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Viability of Toxoplasma gondii in cattle semen cryopreserved with different concentrations of cryoprotectant

Viabilidade do Toxoplasma gondii em sêmen de bovino criopreservado em diferentes concentrações de crioprotetores

ABSTRACT:

Toxoplasma gondii can be eliminated in bovine semen. Cryopreserved semen is often used due to the fact that artificial insemination in dairy and beef cattle provides benefits in terms of production. However, little is known regarding the viability and infectivity of T. gondii tachyzoites in cryopreserved bovine semen. In the present study, cattle semen negative for T. gondii were contaminated with 1 x 106 tachyzoites (RH strain) and cryopreserved with and without different cryoprotectants, such as DMSO (concentrations of 2.5%, 5.0%, 7.5%, 8.0% and 10.0%) and glycerol (2.25%, 2.5%, 3.0%, 5.0%, 7.5% and 10.0%), followed by freezing in liquid nitrogen (-196°C). After 24 hours, the samples were thawed and inoculated in 10 mice per cryoprotectant concentration. The mice were evaluated for clinical signs of toxoplasmosis (rough coat, diarrhea, hypoactivity and sudden death) as well as serum titers of IgM and IgG and the presence of tachyzoites in the peritoneal lavage. The results revealed that T. gondii remained infective in all samples. Clinical signs of toxoplasmosis were observed in the mice beginning with the 6th day post-inoculation (DPI) and 100% lethality was found between the 7th and 9th DPI. Viable tachyzoites were recovered from peritoneal exudate of dead mice (except for the control group), with higher mean of tachyzoite counts in the intraperitoneal lavage for 5% DMSO (±3.32 x 106), 8% DMSO (±3.53 x 106), 3% glycerol (±4.75 x 106), 7.5% glycerol (±6.26 x 106) and the absence of cryoprotectant (±3.11 x 106). Seroconversion occurred in the treated groups, with titers of IgG from 1:16 to 1:128 and IgM from 1:16 to 1:512. T. gondii viability and infectivity were maintained in cattle semen during 24 hours of cryopreservation at -196°C with and without cryoprotectant. However, further studies are necessary to determine whether cryopreserved semen contributes to the spread of toxoplasmosis through artificial insemination.

INDEXING TERMS:
Toxoplasma gondii; cattle; cryopreservation; cryoprotectant; DMSO; glycerol; toxoplasmosis; semen samples; RH strain; IFI

RESUMO:

Sabe-se que Toxoplasma gondii pode ser eliminado no sêmen bovino. A inseminação artificial em bovinos leiteiros e de corte proporcionou avanços e benefícios nas produções e para isso o sêmen criopreservado é frequentemente utilizado. No entanto, pouco se sabe sobre a viabilidade e infectividade dos taquizoítos de T. gondii em sêmen bovino criopreservado. Para isso o sêmen bovino, negativo para T. gondii, foi contaminado com 1x106 taquizoítos (cepa RH), criopreservados com ou sem diferentes crioprotetores como DMSO (2.5%, 5.0%, 7.5%, 8.0% e 10.0%) e Glicerol (2.25%, 2.5%, 3.0%, 5.0%, 7.5% e 10.0%) e congelados em nitrogênio líquido (-196°C). Após 24 horas, essas amostras foram descongeladas e inoculadas em 10 camundongos por diluente de concentração de crioprotetor. Os camundongos foram avaliados quanto a sinais clínicos de toxoplasmose (pele áspera, diarreia, hipoatividade e morte súbita), títulos séricos de IgM e IgG e presença de taquizoítos no lavado peritoneal. Os resultados mostraram que T. gondii se manteve infectante em todas as amostras, inclusive naquelas sem crioprotetor. Sinais clínicos de toxoplasmose foram observados nos camundongos a partir do 6º dia pós-inoculação (DPI) e 100% de letalidade foi verificada entre o 7º ao 9º DPI. Nos camundongos mortos, exceto no grupo controle, taquizoítos viáveis foram recuperados do exsudato peritoneal, com maior média de taquizoítos quantificados na lavagem intraperitoneal para DMSO a 5% (±3.32x106), 8% (±3.53x106) e glicerol 3% (±4.75x106), 7,5% (±6.26x106) e livre de crioprotetor (±3.11x106). A soroconversão ocorreu nos grupos tratados com títulos de IgG (1:16 a 1:128) e IgM (1:16 a 1:512). A viabilidade e infectividade do T. gondii no sêmen bovino durante as 24 horas de criopreservação a -196°C foram mantidas com ou sem crioprotetor. No entanto, mais estudos são necessários para verificar se o sêmen criopreservado contribui para a disseminação da toxoplasmose na inseminação artificial.

TERMOS DE INDEXAÇÃO:
Toxoplasma gondii; bovinos; criopreservação; crioprotetores; DMSO; glicerol; toxoplasmose; amostras seminais; cepa RH; RIFI

Introduction

Toxoplasmosis is a global zoonotic disease caused by Toxoplasma gondii (Nicolle & Manceaux 1909), which is an obligatory intracellular parasite with a heteroxenous, facultative life cycle and the ability to infect a variety of animal species, such as sheep, cattle, goats, pigs, horses and poultry (Mello 1910Mello V. 1910. Un cas de toxoplasmose du chien observé à Turin. Bulletin de la Societe de Pathologie 28:359-363., Quinn & McCraw 1972Quinn P.J. & Mccraw B.M. 1972. Current status of Toxoplasma and toxoplasmosis: a review. Can. Vet. J. 13(11):247-262. <PMid:4567530>, Dubey & Beattie 1988Dubey J.P. & Beattie C.P. 1988. Toxoplasmosis of animals and man. CRC Press, Boca Raton, p.1-220., Bresciani et al. 1999Bresciani K.D.S., Costa A.J., Toniollo G.H., Sabatini G.A., Morais F.R., Paulilo A.C. & Ferraudo A.S. 1999. Experimental toxoplasmosis in pregnant bitches. Vet. Parasitol. 86(2):143-145. <https://dx.doi.org/10.1016/S0304-4017(99)00136-3> <PMid:10496698>
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, Tenter et al. 2000Tenter A.M., Heckeroth A.R. & Weiss L.M. 2000. Toxoplasma gondii: from animals to humans. Int. J. Parasitol. 30(12/13):1217-1258. <https://dx.doi.org/10.1016/S0020-7519(00)00124-7> <PMid:11113252>
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, Sawadogo et al. 2005, Fialho et al. 2009Fialho C.G., Teixeira M.C. & De Araújo F.A.P. 2009. Toxoplasmose animal no Brasil. Acta Scient. Vet. 37(1):1-23. <https://dx.doi.org/10.22456/1679-9216.16180>
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, Dubey et al. 2013Dubey J.P., Hotea I., Olariu T.R., Jones J.L. & Darabus G. 2013. Epidemiological review of Toxoplasmosis in humans and animals in Romania. Parasitology 141(3):311-325. <https://dx.doi.org/10.1017/S0031182013001509> <PMid:24553077>
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). The main T. gondii infection route in cattle is the ingestion of sporulated oocysts present in pastures, silage, feed and water. Moreover, sexual transmission of T. gondii (oocysts and tachyzoites) by the seminal route in cattle has been reported for seminal vesicles diagnosed by PCR and bioassays (semen samples) in mice (Scarpelli et al. 2009Scarpelli L., Lopes W.D.Z., Migani M., Bresciani K.D.S. & Costa A.J. 2009. Toxoplasma gondii in experimentally infected Bos taurus and Bos indicus semen and tissues. Pesq. Vet. Bras. 29(1):59-64. <https://dx.doi.org/10.1590/S0100-736X2009000100009>
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).

