microorganisms

Article
Identification of Bull Semen Microbiome by 16S Sequencing
and Possible Relationships with Fertility
Aleksandar Cojkic 1, * , Adnan Niazi 2 , Yongzhi Guo 1 , Triin Hallap 3 , Peeter Padrik 4 and Jane M. Morrell 1

1 Department of Clinical Sciences, Swedish University of Agricultural Sciences (SLU), 75007 Uppsala, Sweden;
[Link]@[Link] (Y.G.); [Link]@[Link] (J.M.M.)
2 SLU-Global Bioinformatics Centre, Department of Animal Breeding and Genetics,
Swedish University of Agricultural Sciences, 75007 Uppsala, Sweden; [Link]@[Link]
3 Estonian University of Life Sciences, 51014 Tartu, Estonia; [Link]@[Link]
4 Animal Breeders’ Association of Estonia, 79005 Raplamaa, Estonia; [Link]@[Link]
* Correspondence: [Link]@[Link]

Abstract: Reports on the use of 16S sequencing for the identification of bacteria in healthy animals
are lacking. Bacterial contamination of bull semen can have a negative effect on the sperm quality.
The aims of this study were threefold: to identify bacteria in the semen of healthy bulls using 16S
sequencing; to investigate the differences in the bacterial community between individual bulls; and
to establish if there was a relationship between the bacteria isolated and bull fertility. Semen from
18 bulls of known fertility was used for the DNA extraction and 16S sequencing; 107 bacterial genera
were identified. The differences in the amplicon sequence variants (ASVs) and the numbers of
genera between bulls were noted. Negative correlations (p < 0.05) between several bacterial genera
with Curvibacter, Rikenellaceae RC9-gut-group and Dyella spp. were seen. Other negatively correlated





 bacteria were Cutibacterium, Ruminococcaceae UCG-005, Ruminococcaceae UCG-010 and Staphylococcus,
all within the top 20 genera. Two genera, W5053 and Lawsonella, were enriched in bulls of low fertility;
Citation: Cojkic, A.; Niazi, A.; Guo,
Y.; Hallap, T.; Padrik, P.; Morrell, J.M.
this is the first time that these bacteria have been reported in bull semen samples. The majority of the
Identification of Bull Semen bacteria were environmental organisms or were species originating from the mucous membranes of
Microbiome by 16S Sequencing and animals and humans. The results of this study indicate that differences in the seminal microbiota of
Possible Relationships with Fertility. healthy bulls occur and might be correlated with fertility.
Microorganisms 2021, 9, 2431. https://
[Link]/10.3390/microorganisms9122431 Keywords: bull semen; bacteria; 16S sequencing; sperm quality; fertility

Academic Editor: Paolo Calistri

Received: 9 November 2021 1. Introduction

Accepted: 22 November 2021
One of the main reasons for introducing artificial insemination into animal breeding
Published: 25 November 2021
was to prevent the spread of infectious diseases between males and females [1]. Apart
from the urogenital tract itself as a primary source of pathogens [2], there are critical
Publisher’s Note: MDPI stays neutral
points in semen processing where sperm samples can be contaminated with different types
with regard to jurisdictional claims in
published maps and institutional affil-
of microbiota [3–5]. Therefore, antibiotic use in semen for artificial insemination (AI) is
iations.
considered to be essential. Different types of antibiotics, singly and in combination, are
added to semen worldwide with the aim of decreasing or preventing microbial growth.
Antibiotics used in bull semen cryopreservation are mainly based on the regulations given
by government directives, e.g., the European Union [6]. However, despite the addition of
antibiotics, bacteria can still be isolated from bull semen after thawing [7]. In addition, there
Copyright: © 2021 by the authors.
is considerable concern about the emergence of antibiotic-resistant bacteria, particularly
Licensee MDPI, Basel, Switzerland.
methicillin-resistant strains [8]. The identification of bacteria in semen samples is a primary
This article is an open access article
distributed under the terms and
step to the rational use of antibiotics and could be helpful in developing the next steps in
conditions of the Creative Commons
the control of microbial contamination of bull semen [3].
Attribution (CC BY) license (https:// The presence of bacteria in semen does not equate with infection and limited micro-
[Link]/licenses/by/ bial activity does not necessarily affect sperm quality [9]. However, bacteria can have a
4.0/). direct negative affect on sperm quality, depending on the species and number. Negative

