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Control Methods and Evaluation of Bacterial Growth on Fresh and Cooled Stallion Semen
Journal of Equine Veterinary Science xx (2015) 1–6
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Original Research
Control Methods and Evaluation of Bacterial Growth on Fresh and Cooled Stallion Semen Carlos Ramires Neto, Yame Fabres Robaina Sancler da Silva, Helene Lacerda Resende, Priscilla Nascimento Guasti, Gabriel Augusto Monteiro, Patrícia Melo Papa, José Antônio Dell’aqua Júnior, José Nicolau Próspero Puoli Filho, Marco Antônio Alvarenga*, Frederico Ozanam Papa Department of Animal Reproduction, College of Veterinary Medicine and Animal Science, Univ Estadual Paul (UNESP), Botucatu, São Paulo, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 July 2013 Received in revised form 10 July 2014 Accepted 6 January 2015 Available online xxxx
The penis and prepuce of the stallion have a high bacterial load on its surface, forming a natural microbial flora that contaminates the semen during ejaculation. Bacterial growth in semen may cause a decline on sperm quality, viability, and fertility and predisposes the occurrence of endometritis in inseminated mares. Thus, the aim of this study was to evaluate the effect of penile wash before semen collection, the addition of different commercial skim milk–based extenders containing antibiotics (BotuSemen and INRA96), and the removal of seminal plasma by filtration on the quality, viability, and bacterial proliferation on fresh and cooled stallion semen. Animals that were never submitted to penile wash before semen collection tended to have lower bacterial contamination in the ejaculate. Semen samples extended in BotuSemen showed superiority in total motility, progressive motility, average path velocity, and rapid sperm and lower bacterial contamination in relation to semen samples extended in INRA96 after 24 hours of cooling. No difference was found in these parameters between the storage temperatures (5 C and 15 C). Furthermore, the removal of seminal plasma by filtration reduced the bacterial load in semen after cooling. In conclusion, the penile wash before semen collection tended to reduce the bacterial growth in fresh semen. The use of a semen extender with appropriate antibiotics and removal of seminal plasma by filtration were effective in reducing the bacterial contamination and preserved the quality of cooled stallion semen. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Semen Stallion Bacteria Filtration Penile wash
1. Introduction The penis and prepuce have a permanent natural microbial flora with a high amount of bacteria [1] that can be transferred to the semen after collection [2]. Therefore, the equine ejaculate naturally exhibits a large bacterial contamination and is mainly composed of nonpathogenic bacteria [3]. * Corresponding author at: Marco Antônio Alvarenga, Department of Animal Reproduction, College of Veterinary Medicine and Animal Science, Univ Estadual Paul (UNESP), 18610-970 Botucatu, São Paulo, Brazil. E-mail address: [email protected] (M.A. Alvarenga). 0737-0806/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2015.01.014
An imbalance in the microbial environment of the external genitalia can result in the proliferation of opportunistic microorganisms [4], such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Taylorella equigenitalis, and Streptococcus b-hemolytic. These microorganisms may cause endometritis in inseminated mares [1], decrease in fertility rates [5], and affect the semen quality [6]. Methods to reduce bacterial contamination and proliferation in equine semen are largely described, such as washing the stallion penis before semen collection [7], the use of an open-ended artificial vagina [4], the use of semen extenders containing antibiotics [1,4], and the reduction of semen storage temperature [4].
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Table 1 Mean values and standard deviations of total motility (TM), progressive motility (PM), velocity of trajectory (VAP), curvilinear velocity (VCL), rapid sperm (RAP) before (M0) and after (M24) cooling the semen of animals with (W) and without (NW) to wash penis before collection. Groups
TM (%)
W (M0) NW (M0) W (M24) NW (M24)
85.2 75.0 62.8 52.6
VAP (mm/s)
PM (%) 6.9 14.5 15.2 28.3
39.8 42.0 29.4 20.4
5.3 15.1 15.7 12.4
123.3 116.4 102.8 104.4
VCL (mm/s)
18.9 18.6 21.3 22.6
216.2 201.2 194.7 193.8
21.6 22.5 28.7 37.3
RAP (%) 74.8 65.6 51.2 44.6
9.7 17.6 22.2 28.4
Different letters indicate statistical difference between the groups at the same column (P < .05). Same letters or absence of letters indicate no statistical difference.
diacetate and propidium iodide [8], and 100 sperm cells were counted.
To achieve new methods of control of bacterial growth in equine semen, the aims of this study were (1) to determine the effect of washing the stallion penis before collection and (2) to compare different semen extenders and the seminal plasma removal using filtration on quality, viability, and bacterial growth on fresh and cooled stallion semen.
2.4. Bacteria Counting and Identification Isolation of bacterial colonies was based on macroscopic and microscopic characterization (Gram stain method) and biochemical tests [9]. For the count of colony-forming units (CFUs), 0.1 mL of seminal sample was diluted in 9.9 mL of saline. Then, 0.1-mL aliquot of this dilution was plated in the following culture mediums: blood agar base with 7% sterile defibrinated sheep blood, MacConkey agar base, and Sabouraud agar base. The seeded assay plates were incubated 37 C and analyzed for 24, 48, 72, and 96 hours. The total of counted colonies was multiplied by 103 CFUs in accordance with the dilution used in this study. The identification and the CFU count were performed on fresh semen in experiments 1 and 3 and on cooled semen in experiments 2 and 3.
2. Materials and Methods 2.1. Semen Collection Semen collection was performed using an artificial vagina. Initially, three ejaculates from each stallion were collected with an interval of 2 days to eliminate possible damaged cells and stabilize the sperm parameters. 2.2. Semen Cooling and Storage After collection and processing, semen samples were stored in a container of passive cooling (BotuFlex; Botupharma, Sao Paulo, Brazil) at 15 C for 24 hours, for experiments 1, 2 and 3. For experiment 2, samples were also stored at 5 C for 24 hours.