Artificial insemination in beef and dairy cattle using cryopreserved semen is a common management strategy to improve herd genetics and consequently enhance productivity (Boni 2012Boni R. 2012. Ovum pick-up in cattle: a 25 yr retrospective analysis. Anim. Reprod. 9(3):362-369.). Cryopreservation enables the storage of semen for an indefinite period, reducing the risks and costs related to the acquisition and transportation of breeders and facilitating the diffusion of genetic material between distant sites, with the further advantage of reducing the risk of disease transmission (Vishwanath 2003Vishwanath R. 2003. Artificial insemination: the state of the art. Theriogenology. 59(2):571-584. <https://dx.doi.org/10.1016/S0093-691X(02)01241-4>
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, Castelo et al. 2008Castelo T.S., Frota T.R. & Silva A.R. 2008. Considerações sobre a criopreservação do sêmen de caprinos. Acta Vet. Bras. 2(3):67-75. <https://dx.doi.org/10.21708/avb.2008.2.3.885>
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, Rodgers et al. 2012Rodgers J.C., Bird S.L., Larson J.E., DiLorenzo N., Dahlen C.R., DiCostanzo A. & Lam G.C. 2012. An economic evaluation of estrous synchronization and timed artificial insemination in suckled beef cows. J. Anim. Sci. 90(11): 4055-4062. <https://dx.doi.org/10.2527/jas.2011-4836> <PMid:22585820>
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).

Cryoprotectants used for sperm preservation include glycerol, ethylene glycol, dimethyl sulfoxide (DMSO), acetamide, lactamide and dimethylacetamide (Fickel et al. 2007Fickel J., Wagener A. & Ludwig A. 2007. Semen cryopreservation and the conservation of endangered species. Eur. J. Wildl. Res. 53:81-89. <https://dx.doi.org/10.1007/s10344-007-0089-z>
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, Kashiwazaki et al. 2006Kashiwazaki N., Okuda Y., Seita Y., Hisamatsu S., Soroki S., Shino M., Masaoka T. & Inomata T. 2006. Comparison of glycerol, lactamide and dimethyl sulfoxide as cryoprotectants of Japanese white rabbit spermatozoa. J. Reprod. Develop. 52(4):511-516. <https://dx.doi.org/10.1262/jrd.18008> <PMid:16679717>
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). Glycerol is the most common cryoprotective agent used for freezing bovine semen, but can be toxic to sperm cells and cause contraceptive effects (Becker et al. 1977Becker W.C., Senger P.L., Aalseth E.P. & Marshall C.E. 1977. Influence of glycerol levels, diluent and post-thaw temperature on motility and acrossomal maintenance in bovine semen frozen in plastic straws. J. Anim. Sci. 44(6):1067-1071. <https://dx.doi.org/10.2527/jas1977.4461067x> <PMid:873862>
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). DMSO at concentrations of 4 and 6% constitutes an alternative to glycerol (Snedeker & Gaunya 1970Snedeker W.H. & Gaunya W.S. 1970. Dimethyl sulfoxide as a cryoprotective agent for freezing bovine semen. J. Anim. Sci. 30(6):953-956. <https://dx.doi.org/10.2527/jas1970.306953x> <PMid:5463531>
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).

As literature on T. gondii cryopreserved in semen is scarce, the aim of the present study was to assess the viability of T. gondii tachyzoites in cryopreserved cattle semen with different concentrations of DMSO and glycerol.

Materials and Methods

All experimental procedures received approval from the Ethics Committee on Animal Use of the “Faculdade de Ciências Agrárias e Veterinárias” (School of Agrarian and Veterinary Sciences - FCAV) of “Universidade Estadual Paulista ‘Júlio de Mesquita Filho’” (Unesp) (certificate number: 026341/13). The experiment was conducted at the Animal Health Research Center and Department of Preventive Veterinary Medicine and Animal Reproduction, Unesp, in the municipality of Jaboticabal/SP, Brazil, and Laboratory of Molecular Diagnosis Applied to Zoonoses of the Department of Veterinary Hygiene and Public Health, “Faculdade de Medicina Veterinária e Zootecnia” (School of Veterinary Medicine and Zootechnics - FMVZ), Unesp, in the municipality of Botucatu/SP, Brazil.