Microorganisms 2021, 9, 2431. [Link] [Link]

Microorganisms 2021, 9, 2431 2 of 12

correlations were reported between certain bacteria and sperm motility [10], viability, mem-
brane integrity and acrosome reaction [11], sperm DNA fragmentation rates [12,13] and
the total number of spermatozoa [14,15]. Givens and Marley [16] reported the presence of
microorganisms that cause infertility and/or are transmitted via semen. This knowledge
can facilitate the identification and exclusion of subclinically infected bulls from production.
Bacteria present in the urogenital tract can influence fertility in cows [17]. Postpartum
uterine disease caused by pathogenic bacteria not only reduces fertility but also decreases
productivity [18]. Postpartum metritis and clinical endometritis caused subfertility in
cows by increasing the time to first insemination, delayed conception and increasing the
calving to conception interval [19,20]. Furthermore, postpartum clinical endometritis can
cause infertility and consequently results in involuntary culling [21]. Marey et al. [22]
reported that infections extending from the uterus to the oviduct induce an immune system
disbalance that interferes with fertilization. Apart from an effect on the uterus itself, a
uterine infection can lead to disruption in the secretion of gonadotropins [23], disruption
in ovarian follicle growth and function [24] and a reduction in the oocyte quality [25] (cited
from Sheldon and Owens [18]).
In contrast to these extensive studies in females, the presence and influence of bacteria
on sperm quality and fertility in bulls have not been investigated to any great extent.
However, the types of bacteria present in semen in various species such as stallions [2,26],
boars [27] and rams [28] have been described as well as reports of the influence of bacteria
on fertility and sperm quality parameters in humans [29,30]. Interest in the isolation and
identification of the main bacteria in bull semen causing infertility began decades ago [31]
using culture and identification by means of appearance and biochemical properties; other
methods of identification were introduced as the science developed. In general, reports
on the use of 16S sequencing for the identification of bacteriospermia in healthy animals
are lacking, especially in bulls. The identification of individual opportunistic bacteria is of
interest due to the possible transmission between bulls and from bulls to cows, causing
economic losses to cattle production. However, there are few studies in which a full
microbiological profiling of bull semen has been achieved.
The aims of the present study were to identify the bacteria in bull semen via 16S rRNA
sequencing and to investigate the individual differences in bacterial type and number in
bulls at a semen collection station. An additional aim was to examine the relationships
between the bacterial community and the overall fertility of the bulls.

2. Materials and Methods

2.1. Animals and Semen Collection
Ejaculates were obtained from 18 Holstein bulls at an AI center (Animal Breeders’
Association of Estonia, Raplamaa, Estonia) where animals were kept according to national
and international regulations. The age of the bulls ranged from 3 to 10 years. The bulls
were kept on dry bedding (new sawdust added 4× /24 h) and, if necessary, cleaned
by brushing before the semen collection. Semen collection with an artificial vagina is a
routine agricultural practice and, therefore, does not require ethical approval according
to Estonian law [32]; the bulls at the AI station were not considered to be experimental
animals. Therefore, no special ethical permission was required.
The bulls were prepared for semen collection by allowing them to follow each other
in a circular chute for 30–40 min; the bulls were able to make false mounts by jumping
on the bull in front but the chute was not wide enough for the bulls to turn round. A
sterilized artificial vagina lubricated with Bovivet Gel (Jørgen Kruuse A/S, Langeskov,
Denmark) and a sterilized graduated collecting tube were used for the semen collection,
which took place approximately 10 m from the laboratory separated by a glass wall. Within
a minute after the collection, the sterile graduated tube containing the collected ejaculate
was separated from the artificial vagina and passed to the laboratory personnel through
a small hatch in the glass wall. All semen collection procedures were performed with
sterile equipment and aseptic measures to avoid semen contamination. In the laboratory,
Microorganisms 2021, 9, 2431 3 of 12