2.5. Experiments 2.5.1. Experiment 1dEffect of Penile Washing on Contamination and Quality of Stallion Semen An ejaculate from each of 10 healthy stallions of different breeds (Mangalarga Marchador and Quarter Horse) aged 7–15 years old was used. All stallions were used in breeding programs that the semen collection was performed by artificial vagina. The animals were divided into two groups: stallions that were never submitted to penile washing before semen collection, composed by three Mangalarga Marchador and two Quarter Horse stallions (group NW; n ¼ 5; 10 3.2 years old), and stallions that are routinely (three times per week) submitted to penile washing with running water at 30 C before semen collection, composed by three Mangalarga Marchador and two Quarter Horse stallions (group W; n ¼ 5; 10.4 2.7 years old). After semen collection, the ejaculate was filtered to remove the gel fraction and diluted in a skim milk–based extender (Botusemen; Botupharma) to a sperm
2.3. Semen Analysis The kinetic sperm parameters were evaluated immediately after semen collection (0 hours) and 24 hours after semen cooling. The samples were previously placed in a dry water bath at 37 C for 5 minutes and evaluated by computer-assisted sperm analysis (HTM-IVOS 12; Hamilton Thorne Research, MA). Three different fields in Makler Counting Chamber (Sefi Medical Instruments Ltd, Israel) per sample were considered for total motility (%), progressive motility (PM, %), average path velocity (mm/s), curvilinear velocity (mm/s), and rapid sperm (%). Plasma membrane integrity (PMI, %) analysis was assessed through an epifluorescence microscopy (400 magnification) using the fluorescent probes 6-carboxyfluorescein
Table 2 Identification of the bacteria in the culture of cooled stallion’s semen with and without to wash penis before collection. Stallions with wash penis before collection
Stallions without wash penis before collection
1 2 3 4 5
6 7 8 9 10
Staphylococcus spp. Streptococcus spp./Staphylococcus spp.
Klebsiella oxytoca/Escherichia coli Streptococcus b-hemolytic/Staphylococcus b-hemolytic Bacillus spp. Streptococcus a-hemolytic Staphylococcus spp.
C. Ramires Neto et al. / Journal of Equine Veterinary Science xx (2015) 1–6
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Table 3 Mean values and standard deviations of total motility (TM), progressive motility (PM), velocity of trajectory (VAP), curvilinear velocity (VCL), rapid sperm (RAP), plasma membrane integrity (PMI), and bacteria colony-forming unit (CFU) of stallion’s semen diluted with commercial extender Botusemen (BS) and INRA96 (INRA) before (M0) cooling. Groups
TM (%)
PM (%)
VAP (mm/s)
VCL (mm/s)
RAP (%)
PMI (%)
CFU
BS (M0) INRA (M0)
67.4 14.3a 73.1 12.4a
33.1 7.2a 32.6 8.9a
141.3 18.9a 143.7 18.4a
249.0 35.3a 251.7 37.3a
67.5 11.7a 67.4 14.3a
65.2 7.6a 66.6 9.7a
56.2 7225a 6,538.6 5,331.4b
Different superscript letters indicate difference between the groups.
Table 4 Mean values and standard deviations of total motility (TM), progressive motility (PM), velocity of trajectory (VAP), and rapid sperm (RAP) of stallion’s semen diluted with commercial extender Botusemen (BS) and INRA96 (INRA) after (M24) cooling at 5 C and 15 C. Groups
TM (%)
5 C BS INRA
VAP (mm/s)
PM (%)
15 C Aa
65.4 16.8 54.3 19.0Ba
5 C Aa
69.0 15.0 60.7 17.0Ba
15 C Aa
37.0 14.4 26.3 12.0Ba
RAP (%)
5 C Aa
32.7 13.8 23.8 8.9Bb
5 C
15 C Aa
128.1 10.4 116.7 15.4
Ba
134.7 15.2 130.7 16.5
Aa Ba
15 C Aa
59.5 17.6 47.9 19.4
Ba
64.3 15.7Aa 54.1 17.8 Ba
Different superscript uppercase letters indicate statistical difference between columns, and different superscript lowercase letters indicate difference between the lines for the same parameter.
concentration of 40 106 cells/mL and then submitted to cooling at 15 C. 2.5.2. Experiment 2dEffect of Semen Extender on Sperm Kinetic Parameters and Bacterial Growth of Cooled Stallion Semen Two ejaculates were collected from each of 15 healthy stallions of different breeds (Mangalarga Marchador, Quarter Horse, and Thoroughbred), aged 4–6 year old. The semen was collected without penile washing. The gel fraction was removed and a total of 4 109 sperm cells were equally divided between two groups. In BS group, semen samples were added to a commercial skim milk– based extender (BotuSemen; Botupharma) containing gentamicin sulfate (1 mg/mL) and penicillin (1 mg/mL), as indicated by the manufacturer, to a sperm concentration of 40 106 cells/mL; and in INRA group, semen samples were added to a commercial skim milk–based extender (INRA96; IMV, MN) containing penicillin (0.038 mg/mL), gentamicin (0.105 mg/mL), and amphotericin B (0.315 mg/mL) [10,11] to a sperm concentration of 40 106 cells/mL. Samples from the two groups were submitted to cooling at 5 C and 15 C in a cooling passive container (BotuFlex; Botupharma). 2.5.3. Experiment 3dEffect of Seminal Plasma Removal on Sperm Viability and Bacterial Growth of Fresh and Cooled Stallion Semen One ejaculate from each of 10 healthy stallions of different breeds (Mangalarga Marchador, Quarter Horse, and Thoroughbred) aged 7–15 years old was collected. The
gel fraction was removed, and the semen was diluted at a 1:1 ratio with a skim milk–based extender (BotuSemen; Botupharma). Semen samples were divided into two groups according to the presence or not of seminal plasma. In the seminal plasma group (SP), semen samples were extended to a sperm concentration of 40 106 cells/mL. In the filtered group (FL, without seminal plasma), semen was filtered through synthetic hydrophilic membrane (SpermFilter; Botupharma) to remove seminal plasma [13]. Then, sperm were resuspended in semen extender to a sperm concentration of 40 106 cells/mL. Semen samples from the two groups were submitted to cooling at 15 C in a cooling passive container (BotuFlex; Botupharma). 2.6. Statistics Statistical analyses were performed using InStat GraphPad 3 software (GraphPad Software, CA). In experiments 1 and 3, data were statistically analyzed using the Student t test. For the experiment 2, a two-way analysis of variance and Tukey test were carried out to compare the groups and the moments. The significance level was set at P < .05. 3. Results 3.1. Experiment 1 No difference was observed (P > .05) in sperm kinetics parameters between the NW group and W group before
Table 5 Mean values and standard deviations of plasma membrane integrity (PMI) and bacteria colony-forming unit (CFU) of stallion’s semen diluted with commercial extender Botusemen (BS) and INRA96 (INRA) after (M24) cooling at 5 C and 15 C. Groups
BS INRA
PMI (%)
CFU
5 C
15 C
5 C
15 C
58.4 7.8Aa 58.1 9.5Ba
61.0 11.6Aa 61.5 10.2Ba
280.6 738.7Aa 3,352.2 4,315.1Ba
122 464.2Aa 3,593.7 4,088.1Bb
Different superscript uppercase letters indicate statistical difference between columns, and different superscript lowercase letters indicate difference between the lines for the same parameter.
