PCR DETECTION OF GENITAL INFECTIONS IN BULL SEMEN FROM DIFFERENT REGIONS OF UKRAINE , 2013-2016

Th e most important infectious diseases that aff ect fertility of the bull via semen are reviewed in this article. Th e article describes the results of a study of cryopreserved semen samples from diff erent regions of Ukraine for the period 2013 to 2016. Th e strategy of regular testing of semen donors under offi cial veterinary supervision has been adopted by governments worldwide as a means of avoiding the spread of pathogens and reducing excessive contamination of semen by ubiquitous bacteria and viruses. During this period, four hundred fi ft y (n=450) bull semen samples from 10 farms across Ukraine have been tested, PCR diagnostics was performed by the standard method developed in NSC «IECVM». According to the PCR analysis of cryopreserved semen from the bull-sires, 19 samples (6%) from Kharkiv region contained genetic material of Mycoplasma spp., 2 samples from Poltava region (3.5%) were positive for BoHV-1, and 2 samples from Cherkasy region (4.5%) contained genetic material of Chlamydia spp. Based on the obtained results we can conclude that it is necessary to permanently perform PCR screening of bulls semen.


INTRODUCTION
Th e main goal of artifi cial insemination (AI) in cattle is to achieve genetic improvement.However, transmission of infectious diseases by semen (sexual transmission, ST) constitutes a risk that must be avoided.Th e semen used for AI must, therefore, be free of infectious agents (Wentink et al., 2000).Th e bull semen also plays a key role in reproductive excrete in cow herds, acting as a source of infectious diseases.Th e regular testing of semen donors under offi cial veterinary supervision has been adopted by governments worldwide as a means of avoiding the spread of pathogens and reducing excessive contamination of semen by ubiquitous bacteria and viruses (Eaglesome et al., 1997).
Abortion among dairy cows is one of the sources of substantial economic losses in cattle industry.Although the risk of abortion depends on several factors (i.e., genetic abnormalities, heat stress, toxic agents), infectious agents are likely to be one of the most important risk factors associated with abortions.A variety of sexually transmitted infectious agents has been reported to cause bovine abortion throughout the world (Tramuta et al., 2011).Hence, one of the main goals of the present communication is to review major pathogens associated with ST in cattle.

Infectious bovine rhinotracheitis (IBR)
. Th e infection with Bovineherpesvirus, leading to balanoposthitis in bulls and infectious pustular vulvovaginitis (IPV) in cows, has been recognized for several decades.It is a complex, baffl ing and changing disease, giving rise to much controversy, with prophylaxis proving diffi cult to devise and implement.Th e etiological agent, Bovine herpesvirus 1 (BoHV-1) is a double-stranded DNA (dsDNA) virus and is a member of subfamily Alphaherpesvirinae, genus Varicellovirus.Th e virus is not very resistant, and it is transmitted directly by respiratory, venereal or bucco-genital routes or indirectly via contaminated hands of a farmer or a semen collector.Th e infection can occur in both males and females without obvious clinical signs, or can be accompanied by non-specifi c infl ammation, granulation tissue, vesicles and ulceration (which may be due to secondary infection with non-specifi c microorganisms) (Parez et al., 1985).
Bovine viral diarrhea (BVD).Bovine viral diarrhea virus (BVDV) is a pestivirus from the family Flaviviridae (Becher and Th iel, 2011), capable of causing serious clinical disease in cattle.Th e virus is divided into two genotypes (BVDV-1 and BVDV-2) on the basis of antigenic and genetic diff erences (Vilcek et al., 2005).Infection with BVDV is known to have a signifi cant financial impact (Houe, 1999), stemming primarily from the reproductive and immunosuppressive eff ects of acute infection.Th e most common economic losses resulting from BVDV infection are associated with failure in fertilization, abortion, congenital malformation, stillbirth or birth of persistently infected (PI) progeny (Grahn et al., 1984;Rüfenachtet al., 2001).
Chlamydial infections.Recognised for some time as the cause of epidemic abortion in cows in the USA, Chlamydia psittaci in Europe is associated with sporadic abortion (at 3 to 7 months) due to necrotic placentitis and a direct eff ect on the fetus (producing liver lesions).Infertility associated with vaginitis and endometritis was also described.
Chlamydiae are obligate intracellular, gram-negative bacteria with atypical developmental life cycle.During the last ten years, four Chlamydia species have been isolated from humans and from diff erent domestic and wild animals.Infection with chlamydial bacteria, specifi cally C. pecorum, C. abortus, and C. psittaci, are common in cattle.An infection with chlamydia may cause sub-or infertility.Infected bulls may suff er from vesiculitis, or may not be affected at all (Storz et al., 1968).
Mycoplasmoses.Diff erent species of Mycoplasmas can aff ect bovines causing several diseases.Mycoplasmas can cause clinical, subclinical or chronic intramammary infection aff ecting cattle of all ages and at any stage of lactation (Tamiozzo et al., 2014).Th e numerous mycoplasmas involved in diseases of cattle create a complex situation with regard to lesions and the causal agent.Species commonly associated with genital tract infection in Europe are Mycoplasma bovigenitalium, Mycoplasma bovis, Acholeplasma laidlawii and Ureaplasma spp.Organisms of the genera Mycoplasma, Acholeplasma and Ureaplasma have been isolated from the distal part of both male and female bovine genital tract.Mycoplasma bovis, M. bovigenitalium and U. diversum appear to be the most important pathogens of the genital tract, involved in diseases such as decreased sperm motility (M.bovigenitalium), seminal vesiculitis, epididymitis (M.bovis and M. bovigenitalium), endometritis (M.bovis), granular vulvovaginitis, infertility and abortion (U.diversum) (Fish et al., 1985).
Solving the problem of animal pathology is the key to successful management of industrial livestock in Ukraine (Stegniy et. al., 2015).Most common infectious diseases transmitted via bull semen in Ukraine include infectious bovine rhinotracheitis (IBR), bovine viral diarrhea (BVDV), chlamydioses, and mycoplasmoses.Hence, it is important to focus on the control of genetic resources in breeding work in order to eff ectively break the epizootic chain of the diseases.

