The most common contaminants in the semen are mostly the bacterial population of E. coli, Bacillus sp., Pseudomonas sp., Staphylococcus sp., and their increasing load may affect the motility and viability of sperms in the semen (Gupta and Sinha, 2001). Hence, prevention of contamination of semen is very essential for normal reproduction.

The main source of bacteria present in the semen of healthy animals seems to be the prepuce, and the smegma that is mixed with the semen during the ejaculation (Hare, 1985). Other sources of contamination include inflammatory foci in the genital tract, the skin of animals, faulty method of semen collection and equipment cleaning, hygienic status of animal handlers and laboratory personnel, and semen extenders (Kendrick et al., 1975; Reik et al., 1980).

The present study has been undertaken to determine and compare the possible bacterial load in semen of two indigenous breeds of ram/buck from West Bengal namely, Garole rams and Black Bengal bucks.

The semen samples were collected early in the morning by AV method from each of 20 healthy fertile rams and bucks maintained in semi-intensive management at a goat and sheep farm under West Bengal University of Animal and Fishery Sciences at Haringhata, Nadia, West Bengal, during the months of May-July 2003. Before collection of semen, the prepucial area was cleaned, the hairs surrounding the prepuce were clipped and, asepsis was maintained in case of both males and rear portion of the dummies.

To measure the extent of contamination, counting of the microorganism in a sample of fresh semen was done following the method described by O.I.E. (Office International des Epizootics, 1999) with slight modification.

The semen samples were diluted (10⁻¹, 10⁻² and 10⁻³) with PBS (phosphate buffered saline) and then transferred to petridishes. To each petridish, about 15 ml of Tryptic soy agar (TSA) was added and cooled to 45ºC before adding 1 ml of sample dilution to it. It was mixed thoroughly by circular agitation and allowed to solidify on a levelled surface. The surface of the inoculated and cooled medium was then covered with 4ml of white agar, previously cooled to 45ºC to prevent the growth of certain saprophytic bacteria. The dishes were then incubated at 37ºC for 72 hrs.

The colonies were counted following standard procedure of current international standard ISO 4833. The petridishes with the dilutions of 1:100 or 1:1000 having 30-300 colony forming units (CFU)/ml were considered for counting.

In the present study, all the plates were within the countable range with an average count of 8.9±1.5×10² CFU/ml and 7.4 ±1.4×10² CFU/ml in 10⁻² dilution and 2.6±1.2×10³ CFU/ml and 1.2±0.4×10³ CFU/ml in 10⁻³ dilution of semen from rams and bucks, respectively (Table 1). The bacterial contamination at both the dilutions was found to be less in Black Bengal bucks when compared to Garole rams. However, the counts were negligible and well below the acceptable limit for both the species. Therefore, the semen of these experimental animals was found to be of satisfactory grade for further processing and breeding or inseminations.

Bacterial loads of s80.9, 34.5 and 13.2 CFU, respectively, in 10⁻² 10⁻³ and 10⁻⁴ dilutions of pre-wash semen samples has been reported earlier (Naidu et al., 1991). The bacterial load in 337 semen samples of different breeds of goats viz. Barbari, Jamunapari, Marwari, Kutchi, Sirohi and Jakhrana averaged 24.75±1.0×10² CFU/ml, 16.16+0.8×193 CFU/ml and 9.36±0.6×10⁴ CFU/ml, respectively. The bacterial load has been found to be affected significantly by breeds as the semen of Jamunapari bucks showed highest bacterial load of 37.48 CFU/ml, irrespective of seasons followed by Kutchi, Jakhrana, Barbari, Sirohi, Marwari at 10⁻² dilution, age and no significant effect on the bacterial load (Gupta and Sinha, 2001) The season has been reported to have significant effect on bacterial load, which was highest in winter period and lowest in hot-humid and hot-dry periods at 10⁻² dilution. Nevertheless, most of the bacterial contaminants were found to be non-pathogenic in nature (Gupta and Sinha, 2001).

Till date, not much information is available on the regulatory threshold limit of bacterial load in buck semen. Reports on the bacterial load of buck semen are scanty (Naidu et al., 1991; Sharma and Deka, 1986) and no literature is available in this respect on Garole species. Since semen with higher bacterial load impairs the reproductive performance of male and female, the information obtained in the study might be useful in future for cryopreservation of ram and buck semen to be used for artificial insemination.

References