an aliquot of 1 mL semen was transferred to an Eppendorf tube above an alcohol burner

and stored in liquid nitrogen before transfer to −80 ◦ C for storage until the bacterial DNA
extraction was performed. The remainder of the semen samples were processed and used
for the evaluation of the sperm motility by CASA. All samples with a total motility >90%
and a progressive motility >80% were used for the routine inseminations. The CASA results
are presented in Supplementary Table S1. The fertility performance of these bulls was
estimated by non-return rates (NRRs) at 90 days post-AI, based on the outcome from the
first insemination. In total, 48,469 females were inseminated, comprising 34,800 cows and
13,649 heifers. Miglior et al. [33] defined NRRs as the proportion of cows that are not seen
in estrus again after insemination and are, therefore, considered to be potentially pregnant.

2.2. DNA Extraction

The DNA extraction was performed in the Clinical Sciences Research Laboratory at
SLU using an AllPrep DNA/RNA/miRNA Universal Kit Cat No./ID 80224 (GIAGE, Ger-
mantown, Philadelphia, USA) following the manufacturer´s instructions for the protocol
of the simultaneous purification of genomic DNA and total RNA, including miRNA from
cells. In total, 10 µL of semen was used for the DNA extraction to reach the maximum
amount of 1 × 107 cells, spermatozoa and bacteria according to the protocol. The sperm
concentration was evaluated using a Nucleocounter SP100 (ChemoMetec, Allerød, Den-
mark) according to the manufacturer´s instructions. According to our calculations, 10 µL
of semen contained on average 5 × 106 spermatozoa although the bacterial number was
not calculated. All samples were centrifuged; the supernatant was removed and only
pelleted cells were used. The purity and concentration of the DNA were tested using a
NanoDrop 8000 Spectrophotometer (Thermo Scientific, Waltham (HQ), MA, USA). The
DNA purity was considered adequate when the 260/280 ratio was between 1.7 and 1.9 and
the concentrations were between 5.34 and 10.97 ng/µL. The DNA samples were stored at
−80 ◦ C until further preparation.

2.3. 16S rRNA Amplification and Sequencing

A two-step amplification protocol was used for the preparation of the V3–V4 16S gene
region for Illumina sequencing. The details of the primers used and the cycling protocols
are presented in Table 1. The reaction volume of the first step was 15 µL containing 4 ng
of the DNA template, 0.25 µM Pro 341F and the same volume of Pro 805R primers with
Nextera adaptor sequences (Illumina Inc., CA, USA) as well as 1 µg/µL BSA and 1 ×
Phusion Taq ready-to-use mix (New England Biolabs, MA, USA). For the second step,
the reaction volume was 30 µL and contained 3 µL of the purified DNA template from
the first PCR step, 0.20 µM tagged F and R primers with Nextera adaptor sequences and
1 µg/µL BSA and 1 × Phusion Taq ready-to-use mix. Both PCR steps were performed in
duplicate and the reactions were pooled and purified between the steps using SeraMag
Magnetic Carboxylate Modified particles in a ratio of 1:1. An Agilent Bioanalyzer was used
for the quality check. The amplicons were eluted in 10 mM Tris with a pH of 8.5 and stored
at −20 ◦ C. The samples were sent for sequencing to SciLifeLab, SNP&SEQ Technology
Platform, Uppsala University. Paired-end sequencing was performed on a MiSeq system
(Illumina, San Diego, CA, USA) using kit V2. Approximately 40,000 to 60,000 paired-end
reads of 250 bp length were obtained for all samples except the control sample (sterile
water), which yielded only 13 reads after sequencing.

