Acceptable microorganisms concentration in a semen sample for in vitro embryo production

Concentração de microrganismos aceitável em uma amostra de sêmen para produção de embriões in vitro

VETERINARY MICROBIOLOGY

SHORT COMMUNICATION

Acceptable microorganisms concentration in a semen sample for in vitro embryo production

Concentração de microrganismos aceitável em uma amostra de sêmen para produção de embriões in vitro

Magali D'Angelo^I; Danielle L. Pavão^I; Gisele Martins Melo^I; Natali Rojas^I; Rosimeire Joaquim Souza^I; Christiani Athayde^I; Andrea Giannotti GaluppoI,^*; Eliana Scarcelli Pinheiro^II

^ILaboratório de Biologia Celular

^IILaboratório de Doenças Bacterianas da Reprodução; Centro de Sanidade Animal do Instituto Biológico de São Paulo, São Paulo - SP

ABSTRACT

The aim of this study was to report that the acceptable concentration of microorganisms in a semen sample for insemination may not be safe for an in vitro fertilization procedure. It seems that the semen sample should be completely germ-free, because of the excellent microorganism proliferation condition promoted by the in vitro environment.

Key words: semen, microorganisms, in vitro fertilization

RESUMO

O objetivo foi relatar que a concentração de microrganismos aceitável para uma amostra de sêmen utilizada para inseminação pode não ser segura para a realização de fertilização in vitro. Aparentemente a amostra deve ser isenta da presença de contaminantes, pois a condição in vitro promove ambiente favorável para seu crescimento.

Palavras-chave: sêmen, microorganismos, fertilização in vitro

Fundamental procedures currently used in in vitro embryo production (IVP) include transvaginal aspiration and oocytes in vitro maturation; spermatozoa in vitro capacitation and in vitro fertilization (IVF) (4). Infectious agents in IVP systems might reduce the number and the quality of embryos generated and result in transmission of disease to recipients and offspring, or even confound findings for research (3).

Semen is a potential source of non-pathogenic, as well as pathogenic microorganisms. Semen collection can be relatively free of contaminants, but it is not necessarily germ-free (3). Units of semen containing Stenotrophomonas maltophilia, Pseudomonas putida, Pseudomonas aeroginosa, Enterobacter cloacae, Staphylococcus sciuri, Acinectobacter cacloaceticus, Pantoeau agglomerans and Flavobacterium spp have been reported as contaminants in IVF embryo cultures (2). The aim of this work was to report that the acceptable concentration of microorganisms in a semen sample for insemination may not be safe for an IVF.

Routine IVF procedures were performed with oocytes collected from ovaries derived from slaughtered cows, and maturated for 24h in 5% CO₂ atmosphere at 39ºC. The semen samples were thawed at 37ºC/30' and processed in Percoll gradient (Cultilab, Campinas, Brazil) for spermatozoa capacitation. After maturation time, the oocytes were washed in Human Tubal Fluid (HTF - Cultilab, Campinas, Brazil), a fertilization medium, supplemented with 10% bovine fetal serum (BFS) and kept with the processed spermatozoa for fertilization (18-21h). However, after fertilization, in two consecutive IVF procedures, performed with two different semen samples that had presented normal evaluation parameters, the presence of contamination was detected. To locate the main focus of contamination, a new IVF procedure was performed, with the same semen samples, following the pattern protocol (1).

One fertilization dish was used for each semen sample test. They were prepared with four HTF medium drops (50 µL) each. In the first drop a conventional IVF procedure was done (in vitro matured oocytes with processed spermatozoa), in the drop 2 just oocytes were kept and in the drop 3 just processed semen. In the fourth drop HTF medium was left to be used as negative control. The dishes were maintained in 5% CO₂ environment for 18h. The presence of contamination was confirmed in drops 1 and 3 of each dish, showing that probably the semen was the contamination focus. The drops containing the oocytes and the HTF medium were tested in bacterial culture, and presented negative results. However, bacterial culture tests performed in the two semen samples used in the IVF and other seven non-used samples of the same lot indicated the two first semen samples were positive for Acinetobacter spp (5000 cfu/ml), Enterobacter aerogenes (5000 cfu/ml), Escherichia coli (5000 cfu/ml) and Streptococcus spp (uncountable cfu/ml). Among the other seven semen samples, three were negative and four were positive for Acinetobacter spp (300cfu/ml e 1600 cfu/ml), Enterobacter aerogenes (uncountable cfu/ml) and Alcaligenes faecalis (uncountable cfu/ml). The presence of those bacteria in the semen samples makes the IVF procedure not viable.

According to these findings, it is important to consider that new animal reproduction techniques brings new challenges for disease control (4). Therefore, it is important to have in mind that the quality control patterns established for old techniques should not be consider safe for the new ones. This study indicates that apparently microorganisms concentration acceptable in semen samples used for artificial insemination should not be the same accepted for IVF procedure. It should be completely germ-free, because in vitro system promotes an excellent environment for microorganisms proliferation.

ACKNOWLEDGEMENTS

To Cultilab, for financial support.

Submitted: November 01, 2005; Returned to authors for corrections: April 27, 2006; Approved: July 18, 2006

• 1. D'Angelo, M.; Galuppo, A.G.; Barra, S.H.C.; Silva, S.A.C.M.; Melo, G.M.; Zerio, N.M.C. Desenvolvimento de embriões bovinos in vitro com meio definido para embriões humanos. 16Ş Reunião Anual do Instituto Biológico, São Paulo, 2003, n.128.
• 2. Kim, I.H.; Son, D.S.; Lee, H.J.; Yang, B.C.; Lee, D.W.; Suh, G.H.; Lee, K.W.; Jung, S.C. Bacteria in semen used for IVF affect viability but can be removed by stripping cumulus by vortexing. Theriogenology, 50, 293-300, 1998.
• 3. Stringfellow, D.A.; Givens, M.D. Infectious agents in bovine embryo production: hazards and solutions. Theriogenology, 53, 85-94, 2000.
• 4. Stringfellow, D.A.; Givens, M.D.; Waldrop, J.G. Biossecurity issues associated with current and emerging embryo technologies. Reprod. Fertil. Dev, 16, 93-102, 2004.

Publication Dates

• Publication in this collection
  01 Mar 2007
• Date of issue
  Dec 2006

History

• Received
  01 Nov 2005
• Reviewed
  27 Apr 2006
• Accepted
  18 July 2006