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Table 6 Identification of the bacteria in the culture of stallion’s semen diluted with commercial extender with Botusemen (BS) and INRA96 (INRA) before (M0) and after (M24) cooling at 5 C and 15 C. 24 hr/5 C
0 hr
BS
24 hr/15 C
Groups
FS
SP
FL
BS
CFU
236,926 390,888.7a
33 104.4b
3 9.4b
BS
INRA
Stallion/Bacteria
Species Species Species Species
1-Coryne. 2-Coryne. 3-Coryne. 4-Coryne. 5-Rhodo. 6-Coryne. 7-E. coli/Coryne. 8-Coryne. 9-Coryne. 10-Pseudo. 11-Staphyl. 12-Coryne. 13Coryne./Rhodo. 14-Coryne. 15-E. coli/Coryne.
d d d d Rhodo. d d d d d d d d d d
Coryne. Coryne. Coryne. Coryne. Rhodo. Coryne. Coryne. d Coryne. Pseudo. d d Rhodo. Coryne. Coryne.
INRA
d d d d Rhodo. d d d d d d d Rhodo. d Coryne.
Table 8 Average values of bacteria colony-forming unit (CFU) of fresh semen in M0 (FS) and semen with (SP) and without (FL) seminal plasma after 24 hours of cooling.
INRA
0 Coryne. Coryne. Coryne. Rhodo. Coryne. Coryne. d Coryne. Pseudo./Bacillus d d Rhodo. d Enterob.
Species
Different superscript letters in the lines indicate significant differences (P < .05).
d Coryne. d d Rhodo. d d d d d d d d d Coryne.
without seminal plasma (FL; P < .05). The percentage of sperm with intact plasma membrane (PMI) was higher in FL group after cooling 15 C for 24 hours (P < .05; Table 7). The count of CFUs was greater in fresh semen in relation to semen samples with or without seminal plasma after cooling at 15 C for 24 hours (P < .05; Table 8). Different bacterial species were isolated from fresh and cooled semen with or without SP (Table 9). 4. Discussion
Abbreviations: Bacillus, Bacillus spp.; Coryne., Corynebacteruim spp.; E. coli, Escherichia coli; Enterob., Enterobacter cloacae; Pseudo., Pseudomonas aeruginosa; Rhodo., Rhodococcus equi; Staphyl., Staphylococcus spp.
The surface of the stallion penis and prepuce is colonized by a high variety of commensal bacteria [11,12,14,15] that contaminate the ejaculate and consequently the mare reproductive tract [11,12]. The penile wash with running water before semen collection promotes the mechanical removal of debris and reduces bacterial contamination in semen [14,17]. This fact may justify the results in this study, in which the count of CFUs in the ejaculate tended to be superior in stallions that were never submitted to penile wash in relation to stallions that were routinely submitted to penile washing with water before semen collections (experiment 1). These microorganisms found in penis and prepuce may cause endometritis in inseminated mares [1], decrease fertility rates [5], and affect the semen quality [6]. Althouse et al [18] described that bacterial contamination of extended porcine semen has been associated with deleterious effects on semen quality. The authors reported that all isolated bacteria were resistant to gentamicin, the most common antibiotic agent in commercial porcine semen extenders. The bacteria found on penile microbial flora in our study were similar with that described by Corona et al [19]. These bacteria constitute the normal microflora of the stallion’s external genitalia and rarely produce genital infections [16]. In cooled–stored semen, Staphylococcus spp. is one of the most common organisms regarding its survival characteristics [20], as seen in our results. In the present study, no differences between the groups NW and W were observed regarding the parameters of
and after semen cooling (Table 1). However, CFU counts tended to be superior (P ¼ .089) in NW group compared with W group (CFUs/mL, 272,636 375,764a vs. 1,216 2,674b, respectively; Different superscript letters indicate difference between the groups). The bacterial identification of both groups is described in Table 2. 3.2. Experiment 2 Before cooling, no difference was found (P > .05) between semen extenders in the sperm kinetic parameters and PMI. However, the CFU count (P < .05) was higher for the INRA group compared with BS group (Table 3). After 24 hours of cooling, there was difference (P < .05) in all sperm motility parameters and CFUs between BS group and INRA group, however, no difference was found on PMI. When the different cooling temperatures were analyzed, no difference (P > .05) was found in the BS group in the same evaluated parameters, and in the INRA group, PM showed lower values at 15 C than 5 C and greater CFU count than BS group (P < .05; Tables 4 and 5). The bacterial isolation in each group is described in Table 6. 3.3. Experiment 3 Before cooling, PM was greater in semen samples with seminal plasma (SP) compared with semen samples
Table 7 Mean values and standard deviations of total motility (TM), progressive motility (PM), path velocity (VAP), progressive linear velocity (VSL), curvilinear velocity (VCL), rapid spermatozoa (RAP), and plasma membrane integrity (PMI) of semen with (SP) and without (FL) seminal plasma, before (M0) and after (M24) refrigeration. Groups
TM (%)
SP (M0) FL (M0) SP (M24) FL (M24)
79.6 73.9 56.5 60.9
VAP (mm/s)
PM (%) 11.7 15.4 22.4 15.8
39.2 30.3 23.4 24.8
11.2a 11.3b 14.3 10.3
123.4 117.5 103.5 105.6
17.6 23.1 20.8 38.5
VCL (mm/s) 214.3 214.2 193.6 186.4
25.0 38.0 31.7 29.9
Different superscript letters in the columns indicate significant differences at the same moment (P < .05).