Obtention of Toxoplasma gondii tachyzoites. The RH strain (Sabin 1941Sabin A.B. 1941. Toxoplasmic encephalitis in children. J. Am. Med. Assoc. 116(9):801-807. <https://dx.doi.org/10.1001/jama.1941.02820090001001>
https://doi.org/10.1001/jama.1941.028200...
) was used in the present study. T. gondii tachyzoites were maintained by periodic passages in the peritoneal cavity of Swiss mice (Mus musculus) approximately one month of age (weight: 20-25g). Tachyzoites were obtained from the peritoneal lavage (400µL of peritoneal exudate; approximately 106 tachyzoites) of previously infected mice on the second day after infection (Mineo et al. 1980Mineo J.R., Camargo M.E. & Ferreira A.W. 1980. Enzyme-linked immunosorbent assay for antibodies to Toxoplasma gondii polysaccharides in human toxoplasmosis. Infect. Immun. 27(2):283-287. <https://dx.doi.org/10.1128/iai.27.2.283-287.1980> <PMid:7380534>
https://doi.org/10.1128/iai.27.2.283-287...
). The mice were euthanized by cervical dislocation following the guidelines of the National Council for the Control of Animal Experimentation (CONCEA) and positioned dorsally for the injection of 3mL of sterile saline solution into the cavity. Abdominocentesis was then performed for the recovery of T. gondii tachyzoites. A drop of the peritoneal lavage was placed on a slide and observed under an optical microscope (objective: 40x) for the subjective analysis of tachyzoite morphology and viability (Costa et al. 2011Costa G.H.N., Costa A.J., Lopes W.D.Z., Bresciani K.D.S., Santos T.R., Esper C.R. & Santana A.E. 2011. Toxoplasma gondii: infection natural congenital in cattle and an experimental inoculation of gestating cows with oocysts. Exp. Parasitol. 127(1):277-281. <https://dx.doi.org/10.1016/j.exppara.2010.08.005> <PMid:20736009>
https://doi.org/10.1016/j.exppara.2010.0...
). Lavages with a smaller number of somatic cells (red blood cells, leukocytes, desquamated epithelial cells) and higher quantity of tachyzoites (1 x 106) were chosen for semen infection (adapted from Camargo 1964Camargo M.E. 1964. Improved technique of indirect immunofluorescence for serological diagnosis of toxoplasmosis. Revta Inst. Med. Trop. São Paulo 6:117-118. <PMid:14177810>). The selected intraperitoneal lavages were centrifuged twice - once at 1000rpm for 30 minutes for the sedimentation of impurities and then at 2500rpm for eight minutes. The pellet was resuspended in 2.0mL of phosphate-buffered saline (PBS). T. gondii tachyzoites (1 x 106) were submitted to the Trypan Blue exclusion test to assess cell viability and counted in a Neubauer chamber with a volume of 10µL (Mitsuka et al. 1998Mitsuka R., Beckner Da Silva A.C., Navarro I.T., Breganó J.W. & Vidotto O. 1998. Toxoplasma gondii: I. Avaliação da virulência de oito amostras. Braz. J. Vet. Res. Anim. Sci. 35(1):29-31. <https://dx.doi.org/10.1590/S1413-95961998000100005>
https://doi.org/10.1590/S1413-9596199800...
).

Selection of bulls, andrological examination and assessment of semen characteristics. Blood samples from 23 bulls in reproductive age (≥24 months) from the municipality of Ribeirão Preto in the state of São Paulo, Brazil were collected through jugular venipuncture in vacutainer tubes without anticoagulant. The material was centrifuged at 1000g for 10 minutes and sera were stored at -20°C until processing. All samples were tested for T. gondii (Camargo 1964Camargo M.E. 1964. Improved technique of indirect immunofluorescence for serological diagnosis of toxoplasmosis. Revta Inst. Med. Trop. São Paulo 6:117-118. <PMid:14177810>) and Neospora caninum (Conrad et al. 1993Conrad P., Barr B.C., Sverlow K.W., Anderson M., Daft B., Kinde H., Dubey J.P., Munson L. & Ardans A. 1993. In vitro isolation and characterization of a Neospora sp. from aborted bovine foetuses. Parasitology 106(Pt 3):239-249. <https://dx.doi.org/10.1017/S0031182000075065> <PMid:8488061>
https://doi.org/10.1017/S003118200007506...
, Dubey & Lindsay 1996Dubey J.P. & Lindsay D.S. 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1-59. <https://doi.org/10.1016/S0304-4017(96)01035-7>
https://doi.org/10.1016/S0304-4017(96)01...
, Soares et al. 2009Soares H.S., Ahid S.M.M., Bezerra A.C.D.S., Pena H.F.J., Dias R.A. & Gennari S.M. 2009. Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from Mossoró, Rio Grande do Norte, Brazil. Vet. Parasitol. 160(3/4):211-214. <https://dx.doi.org/10.1016/j.vetpar.2008.10.102> <PMid:19091473>
https://doi.org/10.1016/j.vetpar.2008.10...
) antibodies by indirect antibody immunofluorescence assay (IFA) using a bovine specific anti-IgG conjugate (Sigma Chemical - F7887).

Bulls were considered positive for T. gondii when the serum had titers equal to or greater than 1:64 (Camargo 1964Camargo M.E. 1964. Improved technique of indirect immunofluorescence for serological diagnosis of toxoplasmosis. Revta Inst. Med. Trop. São Paulo 6:117-118. <PMid:14177810>) and positive for N. caninum with titers equal to or greater than 1:50 (Figliuolo et al. 2004Figliuolo L.P.C., Rodrigues A.A.R., Viana R.B., Aguiar D.M., Kasai N. & Gennari S.M. 2004. Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in goat from São Paulo State, Brazil. Small Ruminant Res. 55(1/3):29-32. <https://dx.doi.org/10.1016/j.smallrumres.2003.12.013>
https://doi.org/10.1016/j.smallrumres.20...
). The animals were also tested for antibodies against Brucella abortus using the Card Test (Alton et al. 1988Alton G.G., Jones L.M., Angus R.D. & Verger J.M. 1988. Techniques for the Brucellosis Laboratory. INRA Publications, Paris, p.63-129.) and antibodies against Leptospira spp. (serovar wolffi, hardjo, patoc, pomona, icterohaemorrhagiae and canicola) using the microscopic agglutination test (Centro Panamericano de Zoonoses 1985Centro Panamericano de Zoonoses. 1985. Salud Pública Veterinaria. Ministerio de Salud y Acción Social, Organización Panamericana de la Salud, Organización Mundial de la Salud, Argentina, p.193-204.).

To determine the presence of T. gondii DNA, semen was collected from the 23 bulls through electroejaculation (Mascarenhas & Gomes 1950Mascarenhas H. & Gomes W.V. 1950. Contribuição ao Estudo da Eletroejaculação em Bovinos: novo tipo de elétrodo bipolar e técnica de sua aplicação. Vol.8. Instituto de Zootecnia, Rio de Janeiro. 30p.). DNA was extracted using the Illustra Blood genomicPrep Mini Spin kit (GE Healthcare Life Sciences do Brasil Ltda®, Brazil) according to the manufacturer’s instructions, followed by conventional polymerase chain reaction (cPCR) using the primers TOX4 (5’-CGCTGCAGGGAGGAAGACGAAAGTTG-3’) and TOX5 (5’-CGCTGCAGACACAGTGCATCTGGATT-3’), which amplify a region of 529 bp of 18S rRNA (Homan et al. 2000Homan W.L., Vercammen M., De Braekeleer J. & Verschueren H. 2000. Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. Int. J. Parasitol. 30(1):69-75. <https://dx.doi.org/10.1016/s0020-7519(99)00170-8> <PMid:10675747>
https://doi.org/10.1016/s0020-7519(99)00...
). DNA from the RH strain of T. gondii was used as the positive control. The results were viewed in 1% agarose gel stained with ethidium bromide solution (Fig.1).