Table 1. Primer combination and thermal cycling conditions used to quantify the 16S rRNA.

Primer 16S rRNA Sequences (50 -30 ) Terminal Cycling Reference

(98 ◦ C 3 min); (98 ◦ C 30 s, 55 ◦ C 30 s, 72 ◦ C 40 s) × 25;
341F CCTACGGGAGGCAGCAG
72 ◦ C 10 min; 10 ◦ C hold [34]
(98 C 3 min); (98 ◦ C 30 s, 55 ◦ C 30 s, 72 ◦ C 45 s) × 8;
◦
805R GACTACNVGGGTATCTAATCC
72 ◦ C 5 min; 10 ◦ C hold
Microorganisms 2021, 9, 2431 4 of 12

2.4. 16S Profiling

The analysis of the 16S rRNA sequencing data was performed using Nextflow pipeline
ampliseq v.1.1.2 ([Link] accessed on 24 November 2021).
Briefly, raw sequencing reads were quality checked initially using FastQC [35] followed
by the trimming of the primer sequences from the reads using cutadapt v.2.7 [36]. The
raw sequencing data were cleaned from source contamination by running BLAT against
the cow reference genome Bos taurus 8 available in the UCSC genome browser (https://
[Link]/, accessed on 24 November 2021). The sequencing reads were denoised,
dereplicated and filtered for chimeric sequences using DADA2 [37]. The denoised paired-
end reads were truncated from position 229 (forward) and 215 (reverse) after a manual
visualization of the sequencing error profile; all other reads shorter than the cutoff values
were dropped. The truncated sequences were merged with at least a 20 bp overlap, resulting
in exact amplicon sequence variants (ASVs). These ASVs were taxonomically classified
from the phylum to species level clustered with 99% similarity using the SILVA v.132
database [38] by applying a Naive Bayes classifier implemented in QIIME 2 [39] trained
on the preprocessed database. Following the taxonomic classification of the ASVs, the
ASVs classified as a mitochondria or a chloroplast were removed. Only the ASVs with a
minimum read frequency ≥5 in at least one sample were retained for a further analysis.

2.5. Statistical Analysis

The data analysis was performed using R v.3.3.1 software. Pearson correlation coef-
ficients were calculated between the bacterial genera using the [Link] function in the R
environment, with p < 0.05 being considered significant. The plotting was carried out in
R using corrplot v.0.9. A linear discriminant analysis effect size (LEfSe) analysis of the
microbial abundance between low and high fertility bulls was performed to detect the
differences between the two groups and characterize the biomarkers. The groups were
divided based on the NRR where the NRRs were <51% and >51% for low and high fertility
bulls, respectively.

3. Results
The total amount of DNA in the pool was 1320 ng with a ratio of absorbance at
A260/A280 at 1.89 and A260/A230 at 2.1 and a concentration of 24 ng/µL. In total,
107 bacterial genera were identified in 18 bull semen samples. The 20 most frequently
seen bacterial genera are presented in Figure 1.

Figure 1. Distribution of the 20 most abundant bacteria genera in the semen from 18 bulls (amplicon
sequence variants) identified by 16 rRNA sequencing.
Microorganisms 2021, 9, 2431 5 of 12

The bacteria listed among the top 20 for each bull varied considerably; there was
also a considerable variation between bulls in both the number of bacterial genera present
(ranging from 12 to 89) and in the bacterial count (ranging from 27,827 to 247,273) (Table 2).
The highest number of genera identified was in the sample from bull 12, which contained
89 bacterial genera of which 19 came from the 20 most frequently seen. In contrast, Bull 3
was the least colonized, with only 12 bacterial genera present.

Table 2. Number of top 20 and total genera, counts, total counts and non-return rate (NRR, %)
per bull.

