Infertility in Buffalo Bulls

Subfertility in breeding buffalo bulls is a major reason for unsuitability for breeding [1-4]. As buffalo bulls age increase (up to 10-14 y), they have decreases in semen quality and fertility [5-8]. Furthermore, a key environmental factor reducing fertility is hot weather [9-11], as buffalo bulls exposed to heat stress have reduced scrotal circumference and seminal quality [12-20]. In addition, poor libido often hampers breeding [2,21] (23.7%), as it is associated with lameness, scrotal dermatitis, a wide scrotal neck, and various structural abnormalities [22-25]. Testicular (25.0%) and scrotal (13.1%) pathologies were the most frequent, followed by those involving the epididymis (4.3%), penis and prepuce (0.6%) [26]. In Nili-Ravi buffaloes in Pakistan, 35% of bulls were infertile, most commonly due to testicular problems [27]. In Brazilian buffaloes, 80% of pathologies in infertile buffalo bulls involved testes or epididymis, whereas penis, prepuce and accessory sex glands were rarely affected [28]. Charan and Verma [29] reported that the most common pathologies were those involving the testes (52.6%) followed by the epididymis (29.0%) and urethra (18.4%).

Historically, diagnosis of pathology involving the reproductive organs of male buffaloes was primarily based on observation and palpation in live animals, or histological examination of abattoir-derived specimens [22,29]; however, more recently, ultrasonography has also been used [24,30-32]. Once a diagnosis has been established, therapy of many pathological changes in male buffaloes is often neither possible nor desirable. Reproductive physiology and sexual behavior of male buffalo has been described in a previous chapter [33], whereas causes of infertility are described in the current chapter.

Infertility in Buffalo Bulls

The use of buffalo bulls for natural service is still common, with high conception rates frequently achieved [34]. However, collection of buffalo semen for use in artificial insemination (AI) is increasing. Currently there are 44 semen collection centers in India alone, with annual production of approximately 85 million frozen semen doses (FSDs) for domestic use. According to a news report, a single ejaculate from a champion buffalo bull was worth $3000 [35]. Moreover, since many diseases can be transmitted via semen, the importance of buffalo bull evaluation has increased. Although predictions of bull fertility are based on seminal quality (sperm motility and morphology) [36], perhaps the best evidence is conception rates with AI. It was recently reported that fertile buffalo bull semen yielded conception rates >55% whereas use of semen from sub-fertile buffalo bulls resulted in conception rates of only 28-35% [37]. Notwithstanding, conception rates are affected by many variables, including nutrition, housing system, time of insemination, milk production, body condition score, semen collection personnel, semen quality, semen extenders, and cooling and thawing procedures [38-42]. Although 20-40% of domestic bulls in an unselected population have reduced fertility, few are completely sterile [43], whereas ~35% of buffalo bulls are considered sub-fertile [27]. Mating ability and fertility are important for buffalo bulls used for natural service, as well as those used as sources of semen in AI centers. Mating ability of buffalo bulls is assessed by libido testing and service capacity [32,44], whereas scrotal circumference and semen quality are indicators of potential fertility of bulls [36] and buffalo bulls [28,45]. Pathology of the external and internal genital organs, infectious diseases and body conformation can all affect mating ability and fertility [46]. There are limited reports regarding infertility in buffalo bulls, including some regarding infectious infertility [47], hereditary causes [48] and malformations [49]. There are also seasonal effects on fertility and libido of buffalo bulls. There are certain limitations in the descriptions of pathologies in male genital tracts that follow in this chapter. Most pathological conditions have been studied in abattoir derived male buffalo genitals and the incidence thus described might not be the true incidence.

Similar to cattle [46], infertility in buffalo bulls has been classified into 3 categories [28,50-52]:

1. Reduced to complete lack of libido
2. Impotentia coeundi (Inability to copulate)
3. Impotentia generandi (Inability to fertilize)

Impotentia coeundi involves painful conditions of the locomotor system and abnormalities of the penis and prepuce, whereas impotentia generandi includes abnormalities of the testis, duct system, accessory sex glands, and chromosomal aberrations [52].