MATERIALS AND METHODS
Monitoring studies for ST infections were conducted from 2013 to 2016.During this time, four hundred fi ft y (n=450) bull semen samples from 10 farms across Ukraine have been tested.PCR diagnostics was performed by the standard method developed in National Scientifi c Center "Institute for Experimental and Clinical Veterinary Medicine" (NSC «IECVM») (Stegniy et. al., 2015).Isolation of total DNA was performed using a commercial kit for extraction of nucleic acids "DNA Sorb-B", produced by "Central Research Institute of Epidemiology" (Russian Federation).Reverse transcription was performed using a set of reagents RT-Core production company IsoGene (Russian Federation).
Th e amplifi cation reaction was performed using commercial set «PCR-Core» manufactured by IsoGene systems and primers (Table 1).

RESULTS AND DISCUSSIONS
Th e analysis of research samples of cryopreserved semen from the bullsires for PCR revealed that genetic material of Mycoplasma spp was contained in 19 samples (Fig. 1) (6%), which came from farms in Kharkiv region, genetic material of BoHV-1 pathogen was detected in 2 (3.5%) positive samples from Poltava region, and 2 samples (4.5%) containing genetic material of the pathogen Chlamydia spp were obtained from Cherkasy region.We compared current research results of our study regarding frozen bull semen monitoring (period 2013-2016) with the results for period of 2010-2013.Signifi cant reduction of contamination of semen samples with viruses, chlamydia and mycoplasmosis has been established (Fig. 2).

CONCLUSIONS
According to the results obtained in this study, 19 samples (6%) from Kharkiv region contained genetic material of Mycoplasma spp. in frozen bulls semen samples, 2 samples from Poltava region (3.5%) were positive for BoHV-1, and 2 samples from Cherkasy region (4.5%) contained genetic material of Chlamydia spp.
Based on the monitoring results we can conclude that regular PCR screening of bulls' semen for the most common genital infections is positively affected by the dynamics of the testing rate.At present, there are no uniform requirements for contamination monitoring of sperm for the presence of viruses and Chlamydia.Th ere are national requirements for the evaluation of semen contamination operating in Canada and the United States.Further work is therefore important to improve the system for control ofcontamination of cattle genetic resources.

Figure 1 .
Figure 1.Results for of bull semen PCR detection on gel, positive sample of genetic material of Mycoplasma spp.Track number 1 -positive sample, lane number 3 -100 base pair (bp) DNA ladder plus, lane number 4 -positive control.

Figure 2 .
Figure 2. Comparison of PCR screening studies for the period 2010-2012 with the research conducted in the period 2013-2016.

Table 1 .
Primers positive control samples and the size of amplicon generated for IBR, BVDV, and Chlamydia