Fig.1.
Photography of agarose gel of primary PCR product showing non-bands. Bull semen samples not positive in PCR with primers TOX4 and TOX5. Positive samples should amplify in region of 529 bp (control).

Only bulls serologically negative for the diseases evaluated and without T. gondii in the semen were selected. One of the 23 bulls was randomly selected. This bull (no. 10) was approximately four years of age, was crossbred (Bos taurus taurus x Bos taurus indicus), with a good body condition (4 on a scale ranging from 1-5), good overall appearance and no physical abnormalities detected during the clinical examination. The animal was submitted to the andrological examination to determine its reproductive potential following the guidelines of the Brazilian College of Animal Husbandry (CBRA 2013CBRA. 2013. Manual para exame andrológico e avaliação de sêmen animal. 3ª ed. Colégio Brasileiro de Reprodução Animal, Belo Horizonte. 104p.). Examinations were performed of the external genitals (inspection and palpation of the scrotum, testes, epididymis, spermatic cords, foreskin and penis) and internal genitals (vesicular glands, ampulla of the vas deferens, prostate and bulbourethral gland by rectal palpation). A semen sample was collected and analyzed in terms of physical (volume, whirling, vigor, motility and concentration) and morphological characteristics (CBRA 2013CBRA. 2013. Manual para exame andrológico e avaliação de sêmen animal. 3ª ed. Colégio Brasileiro de Reprodução Animal, Belo Horizonte. 104p.).

Semen dilution, cryoprotectants and contamination with T. gondii tachyzoites. Fifty mL of a single ejaculate were divided into fourteen aliquots of 0.25 mL (containing 20 x 106 spermatozoids) and placed in 15-mL Falcon tubes in a water bath (37°C). Twelve of the fourteen semen samples contaminated with T. gondii tachyzoites obtained from the peritoneal lavage of experimentally infected mice. A T. gondii suspension (0.05 mL, 1 x 106 of viable tachyzoites) was added to each aliquot of semen.

After contamination, TRIS (27g/L) + 14g/L citrate (14g/L citric acid + 10g/L fructose) + egg yolk (20% v/v) as diluent were added to each of the samples containing the different concentrations of cryoprotectant (glycerol and DMSO). One mg of streptomycin was also added to each concentration. Five concentrations of DMSO (2.5%, 5.0%, 7.5%, 8.0% and 10%) and six concentrations of glycerol (2.25%, 2.5%, 3.0%, 5.0%, 7.5% and 10%) were used in the experiment based on studies involving mammalian, fish and chicken sperm (Polge et al. 1949Polge C., Smith A.U. & Parkers A.S. 1949. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164(4172):666. <https://dx.doi.org/10.1038/164666a0> <PMid:18143360>
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, Lovelock & Bishop 1959Lovelock J.E. & Bishop M.W.H. 1959. Prevention of freezing damage to living cells by dimethylsulphoxide. Nature 183(4672):1394-1395. <https://dx.doi.org/10.1038/1831394a0><PMid:13657132>
https://doi.org/10.1038/1831394a0...
, Fahy 1986Fahy G.M. 1986. The relevance of cryoprotectant “toxicity” to cryobiology. Cryobiology 23(1):1-13. <https://dx.doi.org/10.1016/0011-2240(86)90013-1> <PMid:3956226>
https://doi.org/10.1016/0011-2240(86)900...
, Fontgibell & Vidal 1998Fontgibell A. & Vidal A.F. 1998. Primeros resultados en el estudio de los efectos de la congelación de semen de gallo en tres razas catalanas. Arch. Zootec. 47(178/179):335-341., Agca & Critser 2002Agca Y. & Critser J.K. 2002. Cryopreservation of spermatozoa in assisted reproduction. Semin. Reprod. Med. 20(1):15-23. <https://dx.doi.org/10.1055/s-2002-23516> <PMid:11941531>
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, Hubálek 2003Hubálek Z. 2003. Protectants used in the cryopreservation of microorganisms. Cryobiology 46(3):205-229. <https://dx.doi.org/10.1016/S0011-2240(03)00046-4> <PMid:12818211>
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, Ribeiro & Godinho 2003Ribeiro R.I.M.A. & Godinho H.P. 2003. Criopreservação do sêmen testicular do teleósteo piau-açu Leporinus macrocephalus. Arq. Bras. Med. Vet. Zootec. 55(1):75-79. <https://dx.doi.org/10.1590/S0102-09352003000100011>
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, Murgas et al. 2007Murgas L.D.S., Miliorini A.B., Freitas R.T.F. & Pereira G.J.M. 2007. Criopreservação do sêmen de curimba (Prochilodus lineatus) mediante adição de diferentes diluidores, ativadores e crioprotetores. Revta Bras. Zootec. 36(3):526-531. <https://dx.doi.org/10.1590/S1516-35982007000300002>
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, Sanches et al. 2008Sanches E.G. & Oliveira I.R. & Serralheiro P.C.S. 2008. Crioconservação do sêmen da garoupa-verdadeira Epinephelus marginatus. Bioikos 22(2):81-90.). However, one should bear in mind that, despite citations in the literature, there is no standardization of concentrations.

Two controls groups were established, in which the semen samples did not receive tachyzoites of T. gondii, only the diluent (TRIS + citrate + egg yolk) and the cryoprotectants 5% DMSO or 3% glycerol. Another group was established without cryoprotectant and contaminated with T. gondii tachyzoites. Two concentrations were first chosen (5% DMSO and 3% glycerol), as lower concentrations of DMSO offer less toxicity on the intracellular level and glycerol at a concentration of 3% is routinely recommended for the cryopreservation of semen at genetic bovine improvement centers.