Reduced Libido in Buffalo Bulls

Buffalo bulls are generally considered to have inherently lower libido than domestic bulls [52,53]. Libido in buffalo bulls is scored on the basis of reaction time (interval from exposure to the teaser to mounting) [21,32] and aggressiveness [44]. Buffalo bulls with a reaction time <5 min are regarded as having good libido, whereas those with reaction times of 5-15 or >15 min are considered to have poor and very poor libido, respectively [21,32]. Poor libido is a major reason for designating buffalo bulls unsuitable for breeding [3,4], particularly bulls that are 3-5 y of age [54], as libido is significantly correlated with mating ability [44]. There are no apparent substantial differences in libido among buffalo breeds [55]. Kundhi buffalo bulls had good libido [56]. The incidence of very poor, poor and good libido in buffalo bulls (<5 y old) in Punjab was 4.2, 8.3 and 87.5%, but in buffalo bulls aged 5-10 and >10 y the incidence was 13.3 and 6.7% for very poor, 16.7 and 20% for poor, and 70.0 and 73.3% for good libido [21], respectively, indicating that libido declined with advanced age [57]. There was no apparent association of scrotal circumference and libido [21]. However, bulls with lameness, scrotal dermatitis, or a wide scrotal neck often had poor or very poor libido [21]. Ectoparasites on the scrotal skin (Fig.1) cause irritation and reduce libido of buffalo bulls [21,28]. Based on an ultrasonographic evaluation, buffalo bulls with high libido had significantly thinner rump fat (5.3±0.6 mm) and smaller seminal vesicles (diameter 1.5±0.5 cm) compared to low-libido buffalo bulls (7.3±0.9 mm and 1.7±0.1 cm, respectively [32]); therefore, it was suggested that these 2 parameters may be useful to predict libido.

Figure 1. A buffalo bull with scrotal dermatitis. The arrow indicates a lesion attributed to an insect bite. (Photo courtesy Prof. William Vale).

Increased thermal stress has been linked to a decline in libido (during hot summer months) in buffalo bulls [16,57-59], with seasonal alterations in plasma concentrations of steroids [60] and luteinizing hormone (LH) [61], as well as poor thyroid function [62,63], postulated as reasons for poor libido during summer. Conversely, improved libido of Egyptian buffalo bulls during spring was attributed to highly nutritious green fodder [64]. Experimental exposure of buffalo bulls to thermal stress did not affect libido [18,65]; however, direct exposure of buffalo bulls to solar radiation suppressed libido [66]. The most common causes of poor libido in buffalo bulls were lameness, scrotal dermatitis and wide scrotal neck [21], although nutrition and management probably also have roles [52]. Furthermore, certain pharmaceuticals are reported to decrease libido in buffalo bulls. For example, administration of oxytetracycline (once daily IM for 7 d) to 4 buffalo bulls transiently reduced libido [67], whereas administration of acetaminophen (50 mg/kg IM for 5 d) to breeding buffalo bulls significantly decreased libido [68].

Thermal amelioration during the hot season may improve libido of buffalo bulls [66]. Specifically, there were significant improvements in sexual behavior (including libido) and semen quality in buffalo bulls maintained under mist cooling and air circulation with fans during hot, humid summer months [69]. In 1 study, efforts to improve thermal comfort coupled with periodic administration of Vitamins A, D and E did not improve sexual behavior of buffalo bulls [70]. However, in another study, treatment of buffalo bulls with vitamin E (100 IU) plus 5 g zinc oxide administered orally (twice a week for 2 mo) significantly improved sexual desire, testicular measurements and semen quality [71]. A thorough physical evaluation of buffalo bulls and review of their management and frequency of use is required before initiating therapy for poor libido. Buffalo bulls with minor lameness problems can be treated, although those that are severely affected may require exclusion from breeding. Since libido is largely influenced by genetic and environmental factors, hormone therapy is not expected to be very efficacious. For example, weekly IM injections of 100 mg testosterone or treatment with hCG (3000 IU given IM every 15 d) only resulted in marginal improvements [72]. Exogenous administration of 20 mg diethylstilbestrol to adult Kundhi buffalo bulls had little impact on their sexual behavior [73], although giving 12 μg of GnRH IM to pubertal buffalo bulls improved both libido and semen quality [74]. Similar effects were recorded in adult Nili Ravi buffaloes [75]. Intramuscular administration of 30 mg PGF2α to Murrah buffalo bulls 30 min prior to each alternate first ejaculation for 6 wk significantly reduced reaction time, but did not affect sperm number or quality [76].