Top 20 Genera Counts Total Genera Total Counts NRR (%)

Bull 1 7 50,127 13 60,678 48.9
Bull 2 20 101,453 69 149,773 48.3
Bull 3 5 17,157 12 56,692 51.4
Bull 4 17 53,773 47 95,193 55.1
Bull 5 12 16,089 31 27,827 61.7
Bull 6 16 188,695 47 247,273 45.9
Bull 7 16 61,305 37 92,055 48.9
Bull 8 17 74,382 49 129,282 52.1
Bull 9 10 53,248 24 70,528 50.4
Bull 10 12 95,853 26 114,379 62.1
Bull 11 18 95,136 82 175,372 51.8
Bull 12 19 70,932 89 130,601 55.2
Bull 13 18 115,553 77 183,004 50.5
Bull 14 18 64,435 76 118,757 47.6
Bull 15 18 102,459 83 170,084 54
Bull 16 19 146,754 53 198,532 52.6
Bull 17 18 68,109 86 141,152 52.3
Bull 18 9 116,226 16 137,258 37
Top 20 genera: 20 most frequently seen genera in all samples; counts: read counts of genera per bull present in the
top 20; total genera: number of identified genera per bull; total counts: read counts of genera per bull of all genera
per bull; NRR: non-return rate.

The 20 most frequently seen bacterial genera, starting with the most frequent, were:
Porphyromonas, Fusobacterium, Ruminococcaceae UCG-010, Fastidiosipila, Ruminococcaceae
UCG-005, Cutibacterium, Histophilus, Oceanivirga, Corynebacterium 1, Campylobacter, W5053,
Dyella, Staphylococcus, Lawsonella, Helcococcus, Bacteroides, Capnocytophaga, Curvibacter,
Kingella and Enhydrobacter, which are presented in Figure 1 as the relative ASV abundance.
There were negative correlations between several bacterial genera (Figure 2). The
majority of the bacteria that showed negative correlations were from the top 20 gen-
era (Figure 1) and included Curvibacter, Cutibacterium, Dyella, Ruminococcaceae UCG-005,
Ruminococcaceae UCG-010 and Staphylococcus spp. In addition, Curvibacter, Rikenellaceae
RC9-gut-group and Dyella spp. (brown spots) were negatively correlated in most cases.
In the bar plot (Figure 3), the taxa (including the genus and family) with significant
differences between the groups were detected by a LEfSe analysis with a log-10 transformed
LDA (linear discriminant analysis) threshold score of 2.0 and a significant p-value <0.05.
The ASV with a higher LDA score indicated that the ASV was more important according
to the LEfSe in discriminating between the low and high individuals. Two genera, W5053
and Lawsonella, were enriched in the low fertility group.
Microorganisms 2021, 9, 2431 6 of 12

Figure 2. Correlation plot of the bacteria in bull semen identified by 16S sequencing in a correlation
matrix showing the significant correlations (p < 0.05) between genera: positive (purple) and negative
(brown). Blank cells indicate non-significant correlations.

Figure 3. Linear discriminant analysis effect size plot. This plot shows the enriched taxa that were
significantly different between the high fertility (red) and low fertility (green) bulls.

4. Discussion
In this study, 16S sequencing of bull semen microbiomes was performed with the
aim of identifying the most common bacteria in the semen of healthy bulls. The determi-
nation of the most common bacteria genera can enable the development of appropriate
control methods.
The isolation and identification of bacteria by commercial microbiological methods
are more difficult compared with metagenomics analyses. First, it may not be possible to
isolate all the bacteria in a sample. The isolation and identification of bacteria by culture-
dependent methods require different culture conditions for different type of bacteria such
as the media, temperature, presence or absence of oxygen and time of incubation. The
Microorganisms 2021, 9, 2431 7 of 12

growth of a few bacteria may be inhibited by competition between bacterial species or

