The objectives of this trial were to estimate prevalence of bacteriospermia, to determine the bacterial load, and to isolate the types of bacteria as well as to assess the association between bacterial load and sperm quality traits in cryopreserved bull semen in field conditions in the South Wollo Zone. A total of 309 cryopreserved straws of semen from the Holstein Friesian (HF)-cross bull (n = 180 straws) and pure Jersey bull (n = 129 straws) were investigated. Bacteriological assessments of the presence of aerobic bacteria, estimation of bacterial count and bacterial isolation, as well as semen quality were performed. Aerobic bacterial contamination was prevalent in 38.8% of the semen straws. No significant difference in the prevalence of bacteriospermia was observed among bulls although the HF-cross bull had a higher prevalence (40.0%). But, significant difference in prevalence of bacteriospermia was found among semen ejaculates of the same bull. The risk of bacteriospermia in the HF-cross bull was higher (Odds ratio = 1.86, 95% CI = 0.168–20.26) compared to Jersey although not significant. Overall average bacterial load of 50.38 ± 16.29 colony-forming units (CFU)/ml (from nil to 1318.20 CFU/ml) was found. No significant difference in bacterial count among bulls and their ejaculates was observed. Moreover, correlation analysis revealed that the proportions of motility, live, and normal morphology were negatively influenced by an increase in the bacterial contamination of semen. In this study, three isolates of coagualse-negative Staphylococcus species and one isolate of Corynebacterium species were found. Average percentages of sperm motility (48.35 ± 1.23), live (66.08 ± 1.0), and normal morphology (80.62 ± 1.24) were observed. It was concluded that cryopreservation does not guarantee the quality of semen from bacterial contamination. Hence, meticulous care should be adopted to prevent contamination of semen by bacteria during collection, transportation, processing, and storage times.
1. Introduction
Ethiopia inhabits significant proportion of cattle population in Africa with its 56.71 million heads of cattle [1]. Indigenous cattle are the main livestock species used for dairy production in Ethiopia, which contributes around 81.2% of the total national annual milk yield [2]. Although the country has a huge potential for milk production, the genetic potential and productivity of local breeds, as a matter of fact, is very low [3]. Consequently, the direct contribution of the dairy sector to the national economy is inadequate [1]. More recently, the country has designed a dairy cattle improvement plan to enhance productivity of local breeds through genetic selection, breeding, and artificial insemination (AI) programs [4]. So as to realize this, accurate prediction of bull fertility is essential because it determines the economic success and sustainability of the dairy industry. This in turn is reliant on obtaining high conception rates after insemination of cows using frozen-thawed semen [5].
AI is the oldest and most popular assisted reproductive biotechnology allowing the use of genetically superior males from elsewhere for genetic improvement of livestock [6]. Long-term storage of semen in liquid nitrogen (−196°C), through cryopreservation, is indispensable in order to realize many of the potential advantages of AI. Cryopreservation halts the metabolic activities of spermatozoa, which allows unlimited storage without substantial loss of fertility. Hence, quality of frozen-thawed semen is an important factor that predicts bull fertility and the sustainability of AI as well as investment of bulls with high genetic merits [7]. However, quality of semen can be altered, among others, by the process of cryopreservation [8–13], types of extender used [14], and bacterial contamination [15–18].
Prevalence of bacteria within sperm “bacteriospermia” had detrimental effects on sperm cell function by reducing sperm motility, viability, and abnormal morphology as well as premature acrosome reaction. Other ways by which bacteriospermia affects fertility is by altered mitochondrial function, which provokes formation of reactive oxygen species thereby increasing DNA fragmentation [15, 19]. These effects of bacteria have been reported to negatively impact the viability and fertility of spermatozoa, thereby resulting in total breeding failure. Thus, one of the main factors contributing to AI failure is the contamination of the germplasm with pathogens, which eventually cause loss of fertility [20]. Most common microorganisms that contaminated bull semen were from species of Corynebacterium, Staphylococcus, Micrococcus, Bacillus, Escherichia, Proteus, Pseudomonas, Klebsiella, Streptococcus, Citrobacter, Enterobacter, and Stenotrophomonas [21–25]. These pathogens, in one way or other, may infect inseminated females or contribute to a rapid deterioration in sperm quality [26]. Nevertheless, scientific evidence based on published data concerning the major risk pathogens or bacterial contamination of the germplasms used in AI operations in Ethiopia has been lacking until recently.
In Ethiopia, AI has been applied for over five decades to crossbred indigenous cattle with bulls of known genetic merits imported from different countries. Although AI in Ethiopia has a long history, the success rate of AI in Ethiopia has been and is generally poor. The National AI Centre (NAIC) is the chief source of semen and other consumables for the procedures of AI throughout the whole country. However, many constraints have been reported associated with the source, the selection procedures, and health status of the AI bulls at NAIC [27]. This was further revealed by the unsatisfactory success of AI in regional and district AI centres due to different factors. The major constraints have been linked to lack of infrastructure, lack of AI technician’s experience, or due to heat detection problems, management problems, and disease problems [28, 29]. These factors coupled with environmental causes, improper handling, transportation, and storage of semen that deteriorates semen quality including motility, thereby affecting subsequent fertility [30]. Nevertheless, the effects of microbiological contaminants such as bacteria in bull (fresh or frozen) semen have not been assessed in Ethiopia, yet. Thus, this research explores the prevalence of bacteriospermia and semen quality of cryopreserved semen of bulls used in AI of cows in the South Wollo Zone, Ethiopia. Moreover, bacterial load and the types of bacterial contaminants of cryopreserved semen were investigated for the first time in Ethiopia.
2. Materials and Methods
2.1. Description of Study Area
For this study, cryopreserved French ministraws of bull semen were purchased from South Wollo Zonal Liquid Nitrogen Production and Semen Distribution Centre (SWLNPSDC), Dessie, Ethiopia. Dessie, the largest city in South Wollo Zone of the Amhara Region, is located in the north central part of the country at a distance of 401 km north of Addis Ababa. This city is placed at latitude of 11′8°N and longitude of 39′38°E with an altitude range of 2470 to 2550 meters above sea level. The area has an average annual rainfall of 1145 mm and a mean annual temperature of 15.2°C. A combination of crop and livestock production is the main farming system of the precinct. The total cattle population of the district was estimated at 1.75 million cattle [1]. Both natural service and artificial insemination (AI) are used for crossbreeding of dairy cows for genetic improvement of local breeds despite its low achievement. Semen and other accessories for AI is obtained from the NAIC, Kality, Ethiopia, and stored in the SWLNPSDC. Semen and liquid nitrogen from SWLNPSDC for AI are dispatched to the district AI centres located in South Wollo Zone (Figure 1).