RAP (%) 70.3 62.0 47.0 43.3
14.3 22.4 24.8 23.8
PMI (%) 62.3 66.0 31.3 42.8
13.8 15.9 12.7b 21.2a
C. Ramires Neto et al. / Journal of Equine Veterinary Science xx (2015) 1–6
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Table 9 Bacteria identified in microbial cultivation of fresh semen in M0 (FS) and semen with (SP) and without (FL) seminal plasma after 24 hours of cooling. Stallion
FS
1 2 3 4 5 6 7 8 9 10
Staphylococcus spp.
Klebsiella oxytoca/Escherichia coli Streptococcus b-hemolytic Streptococcus spp./Staphylococcus spp. Bacillus spp. Escherichia coli Streptococcus b-hemolytic Staphylococcus spp.
sperm kinetics, corroborating the studies of Cottell et al [21]. The authors evaluated the influence of bacterial contamination on concentration, motility, and morphology of sperm of men with different levels of bacteriospermia (105 organisms/mL, <105 organisms/mL, and negative semen cultures) and found no significant differences among the three groups. Previously, studies evaluated the effects of bacterial load in bull semen [22] and equine semen [4,6], and it was described that there were no differences on sperm motility in samples with low or high bacterial population, as observed in this study. In experiment 2, a higher number of CFUs was observed in INRA group compared with BS group. Probably, the concentration of antibiotic present on the extender of the INRA group was not enough to control bacterial growth in semen after cooling. Similar results have been reported [23], and the addition of 1 mg/mL ticarcillin in the same extender was suggested. Other studies [11,12] described there was no difference in bacterial growth in semen diluted with INRA96 and INRA96 Plus (1 mg/mL ticarcillin– clavulanic acid); yet, the sperm velocity was greater in the diluent with extra antibiotic. The control of bacterial growth using a semen extender can reduce bacterial proliferation in the genital tract of the mare, reducing risks to female [24]. Although it is reported that most of the bacteria present in the ejaculate are not pathogenic to healthy mares, however, in case of imbalance of the male genitalia microbial environment, there is proliferation of pathogenic bacteria [25,26], such as K. pneumoniae and P. aeruginosa, which can cause endometritis and decreased fertility in mares [26]. In the present study, the extender used in BS group was able to inhibit microbial growth of Pseudomonas spp., and this was not observed in INRA group. The sensitivity of Pseudomonas spp. to gentamicin, described in our results, corroborates with Althouse et al [19]. The difference in the efficiency on controlling Pseudomonas spp. growth can be explained by the different gentamicin concentration between the two extenders evaluated. Aurich and Sperfser [27] reported that 0.25 mg/ mL of gentamicin in the extender was not able to reduce the effects of Pseudomonas spp. on stallion semen. In relation to the storage temperature, the semen stored at 5 C showed lower bacterial growth in relation to semen stored at 15 C. These results are probably because of the effect of temperature on reduction of cellular metabolism [28] of microorganisms present in semen and consequently their proliferation. These findings agree with Kuisma et al [29] that observed that the number of
SP
FL
Staphylococcus spp. Streptococcus b-hemolytic
Staphylococccus b-hemolytic Citrobacter diversus
Staphylococcus spp.
bacteria did not increase in samples stored at 5 C, whereas in samples preserved at 20 C, an increase of three to four times on bacterial growth after 48 hours of storage was observed. Filtered semen maintained kinetic sperm parameters and showed greater membrane integrity during cooled storage (experiment 3), similar to Ramires Neto et al [13]. This indicates that the presence of seminal plasma can be deleterious for the sperm membrane during cooling process, and its removal is beneficial for some stallions as previously described [30–32]. In addition, a good control on bacterial contamination was observed on filtered semen. The filter used to remove seminal plasma (SpermFilter) contains 2-mm pores that allows the passage of SP and keeps the sperm cells retained. Probably, the micropores present in the filter also allowed the passage of bacteria, reducing the bacterial load after filtration. The reduction of bacterial contamination in semen is important to minimize the risk of endometritis in inseminated mares and prevent sperm cell damages. Human [33–35] and equine [4,24] studies have shown that some bacteria species in the ejaculate may lead to the activation of caspases in sperm by excessive production of reactive oxygen species, stimulating the production of proinflammatory cytokines and release of lipopolysaccharide. The results of these effects are reduction in sperm motility, sperm longevity, and apoptosis [4,34]. In conclusion, the use of a semen extender with appropriate antibiotics and removal of seminal plasma by filtration reduced bacterial growth in equine semen. Although penile wash before semen collection did not affect sperm kinetic parameters, it tended to reduce the number of CFUs in fresh semen. These techniques maximize the quality and viability of fresh and cooled stallion semen and may prevent uterine disturbs in mares. References [1] Baumber-Skaife J. Evaluation of semen. In: McKinnon AO, Squires EL, Vaala WE, Varner DD, editors. Equine reproduction. 2nd ed. Wiley-Blackwell: Chichester; 2012. p. 1278–91. [2] Tischner M, Kosiniak K. Bacterial contamination of stallion semen collected by ‘open’ AV. Vlaams Diergeneeskundig Tijdschrift 1986; 55:90–4. [3] Madsen M, Christensen P. Bacterial flora of semen collected from Danish warmblood stallions by artificial vagina. Acta Vet Scand 1995;36:1–7. [4] Ortega-Ferrusola C, Gonzalez-Fernandez L, Muriel A, MacıasGarcıa B, Rodrıguez-Martınez H, Tapia JA, Alonso JM, Pena FJ. Does the microbial flora in the ejaculate affect the freezeability of stallion sperm? Reprod Domest Anim 2009;44:518–22.
