryos.

Experimental collection and transfer of embryos from bovine immunodeficiency virus (BIV) infected cattle.

Three experiments were conducted to determine whether the lentivirus, bovine immunodeficiency virus (BIV) is likely to be transmitted via embryo transfer. In the first experiment, embryos collected from BIV-negative heifers were exposed in vitro to BIV for 24 h, washed and then tested for the presence of the provirus. In the second experiment, embryos obtained from BIV-negative heifers were transferred to the uterine horns of BIV-infected heifers; 24 h later these embryos were recovered and tested for the presence of BIV. In the third experiment, embryos were collected from heifers experimentally infected with BIV and then transferred to BIV-negative recipients. In all three experiments, (BIV) proviral DNA was not detected by PCR in association with any oocytes, embryos, follicular fluid, oviductal or uterine washes. Twelve single embryos collected from BIV experimentally infected donors were transferred to BIV-negative recipients resulting in the birth of 7 calves all of which were also negative for BIV; the recipients remained BIV-negative throughout the experiment. In conclusion, this study demonstrates that it is possible to produce transferrable stage embryos from donors infected with BIV and that such embryos are unlikely to transmit this agent either to the recipients or the resulting offspring.