<p>Artificial insemination (AI), has been used as a powerful tool for livestock improvement by the breeders. AI greatly increases the utilization of proven sires. It ensures improvement in conception rates if semen is handled properly and the animals are bred at proper time. Successful AI largely depends on good quality semen. The frozen semen can be stored for many months to years in liquid nitrogen and has largely supplemented liquid semen for AI.</p><p>In case semen carries microorganisms, cryopreservation can spread diseases among cattle and buffalo population. Pathogens in semen may come from testes or epididymus, the accessory glands, the vas deferens, the urethra, or the prepuce or the penis. Even under careful conditions semen may get contaminated at the time of most collection or subsequent handling. Unhygienic surroundings, repeated entry of penis into artificial vagina (AV), extenders, and straw have been reported to adversely affect the quality of semen by increasing the number of microorganisms. Many bacterial agents that are transmitted through semen affect the fertility of dam leading to repeat breeding amd infertility. Measures taken by the industry and regulatory authorities, to prevent or control microbial contamination of semen, have included maintaining groups of bulls free from specific diseases and adding antimicrobial agents to the extended semen.</p><p>There are four frozen semen banks (FSB) located in organized sector in Haryana that cater to the need of AI programme of the state. However, the literature reveals no published reports on the microbial flora of semen of bulls in these banks. Therefore, the present investigation was carried out: i) to assess the load and type of bacteria in frozen semen of cattle and buffalo bulls of these banks, ii) and to study the antibiotic sensitivity pattern of the isolated bacteria.</p><p>A total of 108 frozen semen samples were received for determining the microbial quality of semen over a period of one year. These samples were brought in sealed straws placed in liquid nitrogen containers from four frozen semen banks F58 located in organized sector in Haryana. Standard plate count method was used to determine the bacterial load by making 10-fold dilution of semen in brain heart infusion broth. The samples were inoculated on nutrient agar, blood agar and MacConkey's lactose agar for primary isolation. The plates were incubated aerobically at 37°C for 48 hours. The isolated organisms in pure culture were identified on the basis of morphological, cultural and biochemical characteristics (<xref ref-type="bibr" rid="bib005">Quinn and Carter, 1994</xref>). All the isolated bacteria were tested for <italic>in vitro</italic> sensitivity against nine commercial antibiotics by single disc diffusion method (<xref ref-type="bibr" rid="bib002">Baeur <italic>et al.</italic>, 1966</xref>).</p><p>All the frozen semen were used for determining the microbial load. Forty eight (44.44 %) of the total samples screened were found sterile. The percentage contamination of semen samples varied between 42.86% at FSB2 to 69.33% at FSB3 (<xref ref-type="table" rid="tab001">Table 1</xref>). The bacterial load in the contaminated samples was in the range of 4 CFU/ml to 3.6 × 105 CFU/ml. The bacterial load in the contaminated samples at individual frozen semen bank was in the range of 10.00–3.60 × 105, 5.10 × 101–9.10 × 104, 4.00–1.20 × 104, and 6.25 × 101–1.28 × 104 at FSB1, FSB2, FSB3, and FSB4, respectively (Tables <xref ref-type="table" rid="tab001">1</xref>, <xref ref-type="table" rid="tab002">2</xref>). <table-wrap id="tab001"><label>Table 1</label><caption><p>Bacterial load in semen samples of various frozen semen banks</p></caption><table><thead><tr><th align="center">Frozen Seamen bank FSB</th><th align="center">Total samples(%)</th><th align="center">Sterile samples(%)</th><th align="center">Bacterial load in contaminated samples (cfu/ml)</th><th align="left">Bacteria isolated</th></tr></thead><tbody><tr><td colspan="5"><hr /></td></tr><tr><td align="center">FSB1</td><td align="center">39</td><td align="center">23 (58.97)</td><td align="center">10.00–3.60 × 10<sup>5</sup></td><td><italic>Bacillus spp., Corynebacterium renale, Staphylococcus aeureus, Pseudomonas aeroginosa</italic></td></tr><tr><td align="center">FSB2</td><td align="center">26</td><td align="center">18 (69.23)</td><td align="center">5.10 × 10<sup>1</sup>−9.10 × 10<sup>4</sup></td><td><italic>Bacillus spp., Corynebacterium renale, Staphylococcus aeureus, Pseudomonas aeroginosa, Corynebacterium spp</italic>.