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Original Research
Control Methods and Evaluation of Bacterial Growth on Fresh and Cooled Stallion Semen Carlos Ramires Neto, Yame Fabres Robaina Sancler da Silva, Helene Lacerda Resende, Priscilla Nascimento Guasti, Gabriel Augusto Monteiro, Patrícia Melo Papa, José Antônio Dell’aqua Júnior, José Nicolau Próspero Puoli Filho, Marco Antônio Alvarenga*, Frederico Ozanam Papa Department of Animal Reproduction, College of Veterinary Medicine and Animal Science, Univ Estadual Paul (UNESP), Botucatu, São Paulo, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 July 2013 Received in revised form 10 July 2014 Accepted 6 January 2015 Available online xxxx
The penis and prepuce of the stallion have a high bacterial load on its surface, forming a natural microbial flora that contaminates the semen during ejaculation. Bacterial growth in semen may cause a decline on sperm quality, viability, and fertility and predisposes the occurrence of endometritis in inseminated mares. Thus, the aim of this study was to evaluate the effect of penile wash before semen collection, the addition of different commercial skim milk–based extenders containing antibiotics (BotuSemen and INRA96), and the removal of seminal plasma by filtration on the quality, viability, and bacterial proliferation on fresh and cooled stallion semen. Animals that were never submitted to penile wash before semen collection tended to have lower bacterial contamination in the ejaculate. Semen samples extended in BotuSemen showed superiority in total motility, progressive motility, average path velocity, and rapid sperm and lower bacterial contamination in relation to semen samples extended in INRA96 after 24 hours of cooling. No difference was found in these parameters between the storage temperatures (5 C and 15 C). Furthermore, the removal of seminal plasma by filtration reduced the bacterial load in semen after cooling. In conclusion, the penile wash before semen collection tended to reduce the bacterial growth in fresh semen. The use of a semen extender with appropriate antibiotics and removal of seminal plasma by filtration were effective in reducing the bacterial contamination and preserved the quality of cooled stallion semen. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Semen Stallion Bacteria Filtration Penile wash
1. Introduction The penis and prepuce have a permanent natural microbial flora with a high amount of bacteria [1] that can be transferred to the semen after collection [2]. Therefore, the equine ejaculate naturally exhibits a large bacterial contamination and is mainly composed of nonpathogenic bacteria [3]. * Corresponding author at: Marco Antônio Alvarenga, Department of Animal Reproduction, College of Veterinary Medicine and Animal Science, Univ Estadual Paul (UNESP), 18610-970 Botucatu, São Paulo, Brazil. E-mail address: [email protected] (M.A. Alvarenga). 0737-0806/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2015.01.014
An imbalance in the microbial environment of the external genitalia can result in the proliferation of opportunistic microorganisms [4], such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Taylorella equigenitalis, and Streptococcus b-hemolytic. These microorganisms may cause endometritis in inseminated mares [1], decrease in fertility rates [5], and affect the semen quality [6]. Methods to reduce bacterial contamination and proliferation in equine semen are largely described, such as washing the stallion penis before semen collection [7], the use of an open-ended artificial vagina [4], the use of semen extenders containing antibiotics [1,4], and the reduction of semen storage temperature [4].
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Table 1 Mean values and standard deviations of total motility (TM), progressive motility (PM), velocity of trajectory (VAP), curvilinear velocity (VCL), rapid sperm (RAP) before (M0) and after (M24) cooling the semen of animals with (W) and without (NW) to wash penis before collection. Groups
TM (%)
W (M0) NW (M0) W (M24) NW (M24)
85.2 75.0 62.8 52.6
VAP (mm/s)
PM (%) 6.9 14.5 15.2 28.3
39.8 42.0 29.4 20.4
5.3 15.1 15.7 12.4
123.3 116.4 102.8 104.4
VCL (mm/s)
18.9 18.6 21.3 22.6
216.2 201.2 194.7 193.8
21.6 22.5 28.7 37.3
RAP (%) 74.8 65.6 51.2 44.6
9.7 17.6 22.2 28.4
Different letters indicate statistical difference between the groups at the same column (P < .05). Same letters or absence of letters indicate no statistical difference.
diacetate and propidium iodide [8], and 100 sperm cells were counted.
To achieve new methods of control of bacterial growth in equine semen, the aims of this study were (1) to determine the effect of washing the stallion penis before collection and (2) to compare different semen extenders and the seminal plasma removal using filtration on quality, viability, and bacterial growth on fresh and cooled stallion semen.
2.4. Bacteria Counting and Identification Isolation of bacterial colonies was based on macroscopic and microscopic characterization (Gram stain method) and biochemical tests [9]. For the count of colony-forming units (CFUs), 0.1 mL of seminal sample was diluted in 9.9 mL of saline. Then, 0.1-mL aliquot of this dilution was plated in the following culture mediums: blood agar base with 7% sterile defibrinated sheep blood, MacConkey agar base, and Sabouraud agar base. The seeded assay plates were incubated 37 C and analyzed for 24, 48, 72, and 96 hours. The total of counted colonies was multiplied by 103 CFUs in accordance with the dilution used in this study. The identification and the CFU count were performed on fresh semen in experiments 1 and 3 and on cooled semen in experiments 2 and 3.
2. Materials and Methods 2.1. Semen Collection Semen collection was performed using an artificial vagina. Initially, three ejaculates from each stallion were collected with an interval of 2 days to eliminate possible damaged cells and stabilize the sperm parameters. 2.2. Semen Cooling and Storage After collection and processing, semen samples were stored in a container of passive cooling (BotuFlex; Botupharma, Sao Paulo, Brazil) at 15 C for 24 hours, for experiments 1, 2 and 3. For experiment 2, samples were also stored at 5 C for 24 hours.