However, in another study, giving 1 mg prostaglandin (fenprostalene) to buffalo bulls 1 h before semen collection had no significant effect on reaction time, semen volume, percent motility, or sperm concentration [77]. Oral administration of herbal tablets (Tentex Forte, Himalaya Drug Co., Bangalore, India) for 15 d was reported to improve poor libido in buffalo bulls [78]. Finally, proper management, good nutrition and regular exercise were suggested as practical means to improve libido in buffalo bulls [79].

Impotentia coeundi (Inability to Copulate)

Inability to copulate in buffalo bulls could be due to abnormalities affecting the body in general or more specifically the external genital system (i.e. penis and prepuce) [52]. Improper body conformation and inflammatory or traumatic lesions producing pain may prevent a buffalo bull from mounting. Buffalo bulls with arthritis, fractures or wounds and painful foot lesions are reluctant to mount, despite having good libido [52]. Musculo-skeletal defects (e.g. post legs, sickle hocks and knock or bent knees) often decrease libido and cause pain at mounting, which adversely affects mating ability of domestic bulls [80,81] and presumably buffalo bulls.

Abnormalities of the Penis and Prepuce

Inflammatory conditions involving the penis and prepuce are rare in buffalo bulls [26]. In a study involving breeding soundness evaluation of 305 buffalo bulls in Brazil, only 0.7% bulls had inflammatory changes in the prepuce, whereas none had pathology of the penis [26]. Some genital inflammation can occur in buffalo bulls affected with bovine or bubaline herpes virus infection [47] however, clinical cases of posthitis (inflammation of prepuce) or balano-posthitis (inflammation of the glans penis and prepuce) reported in domestic bulls [82,83] have apparently not been reported in buffalo bulls. Urethral abnormalities in male buffaloes have been described, although their association with fertility was not mentioned [84]. Uroliths in the penis and prepuce in buffalo bulls have been reported [85]. A rare congenital malformation of the urethra–hypospadias (abnormal development of penile urethra, scrotum and prepuce) was reported in calves [86,87] and a Mediterranean buffalo [88] and 3 cases of hypospadias in buffalo bulls have been reported [84]. Penile problems such as phimosis (inability to protrude the penis), paraphimosis (inability to withdraw the penis), chronic preputial prolapse, penile hematoma and tumors of penis and prepuce that require surgical correction in domestic bulls [89-92] have not been described for buffalo bulls. Development of a preputial diverticulum is very uncommon in breeding buffalo bulls [93]; symptoms are swelling, phimosis and enlargement during urination (due to urine accumulation) [49]. The expected pathogenesis is that a forceful thrust of the penis against the preputial fold causes swelling and stenosis of the preputial orifice, followed by development of the diverticulum [93].

Although various microbes present in buffalo semen are also harbored in the prepuce [94,95], they seldom cause preputial inflammation. In general, therapy of penile and preputial abnormalities in male buffaloes would be similar to those described for bulls.

Congenital Defects of External Genitalia

Urethral Aplasia

Urethral aplasia, partial or complete absence of the urethra, is rare in male animals [48]. The cause is usually segmental aplasia of mesonephric duct and is a genetic defect [49,96]. The condition can be diagnosed by transrectal examination and by routine semen evaluation. The prognosis is poor and treatment is not very effective, so affected males should be culled [81].

Pigmentation Melanosis

Pigmentation melanosis of the pelvic urethra in buffalo bull has been reported [97]. It is characterized by the entire urethral mucosa having dark brown pigmentation and is associated with other congenital defects, including bilateral segmental gonadal aplasia, bilateral spermiostasis, fusion of ampulla, segmental constriction of ampulla, and fusion of vesicular glands [98].

Impotentia generandi (Inability to Fertilize)

Buffalo bulls with impotentia generandi have normal sexual desire as well as normal ability to copulate, but fertility is suboptimal or absent [52]. Two forms of impotentia generandi have been recognized:

1. Impotentia generandi associated with production of apparently normal semen.
2. Impotentia generandi associated with production of abnormal semen.

