Artificial Insemination

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In the economy of Pakistan the livestock is important financial factor. In livestock buffalo is major dairy animal. Pakistan, being the fifth largest milk producing country of the world, has an annual gross milk production of about 42,199 million tons, with buffalo contributing 71% of the total milk production. Pakistan possesses 29 million heads of buffalo (GOP, 2007-08). Of all the domesticated animals of Asia Nili-Ravi buffalo is most adaptive and versatile animal (Riaz et al., 2008). In Pakistan, the Nili-Ravi breed originated in the valleys of Sutlej and Ravi is dominant in the Punjab province. This breed has now spread over all parts of including NWFP, Sindh, Baluchistan and Kashmir(Afzal et al., 2007). Nili-Ravi buffaloes performed far better than local and crossbred cattle in Muzaffarabad, Azad Jammu and Kashmir (Kuthu, 2007), indicating its adaptation to various environmental conditions.

In tropical countries like Pakistan and Nigeria undergo a steady development in buffalo and cow production rate (Samad, 1985; Pathiraja et al., 1988; Sekoni & Oyedipe, 1995). For genetic improvement and to enhance the milk production Artificial Insemination is most effective technique. Mostly short time storage require for Artificial inemination. As storage media is supportive for microbial growth so it is essential to add the antimicrobial agents in extender which have the ability to inhibit the excessive microbial growth (Shoushtari et al., 2007). Perfectly healthy bull semen mostly considered as microbial contamination free but these contaminations can be derived through testes, epididymis, vas deference, accessory sex glands, urethra, preputial cavity and artificial vagina (Qureshi et al., 1993). Atmosphere, teaser animals, un-sterilized equipment and semen extender may contribute the contamination of semen (Caballo, 1981).

Maintain the fertilizing capacity is the basic purpose of semen preservation and use of superior genetic bull can be maximizing by diluting this semen (Kammisrud et al., 1996). Moreover there is need of effective techniques to reduce the risk of spreading pathogens through the semen (Kapoor, 2003). Fertilizing capacity of spermatozoa can be directly effected by microorganism especially bacteria (Morrell, 2006), these bacteria can induce the acrosome reaction (El-Mulla et al., 1996), can impair the sperm's motility (Panangala et al., 1981; Kauret et al., 1986) and these bateria have the ability to adhere with spermatozoa (Wolff et al., 1993; Diemer et al., 1996). By producing toxins these bacteria can directly effects the fertilizing capacity of spermatozoa (Morrell, 2006).

The fertility potential of Artificial Insemination was significantly increased first time by addition of antibiotic in extender (de Jarnette et al., 2004). Streptomycin and pencillin is the traditional antibiotic combination that is used in buffalo bull semen extender (Sanson et al., 2000). However it is now necessary to look alternatives for the SP-containing extender for buffalo bull semen cryopreservation (Certified Semen Services (CSS®), 2002), because some problem with resistant organisms have been reported. A new combination of antibiotics comprised of gentamycin, tylosin and linco-Spectin has been used successfully to control the certain resistant microorganisms in cryopreserved bull semen (Garner, 1991).

This Study will be conducted to investigate:
(1)- Effect of 3 combinations of different antibiotic in extender on the bacterial count to improve spermatozoa quality of buffalo bull semen.


The importance of controlling bacterial populations in bovine semen used for artificial insemination has been pointed out by Salisbury et al. (1939). A significant relationship has been found between the number of bacteria in diluted semen and its fertility when all types of bacteria were considered (Bush et al., 1950). Pathogenic bacteria isolated from buffalo semen include Pseudomonas aeruginosa (Aleem et al., 1990; Ramaswarmy et al., 2002; Kapoor, 2003), Bacillus spp. (Aleem et al., 1990; Hasan et al., 2001; Ramaswarmy et al., 2002; Kapoor, 2003), Streptococcous pyogenes (Aleem et al., 1990; Kapoor, 2003), Corneybacterium spp. (Ramaswarmy et al., 2002; Kapoor, 2003), Corneybacterium renale (Kapoor, 2003), Esherichia coli (Aleem et al., 1990; Ramaswarmy et al., 2002; Hasan et al.,2001), Staphylococcus spp. (Hasan et al., 2001; Ramaswarmy et al., 2002) Aeromonas spp. and Streptococcus spp. (Ramaswarmy et al.,2002) Pseudomonas putida, Pseudomonas fluorescenas, Pseudomonas testosteroni, Streptococccus aureus, Proteus mirabilis and non-pathogenic bacteria as Micrococcus spp. (Aleem et al., 1990; Hasan et al., 2001), Diphtheroids (Aleem et al., 1990) and Proteus (Hasan et al., 2001). Delapena et al., (1996) isolated gram positive Staphylococcus aureus, Corynebacterium spp., Staphylococcus spp., Bacillus cereus, and B. subtilis, and gram negative Klebsiella pneumoniae and Pseudomonas aeruginosa from fresh buffalo semen. From frozen semen of buffalo containing antibiotic combination streptomycin-pencilline bacterial species of genus Staphylococcus, Pseudomonas (Parbhakar et al., 1993; Hasan et al., 2001), Micrococcus, E. coli (Prabhakar et al., 1993) and Proteus (Hasan et al., 2001) were isolated.