2.5. Experiments 2.5.1. Experiment 1dEffect of Penile Washing on Contamination and Quality of Stallion Semen An ejaculate from each of 10 healthy stallions of different breeds (Mangalarga Marchador and Quarter Horse) aged 7–15 years old was used. All stallions were used in breeding programs that the semen collection was performed by artificial vagina. The animals were divided into two groups: stallions that were never submitted to penile washing before semen collection, composed by three Mangalarga Marchador and two Quarter Horse stallions (group NW; n ¼ 5; 10 3.2 years old), and stallions that are routinely (three times per week) submitted to penile washing with running water at 30 C before semen collection, composed by three Mangalarga Marchador and two Quarter Horse stallions (group W; n ¼ 5; 10.4 2.7 years old). After semen collection, the ejaculate was filtered to remove the gel fraction and diluted in a skim milk–based extender (Botusemen; Botupharma) to a sperm
2.3. Semen Analysis The kinetic sperm parameters were evaluated immediately after semen collection (0 hours) and 24 hours after semen cooling. The samples were previously placed in a dry water bath at 37 C for 5 minutes and evaluated by computer-assisted sperm analysis (HTM-IVOS 12; Hamilton Thorne Research, MA). Three different fields in Makler Counting Chamber (Sefi Medical Instruments Ltd, Israel) per sample were considered for total motility (%), progressive motility (PM, %), average path velocity (mm/s), curvilinear velocity (mm/s), and rapid sperm (%). Plasma membrane integrity (PMI, %) analysis was assessed through an epifluorescence microscopy (400 magnification) using the fluorescent probes 6-carboxyfluorescein
Table 2 Identification of the bacteria in the culture of cooled stallion’s semen with and without to wash penis before collection. Stallions with wash penis before collection
Stallions without wash penis before collection
1 2 3 4 5
6 7 8 9 10
Staphylococcus spp. Streptococcus spp./Staphylococcus spp.
Klebsiella oxytoca/Escherichia coli Streptococcus b-hemolytic/Staphylococcus b-hemolytic Bacillus spp. Streptococcus a-hemolytic Staphylococcus spp.
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Table 3 Mean values and standard deviations of total motility (TM), progressive motility (PM), velocity of trajectory (VAP), curvilinear velocity (VCL), rapid sperm (RAP), plasma membrane integrity (PMI), and bacteria colony-forming unit (CFU) of stallion’s semen diluted with commercial extender Botusemen (BS) and INRA96 (INRA) before (M0) cooling. Groups
TM (%)
PM (%)
VAP (mm/s)
VCL (mm/s)
RAP (%)
PMI (%)
CFU
BS (M0) INRA (M0)
67.4 14.3a 73.1 12.4a
33.1 7.2a 32.6 8.9a
141.3 18.9a 143.7 18.4a
249.0 35.3a 251.7 37.3a
67.5 11.7a 67.4 14.3a
65.2 7.6a 66.6 9.7a
56.2 7225a 6,538.6 5,331.4b
Different superscript letters indicate difference between the groups.
Table 4 Mean values and standard deviations of total motility (TM), progressive motility (PM), velocity of trajectory (VAP), and rapid sperm (RAP) of stallion’s semen diluted with commercial extender Botusemen (BS) and INRA96 (INRA) after (M24) cooling at 5 C and 15 C. Groups
TM (%)
5 C BS INRA
VAP (mm/s)
PM (%)
15 C Aa
65.4 16.8 54.3 19.0Ba
5 C Aa
69.0 15.0 60.7 17.0Ba
15 C Aa
37.0 14.4 26.3 12.0Ba
RAP (%)
5 C Aa
32.7 13.8 23.8 8.9Bb
5 C
15 C Aa
128.1 10.4 116.7 15.4
Ba
134.7 15.2 130.7 16.5
Aa Ba
15 C Aa
59.5 17.6 47.9 19.4
Ba
64.3 15.7Aa 54.1 17.8 Ba
Different superscript uppercase letters indicate statistical difference between columns, and different superscript lowercase letters indicate difference between the lines for the same parameter.
concentration of 40 106 cells/mL and then submitted to cooling at 15 C. 2.5.2. Experiment 2dEffect of Semen Extender on Sperm Kinetic Parameters and Bacterial Growth of Cooled Stallion Semen Two ejaculates were collected from each of 15 healthy stallions of different breeds (Mangalarga Marchador, Quarter Horse, and Thoroughbred), aged 4–6 year old. The semen was collected without penile washing. The gel fraction was removed and a total of 4 109 sperm cells were equally divided between two groups. In BS group, semen samples were added to a commercial skim milk– based extender (BotuSemen; Botupharma) containing gentamicin sulfate (1 mg/mL) and penicillin (1 mg/mL), as indicated by the manufacturer, to a sperm concentration of 40 106 cells/mL; and in INRA group, semen samples were added to a commercial skim milk–based extender (INRA96; IMV, MN) containing penicillin (0.038 mg/mL), gentamicin (0.105 mg/mL), and amphotericin B (0.315 mg/mL) [10,11] to a sperm concentration of 40 106 cells/mL. Samples from the two groups were submitted to cooling at 5 C and 15 C in a cooling passive container (BotuFlex; Botupharma). 2.5.3. Experiment 3dEffect of Seminal Plasma Removal on Sperm Viability and Bacterial Growth of Fresh and Cooled Stallion Semen One ejaculate from each of 10 healthy stallions of different breeds (Mangalarga Marchador, Quarter Horse, and Thoroughbred) aged 7–15 years old was collected. The
gel fraction was removed, and the semen was diluted at a 1:1 ratio with a skim milk–based extender (BotuSemen; Botupharma). Semen samples were divided into two groups according to the presence or not of seminal plasma. In the seminal plasma group (SP), semen samples were extended to a sperm concentration of 40 106 cells/mL. In the filtered group (FL, without seminal plasma), semen was filtered through synthetic hydrophilic membrane (SpermFilter; Botupharma) to remove seminal plasma [13]. Then, sperm were resuspended in semen extender to a sperm concentration of 40 106 cells/mL. Semen samples from the two groups were submitted to cooling at 15 C in a cooling passive container (BotuFlex; Botupharma). 2.6. Statistics Statistical analyses were performed using InStat GraphPad 3 software (GraphPad Software, CA). In experiments 1 and 3, data were statistically analyzed using the Student t test. For the experiment 2, a two-way analysis of variance and Tukey test were carried out to compare the groups and the moments. The significance level was set at P < .05. 3. Results 3.1. Experiment 1 No difference was observed (P > .05) in sperm kinetics parameters between the NW group and W group before
Table 5 Mean values and standard deviations of plasma membrane integrity (PMI) and bacteria colony-forming unit (CFU) of stallion’s semen diluted with commercial extender Botusemen (BS) and INRA96 (INRA) after (M24) cooling at 5 C and 15 C. Groups
BS INRA
PMI (%)
CFU
5 C
15 C
5 C
15 C
58.4 7.8Aa 58.1 9.5Ba
61.0 11.6Aa 61.5 10.2Ba
280.6 738.7Aa 3,352.2 4,315.1Ba
122 464.2Aa 3,593.7 4,088.1Bb
Different superscript uppercase letters indicate statistical difference between columns, and different superscript lowercase letters indicate difference between the lines for the same parameter.