bacterial overgrowth. Traditional culture-dependent morphological identification methods
are time-consuming and do not enable all bacteria to be identified [40]. Although the
development of MALDI-TOF (matrix-assisted laser desorption ionization-time of flight)
mass spectrometry can help in the process of bacterial identification after culturing, the
possibility of identifying isolates depends on the information in the database for the
instrument [2]. In contrast, 16S sequencing does not require a bacterial culture and enables
the identification of large numbers of bacteria present in a sample [26].
To our knowledge, this is the first comprehensive metagenomic study of the seminal
microbiome of healthy bulls. During the introduction of artificial insemination as an animal
husbandry technique, there was a need to inhibit bacterial growth in bull semen [41].
Later, culture-dependent studies were conducted to isolate the bacteria responsible for bull
infertility [42] and this technique was also used in more recent studies of the microbiological
evaluation of frozen semen samples [43]. During the past few years, the identification of
individual bacteria from preputial mucosa [44,45] and bull semen [46] was performed with
metagenomic methods. Interestingly, there were no significant correlations between the
preputial microbial community of bulls of different ages, breed, diet or co-housing [47].
In other species, several studies on culture-independent methods of identification of the
normal microbiota in semen have been reported. Stallion semen microbiota was studied
by conventional methods of identification as well as MALDI-TOF [2] and recently by 16S
sequencing [26]. Al-Kass et al. [26] reported that large numbers of bacterial genera could
be identified in stallion semen using metagenomic analyses by 16S sequencing. Based on
those studies, variations in bacteria genera and their number between individual stallions
and between countries were identified. However, the researchers agreed that the identified
bacteria in both stallions and bulls [26,47] were mostly environmental in origin.
In order to understand the composition of the bull semen bacterial community, it is
important to discuss its potential sources. Three of the most common genera identified in
our study were Porphyromonas, Fusobacterium and Ruminococcaceae UCG-010 respectively.
This finding is in agreement with the study of Wickware et al. [47] where Porphyromonas
and Fusobacterium were the most abundant genera in the preputial microbiota of Hereford
bulls. These two genera, together with Bacterioides, were among the most abundant genera
from the upper respiratory and oral mucosal membranes of healthy calves in the first
month of life [48,49]. In contrast, the anaerobic bacteria Porphyromonas, Fusobacterium and
Fastidiosipila were highly prevalent in most cases of lameness caused by foot lesions [50].
Their presence in semen is probably due to their common occurrence in the environment
and subsequent colonization of mucosal membranes.
In the study of Klein-Jöbstl et al. [49] on the microbiota of newborn calves and their
mothers, Ruminococcaceae was the most abundant type in cow fecal and vaginal samples.
In the same study, Enhydrobacter was the most dominant in the colostrum on the first day
postpartum; this bacterium appeared in our top 20 isolated bacteria, albeit in low numbers.
Ruminococcaceae UCG-005 and Ruminococcaceae UCG-010, ranked in the top 10 isolated
genera in our study, were also present in the samples of healthy skin in the studies of
Bay et al. [50]. Coryneform bacteria Corynebacterium and Cutibacterium are distributed in the
environment in soil and water [51]. These bacteria, as well as Staphylococci, are commensals
and colonizers of the skin and mucous membranes in animals [52]. Dyella, Helicococcus,
Capnocytophaga and Kingella are commensal bacteria isolated from the human respiratory
tract [53] as well as the oral cavity of humans and animals [54] and are part of the skin
flora [55]. Although they are considered to be commensals, all of them were isolated from
a patient with severe clinical symptoms; this is the first report of their occurrence in bull
semen. Their influence on sperm quality parameters is unknown.
Histophilosis is a common disease in North American cattle in the form of septicemia
with a high risk of infection and sudden death in calves. A pathogenic form of Histophila
somni was isolated from the prepuce of a healthy bull and from the vagina of cows with a
clinical manifestation of granular vulvovaginitis and abortion [56]. Bovine genital campy-
Microorganisms 2021, 9, 2431 8 of 12

lobacteriosis caused by Campylobacter fetus veneralis or Campylobacter fetus fetus is a venereal