Earlier studies have shown that either pencillin or streptomycin would stop the growth of bacteria of semen without exerting any harmful effect on survival of spermatozoa (Almquist et al., 1948, 1948a). However the most effective control of bacteria took place when combination of pencillin and streptomycin was used (Almquist et al., 1949). Now researchers are trying to analysis the effect of some new antibiotics like aureomycin, polymyxin and chloromxin and chloromcetin on bacteria of bull semen. Report by Myers et al. (1950) and Sykes and Mixner (1950) and recent papers by foote and Bratton (1950) indicate that aureomycin was good for inhibiting the bacterial growth but it is relatively toxic to the motility of bull spermatozoa. Polymyxin (Foote and Brallon, 1950) nd chloromycetin (Sykes and Mixner, 1950) seem to be less harmful to the spermatozoa and these antibiotics are highly bactericidal. Aureomycin is effective against both gram-positive and gram-negative orgranisms (Paine et al., 1948; Price et al., 1948). Myers and Almquist (1951) found that concentration 12.5 and 25µg per ml of aureomycin did not effect the motility of spermatozoa but that higher level were toxic.

The combination of pencillin and neomycin has been reported to be more effective then the combination of pencillin and streptomycin currently used (sansone, 2000). Shoushtari et al. (2007) studied the effect of 8 different antibiotics on controlling bacterial contamination of buffalo semen and they found that oxytetracycline and pencillin G were effective in controlling seminal bacterial contaminations and because of adverse effect of oxytetracycline on the sperm motility and morphology, it is not suitable for this purpose, they also found that erythromycin in double dose form is effective against the bacteria of buffalo semen.


The research will be conducted at Animal Physiology Laborarty, Pir mher Ali Shah Arid Agriculture University, district Rawalpindi. The semen will be collected from mature Nili-Ravi buffalo bull (Bubalus bubalis) with a history of good fertility in the Semen Production Unit Qadirabad, District Sahiwal, Pakistan by using artificial vagina.
Semen quality will be evaluated by determination of the percentage of sperm progressive motility, viability, abnormal morphology and sperm concentration as well as volume, color and viscosity evaluation of the semen according to the procedure recommended by Ax et al., (2000) and Barth (1997) by using a light microscope with pre-warmed stage (37○C) at x400 magnification. The sample then will be diluted by tris extender, semen quality will be re evaluated to ensure that the dilution will not affect the semen quqlity.


GOP, 2007-08. Economic Survey, Economic Advisor Wing, Finance Division, Govt. Pakistan, Islamabad, Pakistan.

Riaz, M.N., N. A. Malik., F. Nasreen and J. A. Qureshi. 2008. Molecular marker assisted study of kappa-casein gene in nili-ravi (buffalo) breed of Pakistan. Pak. Vet. J., 28(3): 103-106.

Afzal, M., M. Anwar and M. A. Mirza. 2007. some factors affecting milk yield and lactation length in nili ravi buffaloes. Pakistan Vet. J., 2007, 27(3): 113-117.

Kuthu, Z. H., 2007. A study on seasonality of breeding and reproduction in Nili-Ravi buffaloes, indigenous (non-descript) and cross bred (Jersey x indigenous) cows in Muzaffarabad (Azad Jammu and Kashmir). MSc (Hons) Thesis, Allama Iqbal Open University, Islamabad, Pakistan.