Freezing of samples using conventional method. The Falcon tubes containing the semen with or without T. gondii tachyzoites + diluent + with or without cryoprotectant were then placed in two plastic beakers, which were placed in a refrigerator (4.5°C) for four hours (time required for cooling and equilibration of temperature). The samples were then placed in duly labeled 0.25-mL French straws, which were sealed with modeling clay. Eleven straws were filled per tube of semen. The straws were then distributed horizontally, positioned on an appropriate grid, which was placed on a metal platform with a height of 9cm. The set was placed in a polystyrene box (30cm in height x 19cm in width x 39cm in length) containing a 6-cm layer of liquid nitrogen and the straws were maintained 3cm from the nitrogen vapor (-150°C) for 15 minutes. After this period, the platform was removed and the grid, together with the straws, was immediately immersed in liquid nitrogen (-196°C) to complete the freezing process. With the aid of metal tweezers, the straws were then transferred to the cryogenic cylinder for storage until further analysis (adapted from Duarte & Garcia 1997Duarte J.M.B. & Garcia J.M. 1997. Tecnologia da reprodução para propagação e conservação de espécies ameaçadas de extinção, p.228-238. In: Duarte J.M.B. (Ed.), Biologia e Conservação de Cervídeos Sul-americanos: Blastocerus, Ozotoceros e Mazama. FUNEP, Jaboticabal.).

Semen assessment after thawing. For each concentration of DMSO and glycerol, one straw with frozen semen was thawed (water bath at 35°C for 20 seconds) and the semen was immediately submitted to vigor and motility analysis using the methods referenced above.

T. gondii feasibility and infectivity in cryopreserved semen samples. A method adapted from Teale et al. (1982)Teale A.J., Blewett D.A., Miller J.K. & Buxton D. 1982. Experimentally induced toxoplasmosis in young rams: The clinical syndrome and semen secretion of toxoplasma. Vet. Rec. 111(3):53-55. <https://dx.doi.org/10.1136/vr.111.3.53> <PMid:7123822>
https://doi.org/10.1136/vr.111.3.53...
was used to assess the viability and infectivity of T. gondii tachyzoites in cryopreserved (DMSO and glycerol) semen samples. For each of the fourteen semen tubes, 10 straws with semen were thawed (water bath at 35°C for 20 seconds) 24 hours after freezing. Ten mice were inoculated (intraperitoneally) with the total content of one straw (0.25mL) of semen in an equal volume of PBS (Table 1).

Table 1.
Spermatic parameters, post thawing, of bovine semen (20x106 spermatozoids) infected or not with Toxoplasma gondii tachyzoites (1x106) cryopreserved with DMSO and glycerol at different concentrations

The mice were observed daily for 42 days for clinical signs of toxoplasmosis (rough coat, hypoactivity, diarrhea, ascites and eyes half closed), following the method described by Costa et al. (1977)Costa A.J., Araujo F.G., Costa J.O., Lima J.D., Nascimento E. 1977. Experimental infection of bovines with oocysts of Toxoplasma gondii. J. Parasitol. 63(2):212-218. <https://dx.doi.org/10.2307/3280042> <PMid:558305>
https://doi.org/10.2307/3280042...
. Animals that died by the 10th day after inoculation (acute phase of toxoplasmosis) were submitted to necropsy to search for tachyzoites in the abdominal cavity through an analysis of the peritoneal lavage. Mice that survived until the 42nd day after experimental infection were euthanized for blood collection. Serum samples were submitted to IFA to investigate seroconversion. IFA was performed using anti-mouse IgG and IgM conjugate (Sigma Chemical - F0257 and F9259) produced in sheep. Additionally, brain cysts were investigated in seropositive animals and those that died between the 11th and 42nd day after infection (Camargo 1964Camargo M.E. 1964. Improved technique of indirect immunofluorescence for serological diagnosis of toxoplasmosis. Revta Inst. Med. Trop. São Paulo 6:117-118. <PMid:14177810>).

Statistical analysis. The experimental data were tested for independence, normality and homoscedasticity. Differences in tachyzoite counts recovered from the peritoneal exudates of the bioassay with mice were determined using the Kruskal-Wallis test and mean tachyzoite counts were compared using the Scott-Knott test with a 95% confidence interval (p≤0.05). Reciprocal serological concentrations (IgG and IgM class antibodies) in the mice were compared using Fisher’s exact test with a 95% confidence interval. All analyses were performed using the SAS statistical software, version 9.0.

Results

Cryoprotective activity of DMSO and glycerol in semen samples

Semen samples infected with Toxoplasma gondii tachyzoites and with DMSO at concentrations 7.5%, 8.0% and 10% had the greatest vigor (score of 2) and motility (20%). For glycerol, the greatest vigor (score of 4) and motility (35.0%) were found with concentrations of 2.5%, 3.0% and 5.0% (Table 1).

Viability and infectivity of T. gondii in cryopreserved semen samples in mice

The following clinical signs were observed in mice: apathy, rough coat and diarrhea from 6th day post-inoculation (DPI) and 100% lethality on average between days 7 and 9. With the exception of the control group (healthy mice seronegative for toxoplasmosis), all mice that died had viable tachyzoites in the peritoneal lavage, with no significant differences among groups (p>0.05) determined by Tukey’s test (Table 2). However, cluster analysis (Scott-Knott test) revealed a significant difference (p≤0.05) in the mean number of tachyzoites quantified in the intraperitoneal lavage among the groups experimentally inoculated with T. gondii tachyzoites (Fig.2). As the experimental mice that received semen with T. gondii tachyzoites died before the 11th DPI, no brain cysts were found.

Table 2.
Mice mortality, average survival time and Toxoplasma gondii tachyzoites (RH strain) quantifications recovered from peritoneal washing after experimental infection with semen containing or not T. gondii tachyzoites and crioprotectors (DMSO and glycerol)

Fig.2.
Mean Toxoplasma gondii tachyzoites recovered and quantified (Scott-Knott test) with different concentrations of cryoprotectant (glycerol and DMSO).

Scott-Knott cluster analysis demonstrated the highest mean tachyzoite counts recovered from intraperitoneal lavage of experimental mice in the 7.5% glycerol, 3.0% glycerol, 7.5% DMSO and 8.0% DMSO groups (p≤0.05), along with suitable seminal vigor and motility patterns for bovine reproduction procedures (Fig.2).