disease of cattle characterized by infertility, mucopurulent endometritis, early embryonic
death and occasionally abortion in systemically healthy cows. As well as venereal trans-
mission, Campylobacter fetus fetus can be transmitted by AI in contaminated semen as well
as by contaminated instruments. Infections in young bulls can be transient in contrast to
older animals with established chronic infections, which may be due to differences in the
preputial and penile epithelial surfaces of the lumen and within the crypts in the older ani-
mals and the microaerophilic environment that deeper crypts may provide. Campylobacter
is one of the most demanding bacteria to culture due to its requirement for microaerophilic
or anaerobic conditions as well as the need to be cultured immediately after sampling [57].
Therefore, it may be missed when culturing under conventional microbiological conditions.
Cagnoli et al. [11] described a significant negative influence of both Campylobacter fetus
veneralis and Campylobacter fetus fetus bacteria species on bull semen quality parameters.
Metagenomic analyses, especially 16S rDNA sequencing, allows the identification of
bacteria with unusual phenotypic profiles, rare bacteria, slow growing bacteria and bacteria
that cannot be cultured. Furthermore, 16S sequencing can facilitate the definitions of the
etiologies of infectious diseases as well as aiding clinicians to choose the most effective
antibiotics and determining the duration of the treatment. However, the interpretation of
the results can be challenging even for clinical microbiologists [40].
Farahani et al. [58] conducted a systematic review study and meta-analysis of bacteria
identified from fertile and infertile men and their influence on sperm quality and fertility
parameters. Major differences in the bacterial presence of fertile and infertile men were
identified with different sperm quality parameters as well as the negative and positive
effects of the individual bacteria on these parameters. Apparent positive effects of Lacto-
bacillus spp. on the sperm morphology in addition to a protective effect against Pseudomonas
and opportunistic pathogens were highlighted. Boud et al. [9] reported that Pasteurella
spp. abundance was increased in sperm samples with poor motility. Although the latter
study showed that the bacterial content might not have an influence on the fertility of
men, specific bacterial genera had an impact on sperm morphology and motility. There are
few studies on the influence of the bacterial community in semen on sperm quality and
fertility outcomes in veterinary medicine. There are a few studies of bacterial influence
on sperm quality parameters but usually fertility data are lacking [59]. Cryopreservation
does not necessarily reduce the bacterial count. In the study of Reda et al. [60], the bacterial
content of cryopreserved semen was evaluated. Their study showed a negative effect of an
increased bacterial content on sperm motility, viability and morphology with differences
between ejaculates although no differences between the bulls were detected. When boar
semen was stored for five days in the presence of an antibiotic, there was an increase in the
number of certain bacteria, which was associated with a decrease in sperm motility [61].
In this study, we were interested in the potential associations between the bacteria in
the male reproductive tract and the overall fertility of the semen from these bulls. We were
not studying the effects of these bacteria on the sperm quality during subsequent storage,
which is a different topic. Our results showed that two genera, W5053 and Lawsonella,
were enriched in the semen samples from the low fertility bull group. These genera were
also present among the 20 most abundant bacteria. Bell at al. [62] described Lawsonella
spp. as Gram-positive, partially acid-fast, non-spore-forming, anaerobic, catalase-positive
and pleomorphic bacteria. Three strains were isolated from human abscesses, which were
determined to represent a novel genus (Lawsonella clevelandensis gen. nov., sp. nov.). Genus
W5053 is also a novel bacteria; more comprehensive information is lacking. However,
a higher abundance of both genera were seen in HIV-infected patients without chronic
respiratory diseases [63]. The presence of these two bacterial genera in the semen samples
of the bulls with a lower fertility potential is of interest for future research with regard to
the origin and potential microbial influence on the sperm quality.
The diversity, number and interaction between the bacteria found in this study put 16S
rDNA sequencing high on the list of the methods of choice for the diagnostics of bacterial
Microorganisms 2021, 9, 2431 9 of 12