</td></tr><tr><td align="center">FSB3</td><td align="center">28</td><td align="center">12 (42.86)</td><td align="center">4.00–1.20 × 10<sup>4</sup></td><td><italic>Bacillus spp., Corynebacterium renale, Staphylococcus aeureus, Streptococcus pyogenes, Pseudomonas aeroginosa</italic></td></tr><tr><td align="center">FSB4</td><td align="center">15</td><td align="center">07 (46.67)</td><td align="center">6.25 × 10<sup>1</sup>−1.28 × 10<sup>4</sup></td><td><italic>Bacillus spp., Corynebacterium renale, Staphylococcus aeureus, Staphylococcus epidermidis</italic></td></tr><tr><td align="center">Total</td><td align="center">108</td><td align="center">60 (55.56)</td></tr></tbody></table></table-wrap><table-wrap id="tab002"><label>Table 2</label><caption><p>Range of Bacterial load in frozen semen samples</p></caption><table><thead><tr><th rowspan="3" align="left">Range of Bacterial load in frozen semen samples (cfu/ml)</th><th colspan="4" align="center">Number of semen samples in various frozen semen banks</th></tr><tr><th colspan="4"><hr /></th></tr><tr><th align="center">FSB1</th><th align="center">FSB2</th><th align="center">FSB3</th><th align="center">FSB1</th></tr></thead><tbody><tr><td colspan="5"><hr /></td></tr><tr><td>Sterile</td><td align="center">16</td><td align="center">08</td><td align="center">16</td><td align="center">08</td></tr><tr><td>1-10</td><td align="center">01</td><td align="center">Nil</td><td align="center">01</td><td align="center">Nil</td></tr><tr><td>11-100</td><td align="center">Nil</td><td align="center">03</td><td align="center">03</td><td align="center">01</td></tr><tr><td>101-1000</td><td align="center">07</td><td align="center">03</td><td align="center">03</td><td align="center">03</td></tr><tr><td>1001-10000</td><td align="center">10</td><td align="center">09</td><td align="center">04</td><td align="center">02</td></tr><tr><td>10001-100000</td><td align="center">04</td><td align="center">03</td><td align="center">01</td><td align="center">01</td></tr><tr><td>100001-1000000</td><td align="center">01</td><td align="center">Nil</td><td align="center">Nil</td><td align="center">Nil</td></tr><tr><td><bold>Total</bold></td><td align="center"><bold>39</bold></td><td align="center"><bold>26</bold></td><td align="center"><bold>28</bold></td><td align="center"><bold>15</bold></td></tr></tbody></table></table-wrap></p><p>The bacterial load estimated in the semen samples from organized sector semen banks of Haryana was less than reported from organized sector in other states such as Gujarat (<xref ref-type="bibr" rid="bib003">Kher and Dholakia, 1987</xref>), Andhra Pradesh (<xref ref-type="bibr" rid="bib006">Reddy et al., 1971</xref>) and Tamil Nadu (<xref ref-type="bibr" rid="bib004">Naidu et al., 1982</xref>). However, efforts should be made to reduce it further.</p><p>Sixty semen samples of the 108 tested were found positive for one or more than one type of bacteria. Of the positive samples, 43 yielded only a single type of bacteria whereas 17 positive samples yielded more than one type of bacteria. A total of 78 different bacterial isolates were identified. The bacterial isolates in these samples were identified as Bacillus spp. (41.03%), Corynebacterium renale (24.36%), Corynebacterium spp. (2.56%), Staphylococcus aeureus (17.95%), Staphylococcus epidermidis (2.56%), Streptococcus pyogenes (3.85%), Pseudomonas aeroginosa (7.69%), The proportion of Bacillus spp. isolated from the semen samples was highest, which is similar to the findings of other workers (<xref ref-type="bibr" rid="bib004">Naidu et al., 1982</xref>; <xref ref-type="bibr" rid="bib008">Saikia et al., 1987</xref>, <xref ref-type="bibr" rid="bib009">Singh et al, 1995</xref>). The isolation of Corynebacterium renale, Staphylococcus aeureus, and Pseudomonas aeroginosa in the present study is a significant because of their deleterious effects on spermatozoa as has been reported by other workers (<xref ref-type="bibr" rid="bib004">Naidu et al., 1982</xref>; <xref ref-type="bibr" rid="bib008">Saikia et al., 1987</xref>).</p><p>The semen-borne pathogens have been classified into four control-related categories viz. i). Specific pathogen free category ii). Control by surveillance category iii). Control by sanitation and hygiene category iv). Control by addition of antibiotic to the organisms falling under category iii. (<xref ref-type="bibr" rid="bib007">Roberts,1971</xref>). All the above bacteria come under categories iii. and iv. Therefore efforts should be made to improve the hygiene and sanitation at the 4AI centres in states.</p><p>The antibiotic sensitivity using nine different antibiotics on some of these isolates revealed ciprofloxacin (92.46%) and followed by amikacin (82.74%) to be effective. The sensitivity of the isolates to other antibiotics used was norfloxacin (73.28%), ofloxacin (64.98%), chlortetracycline (52.57%), polymyxin B (14.96%), penicillin (17.27%), furaxone (8.29%) and streptomycin (48.09%). Penicillin and streptomycin are the two antibiotics incorporated in the extender to reduce the bacterial load of semen. However, the present study revealed that these two antibiotics are less effective than many of other antibiotics used against the bacterial flora of semen. These findings are in agreement with that of earlier workers (<xref ref-type="bibr" rid="bib008">Saikia <italic>et al.</italic>, 1987</xref>; <xref ref-type="bibr" rid="bib001">Ahmed <italic>et al</italic>, 2001</xref>). It can be concluded that the ciprofloxacin or amikacin in place of penicillin and streptomycin may be used in the semen extender to control the bacteria as has also been suggested by other workers (<xref ref-type="bibr" rid="bib008">Saikia <italic>et al.</italic>, 1987</xref>; <xref ref-type="bibr" rid="bib001">Ahmed <italic>et al.</italic>, 2001</xref>). Moreover, the untoward action of these antibiotics on spermatozoa has to be assessed before recommending these antibiotics for semen.</p></sec></body><back><ack id="ack001"><title>Acknowledgements