IgM and IgG against T. gondii in mice

All experimental mice (except the control group) had seroconversion with titers of IgM (1:16 to 1:512) and IgG (1:16 to 1:128). Analyzing the results with DMSO, all five concentrations led to positive mice for the serological IgM titers (acute phase). The titers with the highest number of positive mice were 1:32 and 1:64, with percentages of 36.0% (18/50) and 36.0% (18/50), respectively. Other IgM titers had the following percentages 1:16 (12.0%, 6/50), 1:128 (8.0%, 4/50), 1:256 (4.0%, 2/50) and 1:512 (4.0%, 2/50). Glycerol concentrations (2.25%, 2.5%, 3.0%, 5.0%, 7.5% and 10.0%) had the highest rates of infected mice with titers 1:64 and 1:32 (40%, 24/60 and 31.66%, 19/60, respectively). The lowest rates were found for titers of 1:16 (15%, 9/60), 1:128 (3.33%, 2/60) and 1:512 (3.33%, 2/60). The serological titer of 1:256 (0.0%) was negative for indirect immunofluorescence in all 60 mice evaluated with different concentrations of glycerol + tachyzoites. For IgG (chronic phase), the highest percentages were 42% (21/50), 40% (20/50) 12% (6/50) for titers 1:64, 1:32 and 1:16, respectively, with all DMSO concentrations tested. Regarding glycerol, the highest rate was found for the titer of 1:32 (36.66%, 22/60) and antibody titers of 1:16 and 1:64 had the same rates (21.66%, 13/60) for all concentrations evaluated (Table 3).

Table 3.
Quantification of anti- Toxoplasma gondii titre (IgM and IgG) in serum of mice experimentally infected with semen containing T. gondii tachyzoites + crioprotectors (DMSO and glycerol) in different concentrations and control groups

Analyzing statistical significance (p≤0.05) by Fisher’s exact test for the IgM antibody, significant differences were found for DMSO concentrations of 5.0% and 7.5%, glycerol concentrations of 2.25%, 2.5%, 3.0%, 5.0%, 7.5% and 10.0% and samples with the absence of cryoprotectant (p≤0.05) regarding serological titers of 1:32 and 1:64 compared to the titers in the control group. Significant differences (p≤0.05) for the IgG antibody were only found with titers of 1:16 and 1:64 in the samples with 2.5% DMSO and 5.0% DMSO. Significant differences (p≤0.05) for the IgG antibody were only found with the titer of 1:16 in the samples with glycerol at concentrations of 2.25%, 3.0% and 5.0%. Samples with glycerol at concentrations of 2.5% and 10.0% had serological reciprocal titers at 1:16 and 1:64 and a significant difference (p≤0.05) was found for 10% glycerol at the titer of 1:32. In the group cryopreserved in liquid nitrogen (-196°C) without the addition of any cryoprotectant, we obtained results of 50% (5/10) for a titer 1:32 and 50% (5/10) for a titer of 1:64. All mice in the control group had negative serological results.

Discussion

The present work offers unprecedented results regarding the viability and infectivity of Toxoplasma gondii in different concentrations of cryoprotectants (DMSO and glycerol) used in animal reproduction. With most concentrations of glycerol and DMSO as well as samples without the addition of cryoprotectant exposed to a low temperature (-196oC in liquid nitrogen) for 24 hours, the quantification of viable tachyzoites was similar, with average survival of 7.6 days among the experimentally infected mice. The most important factor regards seminal parameters (vigor and motility), as the 2.5%, 3.0% and 5.0% glycerol groups had ideal parameters for bovine reproduction. Moreover, the 7.5%, 8.0%, 10% DMSO and 7.5% glycerol groups had close to appropriate parameters. This information is important when we consider the sexual transmission of toxoplasmosis and the small number of experimental trials in the literature involving the use of reproduction biotechnology (insemination artificial) for mammalian species and protozoan etiological agents, such as T. gondii.

The cryopreservation process causes physical, chemical and metabolic harm to sperm cells (Watson 2000Watson P.F. 2000. The causes of reduced fertility with cryopreserved semen. Anim. Reprod. Sci. 60/61:481-492. <https://dx.doi.org/10.1016/s0378-4320(00)00099-3> <PMid:10844218>
https://doi.org/10.1016/s0378-4320(00)00...
). Adding the effects of all processes, an estimated 50% loss of sperm viability occurs from dilution to thawing (Watson 1995Watson P.F. 1995. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fertil. Develop. 7(4):871-891. <https://dx.doi.org/10.1071/rd9950871> <PMid:8711221>
https://doi.org/10.1071/rd9950871...
). The “Colégio Brasileiro de Reprodução Animal” (Brazilian College of Animal Husbandry) (CBRA 2013CBRA. 2013. Manual para exame andrológico e avaliação de sêmen animal. 3ª ed. Colégio Brasileiro de Reprodução Animal, Belo Horizonte. 104p.) determines sperm motility and vigor of at least 30% and a score of 3, respectively, for thawed bovine semen. In the present study, all thawed samples had lower values for both parameters (5.0% to 20% for motility and a score of 1 to 2 for vigor). Therefore, use in artificial insemination programs is considered inappropriate. Motility (%) and vigor (0-5) after the thawing of cryopreserved semen using DMSO were low with all concentrations of the cryoprotectant evaluated. However, thawed semen protected with glycerol had acceptable motility (4%) and vigor (35%), suggesting a good performance in terms of artificial insemination.

DMSO at concentrations ranging from 1 to 32% is widely used for the cryopreservation of mammalian semen and microorganisms (Sherman 1964Sherman J.K. 1964. Dimethyl sulfoxide as a protective agent during freezing and thawing of human spermatozoa. Exper. Biol. Med. 117(1). <https://dx.doi.org/10.3181/00379727-117-29552> <PMid:14223287>
https://doi.org/10.3181/00379727-117-295...
, Zimmerman et al. 1964, O’Shea & Wales 1969O’Shea T. & Wales R.G. 1969. Further studies of the deep freezing of rabbit spermatozoa in reconstituted skim milk powder. Austr. J. Biol. Sic. 22(3):709-720. <https://dx.doi.org/10.1071/BI9690709> <PMid:5801283>
https://doi.org/10.1071/BI9690709...
, Hubálek 2003Hubálek Z. 2003. Protectants used in the cryopreservation of microorganisms. Cryobiology 46(3):205-229. <https://dx.doi.org/10.1016/S0011-2240(03)00046-4> <PMid:12818211>
https://doi.org/10.1016/S0011-2240(03)00...
). In the specific case of T. gondii, Dumas (1974)Dumas N. 1974. Conservation aux basses temperatures de Toxoplasma gondii (Nicolle et Manceaux, 1909): action du dimeethyl-sulfoxyde. Ann. Parasitol. Hum. Comp. 49(1):1-40. <https://dx.doi.org/10.1051/parasite/1974491001>
https://doi.org/10.1051/parasite/1974491...
found that the best concentration of DMSO for parasitic tachyzoite cryoprotection ranged from 5 to 10%, which is in agreement with the results of the present investigation.