Impotentia generandi associated with normal semen may be due to:

1. Infectious diseases
2. Inherited sperm defects
3. Chromosomal aberrations.

Infectious Diseases

Several pathogens can cause infertility in bulls and/or be transmitted via semen [99]. Some directly affect bull fertility by causing disease of the reproductive tract or associating with sperm and interfering with fertilization or subsequent embryonic development [99]. Viral pathogens present in and transmitted by bull semen include bovine herpes virus-1 (BoHV-1), bovine virus diarrhea (BVD), vesicular stomatitis, foot and mouth disease, and rinder pest [100,101]. Bacterial diseases transmitted through semen include brucellosis, campylobacteriosis, leptospirosis, tuberculosis, paratuberculosis, mycoplasma, ureaplasma, chlamydia, mycobacteria, and coxiella [100-102]. Trichomoniasis is a protozoal disease in bulls that is transmitted through semen [101]. Furthermore, organisms causing babesiosis and trypanosomiasis are present in semen, although transmission is not confirmed [101]. For a comprehensive list of various diseases transmitted through semen, readers should refer pertinent literature or the OIE website (www.oie.int/). All of the above-mentioned organisms have been described in buffalo bulls, although not all cause diseases as those described in domestic breeds of bulls [48].

Infectious Bovine Rhinotracheitis (IBR)

Infectious Bovine Rhinotracheitis (IBR) is caused by Bovine Herpes Virus 1 (BoHV-1) which infects respiratory as well as genital tracts of cattle and buffaloes, causing various diseases, including IBR, infectious pustular vulvovaginitis, infectious pustular balanoposthitis, abortion, mastitis, infertility, tracheitis, conjunctivitis-keratoconjunctivitis, encephalitis, and fatal disease in newborn calves [103]. Spread of BoHV occurs through respiratory contact, reproductive and ocular secretions, with the latter route regarded as the most important for entry into herds [104]. Age affects susceptibility for this disease (greater susceptibility with increasing age) [105]. Bulls with IBR may have brief pyrexia, orchitis, azoospermia and pustular balano-posthitis [101]. Furthermore, these bulls may have life-long virus shedding (perhaps intermittently rather than continuously) in their semen [106], causing reduced conception, endometritis and abortion [107]. Virus is shed in semen during the acute phase of the disease and in latent infections [108]. Viral reactivation from the latent state is generally thought to be stress-induced, but can also be induced with exogenous corticosteroids [105]. Since excretion of virus may occur despite a lack of antibody titers, only sero-negative and virus-free bulls should be used for semen collection. Both BoHV-1 and BoHV-5 are known to cause disease in India [109]. There is legislation restricting importation of biological material from countries where the disease is prevalent [110].

Similar to bovine strain (BoHV-1) [111,112] a species-specific bubaline herpes virus-1 (BuHV-1) is widespread among buffaloes, with a high seroprevalence in some areas [113-115]. However, clinical disease was not recorded (Table 1), nor was virus detected in semen from infected buffalo bulls [116], although it was isolated from the prepuce of buffalo bulls [117]. Experimental inoculation of buffaloes with BoHV-1 resulted in infection without clinical disease [118]. Thus, despite a high seroprevalence of BoHV-1 and BuHV-1 in buffalo, clinical disease and transmission of virus by buffalo bulls is yet to be demonstrated; therefore the buffalo may not be the natural host for this virus [118,119].

Foot and Mouth Disease (FMD)

The virus causing FMD can be transmitted by bulls [101] and can survive in cryopreserved semen [101]. Severe degeneration of germinal epithelium in bulls due to foot and mouth disease or vaccination against this pathogen has been reported [123]. During the acute phase of the disease, semen production and quality are severely affected [47]. Mean percent sperm motility is poor and sperm morphological abnormalities are increased significantly (from 34 to 62%) [124]. Similar to findings in domestic bulls [125], FMD vaccination of buffalo bulls significantly decreases sperm concentration and percentage live sperm, and increases percentage of sperm with morphological abnormalities [123,124], although these effects are transient (15-20 d). Transportation of a Murrah buffalo bull from a FMD affected area of Punjab (India) to a new location (Uttar Pradesh, India) caused an outbreak of the disease at the new location [126]. However, neither reproductive effects nor whether the spread involved mating or AI with semen from the affected buffalo bull were described in that report.