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Table 6 Identification of the bacteria in the culture of stallion’s semen diluted with commercial extender with Botusemen (BS) and INRA96 (INRA) before (M0) and after (M24) cooling at 5 C and 15 C. 24 hr/5 C
0 hr
BS
24 hr/15 C
Groups
FS
SP
FL
BS
CFU
236,926 390,888.7a
33 104.4b
3 9.4b
BS
INRA
Stallion/Bacteria
Species Species Species Species
1-Coryne. 2-Coryne. 3-Coryne. 4-Coryne. 5-Rhodo. 6-Coryne. 7-E. coli/Coryne. 8-Coryne. 9-Coryne. 10-Pseudo. 11-Staphyl. 12-Coryne. 13Coryne./Rhodo. 14-Coryne. 15-E. coli/Coryne.
d d d d Rhodo. d d d d d d d d d d
Coryne. Coryne. Coryne. Coryne. Rhodo. Coryne. Coryne. d Coryne. Pseudo. d d Rhodo. Coryne. Coryne.
INRA
d d d d Rhodo. d d d d d d d Rhodo. d Coryne.
Table 8 Average values of bacteria colony-forming unit (CFU) of fresh semen in M0 (FS) and semen with (SP) and without (FL) seminal plasma after 24 hours of cooling.
INRA
0 Coryne. Coryne. Coryne. Rhodo. Coryne. Coryne. d Coryne. Pseudo./Bacillus d d Rhodo. d Enterob.
Species
Different superscript letters in the lines indicate significant differences (P < .05).
d Coryne. d d Rhodo. d d d d d d d d d Coryne.
without seminal plasma (FL; P < .05). The percentage of sperm with intact plasma membrane (PMI) was higher in FL group after cooling 15 C for 24 hours (P < .05; Table 7). The count of CFUs was greater in fresh semen in relation to semen samples with or without seminal plasma after cooling at 15 C for 24 hours (P < .05; Table 8). Different bacterial species were isolated from fresh and cooled semen with or without SP (Table 9). 4. Discussion
Abbreviations: Bacillus, Bacillus spp.; Coryne., Corynebacteruim spp.; E. coli, Escherichia coli; Enterob., Enterobacter cloacae; Pseudo., Pseudomonas aeruginosa; Rhodo., Rhodococcus equi; Staphyl., Staphylococcus spp.
The surface of the stallion penis and prepuce is colonized by a high variety of commensal bacteria [11,12,14,15] that contaminate the ejaculate and consequently the mare reproductive tract [11,12]. The penile wash with running water before semen collection promotes the mechanical removal of debris and reduces bacterial contamination in semen [14,17]. This fact may justify the results in this study, in which the count of CFUs in the ejaculate tended to be superior in stallions that were never submitted to penile wash in relation to stallions that were routinely submitted to penile washing with water before semen collections (experiment 1). These microorganisms found in penis and prepuce may cause endometritis in inseminated mares [1], decrease fertility rates [5], and affect the semen quality [6]. Althouse et al [18] described that bacterial contamination of extended porcine semen has been associated with deleterious effects on semen quality. The authors reported that all isolated bacteria were resistant to gentamicin, the most common antibiotic agent in commercial porcine semen extenders. The bacteria found on penile microbial flora in our study were similar with that described by Corona et al [19]. These bacteria constitute the normal microflora of the stallion’s external genitalia and rarely produce genital infections [16]. In cooled–stored semen, Staphylococcus spp. is one of the most common organisms regarding its survival characteristics [20], as seen in our results. In the present study, no differences between the groups NW and W were observed regarding the parameters of
and after semen cooling (Table 1). However, CFU counts tended to be superior (P ¼ .089) in NW group compared with W group (CFUs/mL, 272,636 375,764a vs. 1,216 2,674b, respectively; Different superscript letters indicate difference between the groups). The bacterial identification of both groups is described in Table 2. 3.2. Experiment 2 Before cooling, no difference was found (P > .05) between semen extenders in the sperm kinetic parameters and PMI. However, the CFU count (P < .05) was higher for the INRA group compared with BS group (Table 3). After 24 hours of cooling, there was difference (P < .05) in all sperm motility parameters and CFUs between BS group and INRA group, however, no difference was found on PMI. When the different cooling temperatures were analyzed, no difference (P > .05) was found in the BS group in the same evaluated parameters, and in the INRA group, PM showed lower values at 15 C than 5 C and greater CFU count than BS group (P < .05; Tables 4 and 5). The bacterial isolation in each group is described in Table 6. 3.3. Experiment 3 Before cooling, PM was greater in semen samples with seminal plasma (SP) compared with semen samples
Table 7 Mean values and standard deviations of total motility (TM), progressive motility (PM), path velocity (VAP), progressive linear velocity (VSL), curvilinear velocity (VCL), rapid spermatozoa (RAP), and plasma membrane integrity (PMI) of semen with (SP) and without (FL) seminal plasma, before (M0) and after (M24) refrigeration. Groups
TM (%)
SP (M0) FL (M0) SP (M24) FL (M24)
79.6 73.9 56.5 60.9
VAP (mm/s)
PM (%) 11.7 15.4 22.4 15.8
39.2 30.3 23.4 24.8
11.2a 11.3b 14.3 10.3
123.4 117.5 103.5 105.6
17.6 23.1 20.8 38.5
VCL (mm/s) 214.3 214.2 193.6 186.4
25.0 38.0 31.7 29.9
Different superscript letters in the columns indicate significant differences at the same moment (P < .05).
