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Microbiological evaluation of fresh semen from captivity kept collared peccary ( Pecari tajacu ) at Brazilian Amazon

This study aimed to identify the existent of microbiota in the fresh semen of collared peccary (Pecari tajacu), kept in captivity, and their susceptibility patterns to antimicrobial drugs. Ten males of this species were submitted into experimental induced ejaculation procedures throughout 14 months. The microorganisms found in the semen were evaluated within the following criteria: morphology, biochemistry, colony forming unit (CFU mL), and antibiotic sensitivity. 225 Gram-positive strains were isolated, out of 88 samples. They were identified as following: Streptococcus (30.2%), Staphylococcus (30.2%), Micrococcus (33.7%), Corynebacterium (2.2%), Enterococcus (1.7%), and Bacillus (1.7%). The cell counting of contaminated semen material was under 300 CFU mL. The evaluation of sensitivity against eleven antibiotics revealed that most of the bacteria found were sensitive to Gentamicin, Cephalothin, Amikacin, Ampicillin, Ceftriaxone, Cephotaxin and Penicillin. During the evaluation period, the presence of microorganisms in the semen here analyzed did not influence the quality of the semen production of the donor animals. Semen contamination is probably coming from the surrounding environment in which the animals were kept, during collection procedures. This study suggests that Gentamicin and Amikacin are better choices over Tetracycline as diluters of collared peccary’s semen.


INTRODUCTION
Collared peccary (Pecari tajacu) is a species of great environmental diversity and adaptation.
In captivity, it represents a highly potential in terms of productivity, when compared to other Amazon mammals (MAYOR et al., 2007;SILVA et al., 2016).
A wide range of bacteria commonly leads to reproductive diseases. Kuster and Althouse (2016) report that the reproductive performance of boar through artificial insemination is threatened by bacteriospermia. They describe a significant list of bacteria registered in semen that come from fecal material, preputial, skin and hair. During the semen collection to be used at in vitro fertilization procedures, it is quite common the occurrence of bacterial contamination coming from either the environment or from the donor animal's body (RIESENBECK et al., 2015). Such contamination is extremely hard to avoid. Therefore, it is crucial the addition of wide range of antibiotics to the semen's diluent (YÁNIZ et al., 2010).  (OLIVEIRA et al., 2007) and humans (WENG et al., 2014). However, there is no report found in the literature regarding the presence of such microorganisms in the semen of collared peccary.
It is suggested that the presence of microorganisms in the semen of collared peccary may interfere on its quality when used on in vitro fertilization procedures. As data regarding this topic is absent, studies focused in this topic are in high demand. The goals of this study were to identify the existent micro flora in fresh semen of collared peccary kept in captivity, as well as to evaluate their sensitivity towards the action of antibiotics.

MATERIAL AND METHODS
This study has followed all the rules established by the National Council for Animal Experiments of Brazil (CONCEA) and approved by Animal Ethics Committee (CEUA) of the Universidade Federal do Pará, registration nº 184/13 CEPAE-UFPA.
10 individuals, males, with average age of 76.8 (±37.8) months and weighing on average 19.8 (±2.7) kg, kept in the scientific captivity area of Embrapa Amazônia Oriental (Belém-PA, Brazil; 01º24'S; 48º20'W) were used. The animals were kept in 36 m 2 collective cubes, grouped by family related individuals of up to 10 animals per cube. The male:female ratios in the groups were 1:2 or 1:3, under natural conditions of temperature, humidity and full darkness-lightness cycle. All the animals received food supplementation composed by pig food based on corn and soy (500 g animal -1 ), diverse species of grass, fruit and water ad libitum. At 24 hours before collecting samples, the animals were isolated and submitted into feast from both food and liquids.
The animal's sedation was done in accordance to the protocol established by Souza et al. (2009) and Kahwage et al. (2010). After sedation, cleaning procedures such as hair removal, prepuce washing, feces removal, and rectus cleaning took place. The external and internal prepuce areas were cleaned up with pre-warmed NaCl 0.9 % solution (35 °C).
The semen collection was done in intervals of 15 days, by rectal electro stimulation, as described by Souza et al. (2009). Aliquots of the collected semen were used for seminal analysis, as well as for microbiological tests.
Samples were first evaluated for the following sperm analysis parameters: general appearance, color, total volume, concentration, pH, sperm's motility and vitality; morphology, viability and defected sperm cells.
For the microbiological assays, sample aliquots were inoculated in duplicates, in Petri dishes containing enriched bacteriological medium agar with 5% sheep blood defibrinated and MacConkey Agar. Petri dishes were then incubated at 37 °C, during 24 to 48 h. After incubation, the colonies were evaluated through their morphology per stain characteristics and biochemical tests were performed for their identification (QUINN et al., 1994).
The CFU counting was performed in accordance to the protocol preconized by World Organization for Animal Health (OIE, 2008) for cattle, with some few modifications.