Bovine Viral Diarrhoea (BVD)

The Bovine Viral Diarrhoea Virus (BVDV) belongs to genus Pestivirus and family Togaviridae [127]. Although the primary host is domestic cattle, the virus has been isolated from buffaloes in many countries [11,112,114,128-132]. This virus replicates in the seminal vesicles, prostate gland and epididymis of the bull [133,134]. Prolonged testicular infection can occur, with detection of virus in testicular tissue for 7 mo following an acute infection [135]. Although BVD can be shed in semen, acute or persistent infections may or may not affect semen quality [133,136]. Reduced conception rates, production of persistently infected calves or birth of clinically normal calves may result from AI with infected semen [135,136]. Despite a frequent seroprevalence of BVD in buffaloes, the pathogenesis of BVDV infection in buffalo bulls is not well understood [129].

Brucellosis

Buffaloes are susceptible to infection with Brucella abortus and Brucella melitensis [114,137]. Brucellosis has worldwide distribution wherever cattle and buffaloes are maintained [138]. It is zoonotic disease [139] and infection in bulls can lead to orchitis, epididymitis, seminal vesiculitis, ampulitis, decreased libido, and infertility [99]. The B. melitensis biovar 3 and B. abortus biovars 1 and 6 are predominant in Italian buffalo herds [114] and seropositivity to Brucella abortus has been reported in several countries [140-144]. Orchitis and epididymitis were described in buffalo bulls in 1 abattoir study, but organisms were not isolated [145]. Contaminated semen can transmit the infection [146] when AI is used, with a lower risk of transmission following natural service [110]. Although transmission of this disease through semen is probably not the most important route [101], buffalo bulls should be tested for brucellosis as mentioned in article 3.2.1.5 of Office International des Epizooties (OIE) and as reported [147]. There was a high seroprevalence to brucellosis in buffalo bulls used for natural service in Gujarat (India) [148], emphasizing the need for testing.

Trueperella pyogenes

Trueperella pyogenes (previously termed Arcanobacterium pyogenes) is commonly present on the nasopharyngeal and preputial mucosa of buffalo bulls [114,137] and is frequently implicated in suppurative lesions [137]. This bacterium is a frequent isolate in bulls with vesiculitis [149], is present in semen [101], and is usually sensitive to penicillin [150]. Because of the potential presence of the organism in preputial mucosa of buffalo bulls, washing of prepuce with normal saline (0.9%) or KMnO4 (0.02%) is suggested before collection of semen. The prepuce should be infused with normal saline and washed once without vigorous shaking.

Campylobacteriosis

Infection of buffaloes with Campylobacter fetus has been reported [114,151]. Campylobacter fetus subsp. venerealis causes a venereal disease in cows that is transmitted through natural service or AI, yet it does not cause clinical signs in bulls [102]. These organisms reside on the preputial epithelium of bulls, with potential for a lifelong infection [99,146]. Shared bulls and failure to test for Campylobacter fetus at AI centers are important factors in spreading the infection [114]. Seven strains of Campylobacter sputorum subsp. Bubulus [152] and Campylobacter fetus, Campylobacter fetus subsp. venerealis and Campylobacter fetus subsp. fetus have been isolated from the prepuce of buffalo bulls [151]. The disease has been reported in India, Malaysia and the former USSR [146]. The incidence of Campylobacter spp. in preputial washings from buffalo bulls was 11.1% [151], 15.2% [152] and 92.7% [153]. In spite of the presence of Campylobacter spp. in preputial washings from buffalo bulls, venereal transmission is not well documented. Control procedure mention that the animals used for the artificial insemination or natural breeding undergo quarantine and should have negative cultures on three consecutive preputial washings collected at intervals of 2 wk [114], with re-examination every 6 mo. Campylobacter fetus isolated from aborted buffaloes had 100% sensitivity towards gentamicin (and 71.4% towards streptomycin [154]); therefore, gentamicin should be considered for use in semen extenders.

Leptospirosis

Leptospires are present in the renal tubules and/or genital tracts. The main pathologic effects caused by Leptospira hardjo in cattle are abortion, stillbirth and agalactia. Leptospiral infection in buffaloes has been reported in many countries [155], with antibodies against many Leptospira spp. [146]. These microorganisms can reduce fertility, cause abortion and survive in frozen semen [102]. Particular attention has to be paid to the breeding bulls that are used to produce semen for AI. Accessory sex glands of the bull, e.g. seminal vesicles, are a major localization site for Leptospira interrogans serovar hardjo. In addition, Leptospira pomona, Leptospira canicola and Leptospira hardjo serotypes have been isolated from fetal buffalo kidneys [146]. The seroprevalence of Leptospira in buffalo bulls varied from 10.4 to 56.0% [156-159]; however, no clinical disease was reported.