RAP (%) 70.3 62.0 47.0 43.3
14.3 22.4 24.8 23.8
PMI (%) 62.3 66.0 31.3 42.8
13.8 15.9 12.7b 21.2a
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Table 9 Bacteria identified in microbial cultivation of fresh semen in M0 (FS) and semen with (SP) and without (FL) seminal plasma after 24 hours of cooling. Stallion
FS
1 2 3 4 5 6 7 8 9 10
Staphylococcus spp.
Klebsiella oxytoca/Escherichia coli Streptococcus b-hemolytic Streptococcus spp./Staphylococcus spp. Bacillus spp. Escherichia coli Streptococcus b-hemolytic Staphylococcus spp.
sperm kinetics, corroborating the studies of Cottell et al [21]. The authors evaluated the influence of bacterial contamination on concentration, motility, and morphology of sperm of men with different levels of bacteriospermia (105 organisms/mL, <105 organisms/mL, and negative semen cultures) and found no significant differences among the three groups. Previously, studies evaluated the effects of bacterial load in bull semen [22] and equine semen [4,6], and it was described that there were no differences on sperm motility in samples with low or high bacterial population, as observed in this study. In experiment 2, a higher number of CFUs was observed in INRA group compared with BS group. Probably, the concentration of antibiotic present on the extender of the INRA group was not enough to control bacterial growth in semen after cooling. Similar results have been reported [23], and the addition of 1 mg/mL ticarcillin in the same extender was suggested. Other studies [11,12] described there was no difference in bacterial growth in semen diluted with INRA96 and INRA96 Plus (1 mg/mL ticarcillin– clavulanic acid); yet, the sperm velocity was greater in the diluent with extra antibiotic. The control of bacterial growth using a semen extender can reduce bacterial proliferation in the genital tract of the mare, reducing risks to female [24]. Although it is reported that most of the bacteria present in the ejaculate are not pathogenic to healthy mares, however, in case of imbalance of the male genitalia microbial environment, there is proliferation of pathogenic bacteria [25,26], such as K. pneumoniae and P. aeruginosa, which can cause endometritis and decreased fertility in mares [26]. In the present study, the extender used in BS group was able to inhibit microbial growth of Pseudomonas spp., and this was not observed in INRA group. The sensitivity of Pseudomonas spp. to gentamicin, described in our results, corroborates with Althouse et al [19]. The difference in the efficiency on controlling Pseudomonas spp. growth can be explained by the different gentamicin concentration between the two extenders evaluated. Aurich and Sperfser [27] reported that 0.25 mg/ mL of gentamicin in the extender was not able to reduce the effects of Pseudomonas spp. on stallion semen. In relation to the storage temperature, the semen stored at 5 C showed lower bacterial growth in relation to semen stored at 15 C. These results are probably because of the effect of temperature on reduction of cellular metabolism [28] of microorganisms present in semen and consequently their proliferation. These findings agree with Kuisma et al [29] that observed that the number of
SP
FL
Staphylococcus spp. Streptococcus b-hemolytic
Staphylococccus b-hemolytic Citrobacter diversus
Staphylococcus spp.
bacteria did not increase in samples stored at 5 C, whereas in samples preserved at 20 C, an increase of three to four times on bacterial growth after 48 hours of storage was observed. Filtered semen maintained kinetic sperm parameters and showed greater membrane integrity during cooled storage (experiment 3), similar to Ramires Neto et al [13]. This indicates that the presence of seminal plasma can be deleterious for the sperm membrane during cooling process, and its removal is beneficial for some stallions as previously described [30–32]. In addition, a good control on bacterial contamination was observed on filtered semen. The filter used to remove seminal plasma (SpermFilter) contains 2-mm pores that allows the passage of SP and keeps the sperm cells retained. Probably, the micropores present in the filter also allowed the passage of bacteria, reducing the bacterial load after filtration. The reduction of bacterial contamination in semen is important to minimize the risk of endometritis in inseminated mares and prevent sperm cell damages. Human [33–35] and equine [4,24] studies have shown that some bacteria species in the ejaculate may lead to the activation of caspases in sperm by excessive production of reactive oxygen species, stimulating the production of proinflammatory cytokines and release of lipopolysaccharide. The results of these effects are reduction in sperm motility, sperm longevity, and apoptosis [4,34]. In conclusion, the use of a semen extender with appropriate antibiotics and removal of seminal plasma by filtration reduced bacterial growth in equine semen. Although penile wash before semen collection did not affect sperm kinetic parameters, it tended to reduce the number of CFUs in fresh semen. These techniques maximize the quality and viability of fresh and cooled stallion semen and may prevent uterine disturbs in mares. References [1] Baumber-Skaife J. Evaluation of semen. In: McKinnon AO, Squires EL, Vaala WE, Varner DD, editors. Equine reproduction. 2nd ed. Wiley-Blackwell: Chichester; 2012. p. 1278–91. [2] Tischner M, Kosiniak K. Bacterial contamination of stallion semen collected by ‘open’ AV. Vlaams Diergeneeskundig Tijdschrift 1986; 55:90–4. [3] Madsen M, Christensen P. Bacterial flora of semen collected from Danish warmblood stallions by artificial vagina. Acta Vet Scand 1995;36:1–7. [4] Ortega-Ferrusola C, Gonzalez-Fernandez L, Muriel A, MacıasGarcıa B, Rodrıguez-Martınez H, Tapia JA, Alonso JM, Pena FJ. Does the microbial flora in the ejaculate affect the freezeability of stallion sperm? Reprod Domest Anim 2009;44:518–22.
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