IJCMIID

Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases

0970-93200974-0147

Indian Association of Veterinary Microbiologists, Immunologists and Specialists in Infectious Diseases

ijcmiid-24-2-021

Short Communication

Isolation of aerobic bacteria in frozen semen of cattle and buffaloes

Kapoor

Department of Veterinary Microbiology CCS Haryana Agricultural University, Hisar–125 004 (Haryana)

7-122003

2422072082432004

<p>Artificial insemination (AI), has been used as a powerful tool for livestock improvement by the breeders. AI greatly increases the utilization of proven sires. It ensures improvement in conception rates if semen is handled properly and the animals are bred at proper time. Successful AI largely depends on good quality semen. The frozen semen can be stored for many months to years in liquid nitrogen and has largely supplemented liquid semen for AI.</p><p>In case semen carries microorganisms, cryopreservation can spread diseases among cattle and buffalo population. Pathogens in semen may come from testes or epididymus, the accessory glands, the vas deferens, the urethra, or the prepuce or the penis. Even under careful conditions semen may get contaminated at the time of most collection or subsequent handling. Unhygienic surroundings, repeated entry of penis into artificial vagina (AV), extenders, and straw have been reported to adversely affect the quality of semen by increasing the number of microorganisms. Many bacterial agents that are transmitted through semen affect the fertility of dam leading to repeat breeding amd infertility. Measures taken by the industry and regulatory authorities, to prevent or control microbial contamination of semen, have included maintaining groups of bulls free from specific diseases and adding antimicrobial agents to the extended semen.</p><p>There are four frozen semen banks (FSB) located in organized sector in Haryana that cater to the need of AI programme of the state. However, the literature reveals no published reports on the microbial flora of semen of bulls in these banks. Therefore, the present investigation was carried out: i) to assess the load and type of bacteria in frozen semen of cattle and buffalo bulls of these banks, ii) and to study the antibiotic sensitivity pattern of the isolated bacteria.</p><p>A total of 108 frozen semen samples were received for determining the microbial quality of semen over a period of one year. These samples were brought in sealed straws placed in liquid nitrogen containers from four frozen semen banks F58 located in organized sector in Haryana. Standard plate count method was used to determine the bacterial load by making 10-fold dilution of semen in brain heart infusion broth. The samples were inoculated on nutrient agar, blood agar and MacConkey's lactose agar for primary isolation. The plates were incubated aerobically at 37°C for 48 hours. The isolated organisms in pure culture were identified on the basis of morphological, cultural and biochemical characteristics (<xref ref-type="bibr" rid="bib005">Quinn and Carter, 1994</xref>). All the isolated bacteria were tested for <italic>in vitro</italic> sensitivity against nine commercial antibiotics by single disc diffusion method (<xref ref-type="bibr" rid="bib002">Baeur <italic>et al.</italic>, 1966</xref>).