Few studies have been conducted on the use of DMSO as a cryoprotectant for bovine semen. Snedeker & Gaunya (1970)Snedeker W.H. & Gaunya W.S. 1970. Dimethyl sulfoxide as a cryoprotective agent for freezing bovine semen. J. Anim. Sci. 30(6):953-956. <https://dx.doi.org/10.2527/jas1970.306953x> <PMid:5463531>
https://doi.org/10.2527/jas1970.306953x...
studied the cryopreservation of bovine semen with different concentrations of DMSO alone or in combination with glycerol. Evaluating the isolated action of DMSO in homogenized whole milk diluent, the authors found semen motility of 11%, 20%, 24% and 18% after thawing using DMSO at concentrations of 2.0%, 4.0%, 6.0% and 8.0%, respectively. These figures are very similar to those obtained in the present study, as 20% semen motility was found for different concentrations of DMSO (7.0%, 8.0% and 10%). Although DMSO has been used with relative success for several animal species, its use in bovine semen cryopreservation is not as effective as glycerol due to its insufficient cryoprotective activity.

Strains of T. gondii are classified in terms of virulence based on the inoculation of laboratory animals with known doses of the parasite (Willians et al. 1978Willians D.M., Grumet F.C. & Remington J.S. 1978. Genetic control of murine resistance to Toxoplasma gondii. Infect. Immun. 19(2):416-420. <https://dx.doi.org/10.1128/iai.19.2.416-420.1978> <PMid:631881>
https://doi.org/10.1128/iai.19.2.416-420...
). Darde et al. (1988)Darde M.L., Bouteille B. & Pestre-Alexandre M. 1988. Izoenzymic characterization of seven strains of Toxoplasma gondii by electrofocussing in polyacrilamid gels. Am. J. Trop. Med. Hyg. 39(6):551-558. <https://dx.doi.org/10.4269/ajtmh.1988.39.551> <PMid:2462794>
https://doi.org/10.4269/ajtmh.1988.39.55...
found 100% lethality in Swiss mice inoculated with tachyzoites of the RH strain. Regardless of the dose, the longest survival period was found in mice inoculated with 104 tachyzoites (8.67 days on average). Derouin & Garin (1991)Derouin F. & Garin Y.J.F. 1991. Toxoplasma gondii: blood and tissue kinetics during acute and chronic infections in mice. Exp. Parasitol. 73(4):460-468. <https://dx.doi.org/10.1016/0014-4894(91)90070-d> <PMid:1959573>
https://doi.org/10.1016/0014-4894(91)900...
reported average survival of 6.5 days, with death occurring between the 6th and 8th DPI; however, the inoculation route was intraperitoneal. Inoculating 102 tachyzoites (RH strain) subcutaneously, Villard et al. (1995)Villard O., Candolfi E., Despringre J.L., Deroum F., Marcellin L., Viville S. & Kien T. 1995. Protective effectof low doses of an anti-IL-4 monoclonal antibody in a murme model of acute toxoplasmosis. Parasite Immunul. 17(5):233-326. <https://dx.doi.org/10.1111/j.1365-3024.1995.tb01020.x> <PMid:7675509>
https://doi.org/10.1111/j.1365-3024.1995...
reported average survival of 10.83 days, with the death of mice occurring between the 10th and 12th DPI. Regarding the dose of the inoculum, the present study was based on Scarpelli et al. (2009)Scarpelli L., Lopes W.D.Z., Migani M., Bresciani K.D.S. & Costa A.J. 2009. Toxoplasma gondii in experimentally infected Bos taurus and Bos indicus semen and tissues. Pesq. Vet. Bras. 29(1):59-64. <https://dx.doi.org/10.1590/S0100-736X2009000100009>
https://doi.org/10.1590/S0100-736X200900...
, who used a 5 x 106 tachyzoites injected directly into bovines through the subcutaneous route. As we used semen samples in mice (intraperitoneal) with the virulent RH strain and did not know the effects of low temperature (-196oC) on the replication of T. gondii, a dose of 1 x 106 tachyzoites was chosen.

The RH strain is highly pathogenic and is known to cause death in mice during the acute phase (Gravrilesco & Denkers 2001Gravrilesco L.C. & Denkers E.Y. 2001. IFN-gama overproduction and high level apoptosis are associated with high but not low virulence Toxoplasma gondii infection. J. Immunol. 167(2):902-909. <https://dx.doi.org/10.4049/jimmunol.167.2.902> <PMid:11441097>
https://doi.org/10.4049/jimmunol.167.2.9...
, Barragan & Sibley 2002Barragan A. & Sibley L.D. 2002. Transepithelial migration of Toxoplasma gondii is linked to parasite motility and virulence. J. Exp. Med. 195(12):1625-1633. <https://dx.doi.org/10.1084/jem.20020258> <PMid:12070289>
https://doi.org/10.1084/jem.20020258...
). In the present study, average mortality of mice inoculated using the RH strain frozen for 24 hours at -196°C with cryoprotectants (different concentrations of DMSO and glycerol) was 7.6 to 9.2 days and clinical signs were observed beginning with the 6th DPI. All virulent of T. gondii strains behave differently from other genotypes, which have a slow onset and the formation of tissue cysts (Sevá et al. 2006Sevá A.P., Silva R.C., Silva A.V., Castro A.P.B., Menozzi B.D. & Langoni H. 2006. Avaliação da virulência de cepas de Toxoplasma gondii, em camundongos, isoladas de cães com sinais neurológicos, em Botucatu, SP. Vet. Zootec. 13(1):33-43.).

The results of this study indicate that T. gondii maintained its infectivity in all contaminated samples, including in the absence of cryoprotectant. The maintenance of the viability and infectivity of T. gondii tachyzoites was confirmed in the bioassay, as all mice developed acute toxoplasmosis, which was diagnosed based on clinical signs. The virulence of the strain was confirmed by its lethality, with the death of 100% of the animals within 10 days after inoculation. It was not possible to calculate the infective dose effectively administered due to the loss of viable tachyzoites during the experimental procedures, such as semen dilution and thermal stress due to cooling, freezing and thawing.