DISCUSSION
The semen bacterial biodiversity in all animals were similar to found in other studies analyzing semen of several animal groups, such as cattle (HERNÁNDEZ et al., 2002;PRADO;PEREZ, 2005), sheep (SOUZA et al., 2006) pigs (SONE, 1990;CORRÊA et al., 2001), fishes (OLIVEIRA et al., 2007), boar (PRIETO-MARTINEZ et al. 2014), and human (VILVANATHAN et al., 2016 Prado and Perez (2005), in cattle's semen, in which less diluted semen leads to lower CFU mL -1 concentration. However, it corroborates with the results found by Hernández et al. (2002) in bovine's thawed semen. The 1 in 100,000 dilution did not alter the sperms motility. This observation was also reported by Bennermann et al. (2000), as well as in Prado and Pérez (2005) study. However, Pineda and Santander (2007) stated that at 1 in 10,000 dilution, changes in the quality of the semen of pig could happen.
In regard of antimicrobial agents tested in this work, the sensitivity against Gentamicin, Amikacin and Cephotaxin confirmed the results found by Souza et al. (2006) in semen of sheep, and by Hernández et al. (2002) in cattle's semen. Nevertheless, in regard of the microorganisms isolated from semen of pig, the study of Gączarzewicz et al. (2016) presented high inhibitory activity against some antimicrobial agents as Gentamicin. In addition, Tetracycline, a very commonly antibiotic used as semen's dilutor, presented the lowest numbers of sensitivity on antibiotic susceptibility results.
The presence of such microorganisms in the semen has possibly no association or no relation to the efficiency of the male's reproductive system of the donor animal, once these microorganisms have been found in semen of healthy animals (KUSTER; ALTHOUSE, 2016). Therefore, it was observed that the presence of the six microorganisms did not interfere on the quality of semen analyzed. This fact also corroborates with studies of Sannat et al. (2015), and Kuster and Althouse (2016).
According to Riesenbeck et al. (2015), the semen contamination can be related to the fact the ejaculated material gets direct contact with the animal's prepuce secretions and hair. In addition, accidental handling by the technician, indirect contact to aerosol, sampling instruments, as well as the process of semen dilution and storage contribute to semen contamination. So, the presence of saprophytic bacteria in collared peccary semen might be occurring due to contamination during sampling and/or from the external environment in which the animals are raised. Yániz et al. (2010) also found microbiological contamination in studies with boar semen that are from other environment.

CONCLUSION
The presence of microorganisms in the semen of collared peccary during the entire time of this evaluation does not influence on the seminal quality of the animals. Moreover, it is very likely that the ejaculated material have been contaminated by external microbiota from the surrounding environment. Finally, it is recommended to be reevaluated the use of Tetracycline as a common dilutor in the semen. Additionally, it would be very valuable the see more studies to confirm the use of Gentamicin and/or Amikacin as ideal antibiotics to be used as dilutors in the semen of collared peccaries.