</p><p>All the frozen semen were used for determining the microbial load. Forty eight (44.44 %) of the total samples screened were found sterile. The percentage contamination of semen samples varied between 42.86% at FSB2 to 69.33% at FSB3 (<xref ref-type="table" rid="tab001">Table 1</xref>). The bacterial load in the contaminated samples was in the range of 4 CFU/ml to 3.6 × 105 CFU/ml. The bacterial load in the contaminated samples at individual frozen semen bank was in the range of 10.00–3.60 × 105, 5.10 × 101–9.10 × 104, 4.00–1.20 × 104, and 6.25 × 101–1.28 × 104 at FSB1, FSB2, FSB3, and FSB4, respectively (Tables <xref ref-type="table" rid="tab001">1</xref>, <xref ref-type="table" rid="tab002">2</xref>). <table-wrap id="tab001"><label>Table 1</label><caption><p>Bacterial load in semen samples of various frozen semen banks</p></caption><table><thead><tr><th align="center">Frozen Seamen bank FSB</th><th align="center">Total samples(%)</th><th align="center">Sterile samples(%)</th><th align="center">Bacterial load in contaminated samples (cfu/ml)</th><th align="left">Bacteria isolated</th></tr></thead><tbody><tr><td colspan="5"><hr /></td></tr><tr><td align="center">FSB1</td><td align="center">39</td><td align="center">23 (58.97)</td><td align="center">10.00–3.60 × 10<sup>5</sup></td><td><italic>Bacillus spp., Corynebacterium renale, Staphylococcus aeureus, Pseudomonas aeroginosa</italic></td></tr><tr><td align="center">FSB2</td><td align="center">26</td><td align="center">18 (69.23)</td><td align="center">5.10 × 10<sup>1</sup>−9.10 × 10<sup>4</sup></td><td><italic>Bacillus spp., Corynebacterium renale, Staphylococcus aeureus, Pseudomonas aeroginosa, Corynebacterium spp</italic>.</td></tr><tr><td align="center">FSB3</td><td align="center">28</td><td align="center">12 (42.86)</td><td align="center">4.00–1.20 × 10<sup>4</sup></td><td><italic>Bacillus spp., Corynebacterium renale, Staphylococcus aeureus, Streptococcus pyogenes, Pseudomonas aeroginosa</italic></td></tr><tr><td align="center">FSB4</td><td align="center">15</td><td align="center">07 (46.67)</td><td align="center">6.25 × 10<sup>1</sup>−1.28 × 10<sup>4</sup></td><td><italic>Bacillus spp., Corynebacterium renale, Staphylococcus aeureus, Staphylococcus epidermidis</italic></td></tr><tr><td align="center">Total</td><td align="center">108</td><td align="center">60 (55.56)</td></tr></tbody></table></table-wrap><table-wrap id="tab002"><label>Table 2</label><caption><p>Range of Bacterial load in frozen semen samples</p></caption><table><thead><tr><th rowspan="3" align="left">Range of Bacterial load in frozen semen samples (cfu/ml)</th><th colspan="4" align="center">Number of semen samples in various frozen semen banks</th></tr><tr><th colspan="4"><hr /></th></tr><tr><th align="center">FSB1</th><th align="center">FSB2</th><th align="center">FSB3</th><th align="center">FSB1</th></tr></thead><tbody><tr><td colspan="5"><hr /></td></tr><tr><td>Sterile</td><td align="center">16</td><td align="center">08</td><td align="center">16</td><td align="center">08</td></tr><tr><td>1-10</td><td align="center">01</td><td align="center">Nil</td><td align="center">01</td><td align="center">Nil</td></tr><tr><td>11-100</td><td align="center">Nil</td><td align="center">03</td><td align="center">03</td><td align="center">01</td></tr><tr><td>101-1000</td><td align="center">07</td><td align="center">03</td><td align="center">03</td><td align="center">03</td></tr><tr><td>1001-10000</td><td align="center">10</td><td align="center">09</td><td align="center">04</td><td align="center">02</td></tr><tr><td>10001-100000</td><td align="center">04</td><td align="center">03</td><td align="center">01</td><td align="center">01</td></tr><tr><td>100001-1000000</td><td align="center">01</td><td align="center">Nil</td><td align="center">Nil</td><td align="center">Nil</td></tr><tr><td><bold>Total</bold></td><td align="center"><bold>39</bold></td><td align="center"><bold>26</bold></td><td align="center"><bold>28</bold></td><td align="center"><bold>15</bold></td></tr></tbody></table></table-wrap></p><p>The bacterial load estimated in the semen samples from organized sector semen banks of Haryana was less than reported from organized sector in other states such as Gujarat (<xref ref-type="bibr" rid="bib003">Kher and Dholakia, 1987</xref>), Andhra Pradesh (<xref ref-type="bibr" rid="bib006">Reddy et al., 1971</xref>) and Tamil Nadu (<xref ref-type="bibr" rid="bib004">Naidu et al., 1982</xref>). However, efforts should be made to reduce it further.