contamination based on its objectivity and reliability. As most of the bacteria present in
bull semen samples originate from the environment or from the mucosa of animals and
humans, there is a need for a more effective management of the critical control points
during semen collection. However, as 16S sequencing provides information about the
presence of bacterial DNA in samples and not specifically about bacterial viability, it can
only indicate the likely presence of the bacteria rather than the actual cause of a fertility
issue. A possible interaction between the bacteria found in bull semen with a low fertility
potential and host bacteria complex interactions would be of interest in future research.
Braga et al. [64] reported that the bacteria of different genera have an influence
on microbial community modulation. This microbial coexistence occurs via chemical
mediators among bacteria and also between microbes and hosts [65]. Such an interaction
may cause alterations in the host physiology [64].
Deines et al. [66] also reported that a host-bacteria interaction, i.e., a host-environment
and a bacteria–bacteria interaction, influences the coexistence of microbial species. This
study showed that the competitive effect of Curvibacter depends on direct contact and
indicated that rare microbial community members might be relevant for achieving a native
community composition and carrying capacity. Although the genus Curvibacter was first
mentioned by Ding and Yakota [67], who described three species isolated from well water
as the source of origin, there is no previous documentation of its presence in bull semen
or other sources. That Curvibacter was negatively correlated with other bacterial genera
in most cases in this study indicated that this bacterium could be the focus for further
research on the influence of the semen microbiota on the fertility of healthy bulls.
The second most negatively correlated bacteria in this study was Rikenellaceae RC9-
gut-group. It belongs to the Rikenellacea family that are recently identified bacteria described
as being challenging to culture [68]. This bacterial genus was previously identified in the
digestive tract and fecal samples of different animals but not in other types of samples
including bull semen. An increased abundance of this bacteria was found in an inflamed
human digestive tract but there was no direct indication of their association with dis-
ease [69]. The study of Bálingt A et al. [70] showed that Rikenellaceae RC9-gut-group with
other bacteria increased the sensitivity of the gut to inflammation. Based on the fact that
these organisms can be challenging to culture, our current knowledge about these bacteria
is based on the information gained from large scale sequencing studies.
The results of the present study indicated that differences in the bacterial microbiota
of healthy bulls occur and might be associated with the fertility potential of the bull. Most
of the identified bacteria were environmental in origin, indicating that a focus on how bulls
are housed and how the semen is processed could help to reduce the bacterial abundance
in commercial semen doses. The processing of bull semen should always be performed
with a high level of hygiene and microbiological control.

Supplementary Materials: The following are available online at [Link]

.3390/microorganisms9122431/s1: Table S1: CASA results for the sperm samples from 18 bulls.
Author Contributions: Conceptualization, A.C. and J.M.M.; methodology, A.C., A.N., Y.G., T.H., P.P.
and J.M.M.; investigation, A.C., A.N. and Y.G.; data curation, A.C. and A.N.; writing—original draft
preparation, A.C.; writing—review and editing, A.C., A.N., Y.G., T.H., P.P. and J.M.M.; resources,
J.M.M. and P.P.; supervision, J.M.M.; project administration, J.M.M.; funding acquisition, J.M.M. and
A.C. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by FORMAS, Stockholm (project number 2017-00957, awarded
to JMM), KSLA (grant number GFS2020-0058).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The sequencing data generated in this study were deposited in the
European Nucleotide Archive (ENA) under accession number PRJEB47651.
Microorganisms 2021, 9, 2431 10 of 12

Acknowledgments: We thank the personnel at the Kehta cattle breeding station, Animal Breeders’
Association of Estonia, for providing the bull semen samples. Sequencing was performed by the
SNP&SEQ Technology Platform in Uppsala. The facility is part of the National Genomics Infras-
tructure (NGI) Sweden and Science for Life Laboratory. The SNP&SEQ Platform is also supported
by the Swedish Research Council and the Knut and Alice Wallenberg Foundation. We thank SLU
Bioinformatics Infrastructure (SLUBI) for the management and processing of the sequencing data.
Support from NBIS (National Bioinformatics Infrastructure Sweden) is gratefully acknowledged.
This project was supported by the UMBLA platform at SLU.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or
in the decision to publish the results.

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