All tachyzoite-inoculated mice exhibited seroconversion for IgM and IgG. However, different results have been reported in other studies (Camargo et al. 1991Camargo M.E., Silva S.M., Leser P.G. & Granato C.H. 1991. Avidez de anticorpos IgG específicos como marcadores de infecção primária recente pelo Toxoplasma gondii. Revta Inst. Med. Trop. 33(3):213-218. <https://dx.doi.org/10.1590/S0036-46651991000300008>
https://doi.org/10.1590/S0036-4665199100...
, Camargo 1996Camargo M.E. 1996. Toxoplasmosis, p.165-174. In: Ferreira A.W. & Moraes S.L. (Eds), Diagnóstico Laboratorial das Principais Doenças Infecciosas e Auto-imunes. Guanabara Koogan, Rio de Janeiro., Cunha et al. 2004Cunha E.L.P., Silva R.C., Da Silva A.V. & Langoni H. 2004. The protective effects of Baypamun® HK in mice experimentally infected with Toxoplasma gondii. Braz. J. Vet. Res. Anim. Sci. 41(3):169-172. <https://dx.doi.org/10.1590/S1413-95962004000300004>
https://doi.org/10.1590/S1413-9596200400...
, Guimarães et al. 2009Guimarães L.R., Barbaresco A.A. & Castro A.M. 2009. Perfil sorológico de camundongos cronicamente infectados com a cepa cistogênica ME-49 de Toxoplasma gondii antes e após terapêutica específica. Revta Patol. Trop. 38(4):261-266. <https://dx.doi.org/10.5216/rpt.v38i4.8589>
https://doi.org/10.5216/rpt.v38i4.8589...
, Costa-Silva & Pereira-Chioccola 2010Costa-Silva T.A. & Pereira-Chioccola V.L. 2010. Fase aguda da infecção por Toxoplasma gondii: avaliação do parasitismo sanguíneo e resposta humoral em camundongos isogênicos AS/n. Scientia Medica 20(1):88-92.). This may have occurred because the infective dose of tachyzoites inoculated in each animal was higher in the present investigation and different strains were used in other studies. Other differences regarding the virulence of strains likely occur due to successive passages of the parasite in the same animal species, such that the characteristic of virulence may be altered (Jacobs & Melton 1954Jacobs L. & Melton M.L. 1954. Modification in virulence of a strain of Toxoplasma gondii by passage in various host. Am. J. Trop. Med. Hyg. 3(3):447-457. <https://dx.doi.org/10.4269/ajtmh.1954.3.447> <PMid:13158764>
https://doi.org/10.4269/ajtmh.1954.3.447...
). Based on these previous studies, the immunological response with production of anti-T. gondii antibodies (IgG) suggests a possible attenuation of tachyzoites during the cryopreservation process, which enabled greater antigenic stimulation in the infected animals.

Using semen cryopreserved with a commercial formulation containing 3% glycerol and contaminated with 107 tachyzoites of T. gondii frozen in liquid nitrogen and thawed, Consalter et al. (2017)Consalter A., Silva A.F., Frazao-Teixeira E., Matos L.F., Oliveira F.C.R., Leite J.S., Silva F.B.F. & Ferreira A.M.R. 2017. Toxoplasma gondii transmission by artificial in sheep with experimentally contaminated frozen semen. Theriogenology 90:169-174. <https://dx.doi.org/10.1016/j.theriogenology.2016.12.004> <PMid:28166964>
https://doi.org/10.1016/j.theriogenology...
found vigor and motility of 4% and 35%, respectively. To confirm viability, mice were inoculated with tachyzoites of T. gondii (1 x 106) + sperm (20 x 106) by the intraperitoneal route, all of which became ill and died within two weeks. Despite the difference between cattle and sheep semen, the same concentration of glycerol (3%) led to the same vigor and motility results in the present study after storage in liquid nitrogen and a similar period of sickness and death of the mice. Thus, this is likely a standard for the RH strain of T. gondii.

The sexual transmission of toxoplasmosis has been demonstrated in some species, such as sheep (Moraes et al. 2010Moraes E.P.B.X., Batista A.M., Faria E.B., Freire R.L., Freitas A.C., Silva M.A.R., Braga V.A. & Mota R.A. 2010. Experimental infection by Toxoplasma gondii using contaminated semen containing different doses of tachyzoites in sheep. Vet. Parasitol. 170(3/4):318-322. <https://dx.doi.org/10.1016/j.vetpar.2010.02.017> <PMid:20227187>
https://doi.org/10.1016/j.vetpar.2010.02...
, Lopes et al. 2013Lopes W.D.Z., Rodriguez J.D., Souza F.A., Santos T.R., Santos R.S., Rosanese W.M., Lopes W.R.Z., Sakamoto C.A. & Costa A.J. 2013. Sexual transmission of Toxoplasma gondii in sheep. Vet. Parasitol. 195(1/2):47-56. <https://dx.doi.org/10.1016/j.vetpar.2012.12.056> <PMid:23384578>
https://doi.org/10.1016/j.vetpar.2012.12...
), goats (Santana et al. 2013Santana L.F., Rossi G.A.M., Gaspar R.C., Pinto V.M.R., Oliveira G.P. & Costa A.J. 2013. Evidence of sexual transmission of Toxoplasma gondii in goats. Small Rumin. Res. 115(1/3):130-133. <https://dx.doi.org/10.1016/j.smallrumres.2013.08.008>
https://doi.org/10.1016/j.smallrumres.20...
) and rabbits (Liu et al. 2006Liu S.-G., Qin C., Yao Z.-J. & Wang D. 2006. Study on the transmission of Toxoplasma gondii by semen in rabbits. Chinese J. Parasitol. Parasit. Dis. 24(3):166-170. <PMid:17094613>). However, the maintenance of T. gondii tachyzoite viability in cryopreserved bull semen samples confirmed in the present study does not prove the infectivity of the parasite for bovine females through the vaginal route. Thus, future studies should be performed to evaluate whether tachyzoite-infected cryopreserved semen plays a role in T. gondii infection in cattle through the vaginal route.

Conclusion

Toxoplasma gondii tachyzoites remained viable and infectious in cryopreserved bovine semen containing DMSO and glycerol at all concentrations evaluated. Mice (Swiss strain) inoculated with cryopreserved semen contaminated with T. gondii tachyzoites exhibited clinical symptoms and seroconversion with varying titers of IgM and IgG.

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Publication Dates

  • Publication in this collection
    16 Jan 2023
  • Date of issue
    2023

History

  • Received
    27 June 2022
  • Accepted
    20 July 2022
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