</p><p>Sixty semen samples of the 108 tested were found positive for one or more than one type of bacteria. Of the positive samples, 43 yielded only a single type of bacteria whereas 17 positive samples yielded more than one type of bacteria. A total of 78 different bacterial isolates were identified. The bacterial isolates in these samples were identified as Bacillus spp. (41.03%), Corynebacterium renale (24.36%), Corynebacterium spp. (2.56%), Staphylococcus aeureus (17.95%), Staphylococcus epidermidis (2.56%), Streptococcus pyogenes (3.85%), Pseudomonas aeroginosa (7.69%), The proportion of Bacillus spp. isolated from the semen samples was highest, which is similar to the findings of other workers (<xref ref-type="bibr" rid="bib004">Naidu et al., 1982</xref>; <xref ref-type="bibr" rid="bib008">Saikia et al., 1987</xref>, <xref ref-type="bibr" rid="bib009">Singh et al, 1995</xref>). The isolation of Corynebacterium renale, Staphylococcus aeureus, and Pseudomonas aeroginosa in the present study is a significant because of their deleterious effects on spermatozoa as has been reported by other workers (<xref ref-type="bibr" rid="bib004">Naidu et al., 1982</xref>; <xref ref-type="bibr" rid="bib008">Saikia et al., 1987</xref>).</p><p>The semen-borne pathogens have been classified into four control-related categories viz. i). Specific pathogen free category ii). Control by surveillance category iii). Control by sanitation and hygiene category iv). Control by addition of antibiotic to the organisms falling under category iii. (<xref ref-type="bibr" rid="bib007">Roberts,1971</xref>). All the above bacteria come under categories iii. and iv. Therefore efforts should be made to improve the hygiene and sanitation at the 4AI centres in states.</p><p>The antibiotic sensitivity using nine different antibiotics on some of these isolates revealed ciprofloxacin (92.46%) and followed by amikacin (82.74%) to be effective. The sensitivity of the isolates to other antibiotics used was norfloxacin (73.28%), ofloxacin (64.98%), chlortetracycline (52.57%), polymyxin B (14.96%), penicillin (17.27%), furaxone (8.29%) and streptomycin (48.09%). Penicillin and streptomycin are the two antibiotics incorporated in the extender to reduce the bacterial load of semen. However, the present study revealed that these two antibiotics are less effective than many of other antibiotics used against the bacterial flora of semen. These findings are in agreement with that of earlier workers (<xref ref-type="bibr" rid="bib008">Saikia <italic>et al.</italic>, 1987</xref>; <xref ref-type="bibr" rid="bib001">Ahmed <italic>et al</italic>, 2001</xref>). It can be concluded that the ciprofloxacin or amikacin in place of penicillin and streptomycin may be used in the semen extender to control the bacteria as has also been suggested by other workers (<xref ref-type="bibr" rid="bib008">Saikia <italic>et al.</italic>, 1987</xref>; <xref ref-type="bibr" rid="bib001">Ahmed <italic>et al.</italic>, 2001</xref>). Moreover, the untoward action of these antibiotics on spermatozoa has to be assessed before recommending these antibiotics for semen.</p></sec></body><back><ack id="ack001"><title>Acknowledgements

The author gratefully acknowledges the help and facilities provided by Dr S. K. Kalra, Director, ICAR Center of Advanced Studies in Veterinary Microbiology, CCS Haryana Agricultural University, Hisar.

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