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

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.


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 1910, Quinn & McCraw 1972, Dubey & Beattie 1988, Bresciani et al. 1999, Tenter et al. 2000, Sawadogo et al. 2005, Fialho et al. 2009, Dubey et al. 2013).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. 2009).
Artificial insemination in beef and dairy cattle using cryopreserved semen is a common management strategy to improve herd genetics and consequently enhance productivity (Boni 2012).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 2003, Castelo et al. 2008, Rodgers et al. 2012, Baruselli et al. 2017).
Cryoprotectants used for sperm preservation include glycerol, ethylene glycol, dimethyl sulfoxide (DMSO), acetamide, lactamide and dimethylacetamide (Fickel et al. 2007, Kashiwazaki et al. 2006).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. 1977).DMSO at concentrations of 4 and 6% constitutes an alternative to glycerol (Snedeker & Gaunya 1970).
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 1941) 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 10 6 tachyzoites) of previously infected mice on the second day after infection (Mineo et al. 1980).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. 2011).Lavages with a smaller number of somatic cells (red blood cells, leukocytes, desquamated epithelial cells) and higher quantity of tachyzoites (1 x 10 6 ) were chosen for semen infection (adapted from Camargo 1964).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 10 6 ) 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. 1998).
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 1964) and Neospora caninum (Conrad et al. 1993, Dubey & Lindsay 1996, Soares et al. 2009) 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 1964) and positive for N. caninum with titers equal to or greater than 1:50 (Figliuolo et al. 2004).The animals were also tested for antibodies against Brucella abortus using the Card Test (Alton et al. 1988) and antibodies against Leptospira spp.(serovar wolffi, hardjo, patoc, pomona, icterohaemorrhagiae and canicola) using the microscopic agglutination test (Centro Panamericano de Zoonoses 1985).
To determine the presence of T. gondii DNA, semen was collected from the 23 bulls through electroejaculation (Mascarenhas & Gomes 1950).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. 2000).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).
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 2013).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 2013).
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 10 6 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 10 6 of viable tachyzoites) was added to each aliquot of semen.
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 1997).
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) 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).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).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 antimouse 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 1964).
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.

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.
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).
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 (-196 o C 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 2000).Adding the effects of all processes, an estimated 50% loss of sperm viability occurs from dilution to thawing (Watson 1995).The "Colégio Brasileiro de Reprodução Animal" (Brazilian College of Animal Husbandry) (CBRA 2013) 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 1964, Zimmerman et al. 1964, O'Shea & Wales 1969, Hubálek 2003).In the specific case of T. gondii, Dumas (1974) 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) 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. 1978).Darde et al. (1988) 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 10 4 tachyzoites (8.67 days on average).Derouin & Garin (1991) reported average survival of 6.5 days, with death occurring between the 6th and 8th DPI; however, the inoculation route was intraperitoneal.Inoculating 10 2 tachyzoites (RH strain) subcutaneously, Villard et al. (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), who used a 5 x 10 6 tachyzoites injected directly into bovines through the subcutaneous route.As we used semen samples in mice A,B,C Values followed by the same letter on the same row do not differ significantly at Fisher´s Exact test (p≥0.05).
(intraperitoneal) with the virulent RH strain and did not know the effects of low temperature (-196 o C) on the replication of T. gondii, a dose of 1 x 10 6 tachyzoites was chosen.The RH strain is highly pathogenic and is known to cause death in mice during the acute phase (Gravrilesco & Denkers 2001, Barragan & Sibley 2002).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. 2006).
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. 1991, Camargo 1996, Cunha et al. 2004, Guimarães et al. 2009, Costa-Silva & Pereira-Chioccola 2010).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 1954).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 10 7 tachyzoites of T. gondii frozen in liquid nitrogen and thawed, Consalter et al. (2017) found vigor and motility of 4% and 35%, respectively.To confirm viability, mice were inoculated with tachyzoites of T. gondii (1 x 10 6 ) + sperm (20 x 10 6 ) 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. 2010, Lopes et al. 2013), goats (Santana et al. 2013) and rabbits (Liu et al. 2006).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.

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).

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)
* Control groups: euthanazed animals after the 42nd experimental day; A,B Mean values followed by the same letter on the same line do not differ significantly at a Kruskal-Wallis test (p≥0.05);PID = post-infection day.