Chlamydiosis

Chlamydiosis is a zoonotic disease caused by various species of Chlamydiae. Ruminants are infected by 2 species (Chlamydophila abortus and Chlamydophila pecorum), with the former organism causing abortion in small ruminants and buffalo [114]. Chlamydia can cause infection of the reproductive tract of the bull [160], be present in semen and survive cryopreservation [99]. It has been claimed that Chlamydophila pecorum is the main etiological agent responsible for abortion and encephalomyelitis in buffalo cows [146]. Chlamydia psittaci was isolated from buffaloes in India [161-163], with seroprevalence of up to 53% [162]. Antibodies to chlamydia (C. abortus and C. pecorum) were detected in serum and vaginal swabs of 57% of aborting Italian buffaloes [164,165]. Furthermore, both of these organisms were present in buffalo semen (based on polymerase chain reaction) [166].

Mycobacterium Infection

Like cattle, mycobacterium infection can be generalized or localized in the lungs, udder and brain in buffaloes [167]. Prevalence of tuberculosis is higher in buffalo than in cattle in India [168]. Most cultures of tubercle bacilli from buffaloes are Mycobacterium bovis. This organism causes orchitis in buffalo [114,169]; therefore, buffalo bull or its semen that are used for breeding has to be tested before using (and retested annually). Diagnosis is based on the delayed type hypersensitivity reaction following intradermal inoculation of PPD, the presence of granuloma and detection of acid-fast bacilli [47]. Serological tests are unsatisfactory due to low titers and potential for cross reaction with non-tuberculous species of Mycobacteria and Nocardia [170]. Mycobacteria affect the testes and epididymis of buffalo bulls [169] and can be transmitted through semen [99]. Buffaloes with tuberculosis often have scrotal asymmetry, lobulation and increased volume, with fibrotic testes and epididymes [169]. Surti and Murrah buffaloes that were positive on a tuberculin test had poor semen quality (reduced mass activity, ejaculate volume, initial motility and post thaw motility [171]).

Trichomoniasis

Trichomoniasis is caused by Tritrichomonas fetus, a protozoan that causes venereal disease, premature abortions, pyometra and infertility in cows [172], but no clinical symptoms in bulls, although a similar disease has not been reported in buffaloes [47, [173]. Trichomonas fetus infection was reported in the genital tract of buffaloes in India and Egypt, albeit without clinical disease [146]. Presumably buffalo are an unusual host for this protozoa parasite and not generally susceptible to this protozoan infection. Regardless, there is the potential that affected animals could be lifelong carriers. Semen donors must be periodically screened, as trichomonas was recently isolated from aborting Kundhi buffaloes [174].

Mycoplasmosis

Mycoplasma bovigenitalicum and Mycoplasma bovis are frequently present in the prepuce and preputial orifice of bulls [101] without causing lesions. However, testicular involvement in the bull causes lesions with reduced sperm motility [175]. Therefore, the presence of these organisms in prepuce warrants washing and disinfection of these sites before semen collection [176]. Contamination of bull semen with mycoplasma can also originate from use of semen extenders containing egg yolk or milk [177]. Thus care should be exercised at semen collection centers as many antibiotics (gentamicin, tylosin, lincomycin) added to semen do not kill mycoplasma [178]. Transmission of these organisms through semen of bulls is known [101] and severe inflammatory changes can occur in the female via natural service or insemination with contaminated semen. Mycoplasma bovis and Mycoplasma bovigenitalicum were isolated (10%) from vaginal swabs of female buffaloes [178] and semen samples from buffalo bulls [179-182], indicating that these organisms may be important in buffalo.

Prevention of Infectious Diseases in Buffalo Bulls

Breeding buffalo bulls need to undergo screening during pre-quarantine, quarantine, and as residents, in accordance with evaluation of seminal parameters as mentioned in article 3.2 of OIE [147]. Pre-quarantine testing should confirm the bull to be free of brucellosis, tuberculosis, paratuberculosis, BVD, as well as BoHV-1 [47]. Quarantine should commence for at least 28 d when a buffalo bull is brought to a new location. During this interval, bulls should be tested and confirmed negative for brucellosis, BVDV, Trichomonas fetus, Campylobacter fetus, and BoHV-1. Bulls resident at semen collection facilities should be tested at least annually for brucellosis, tuberculosis, BVDV, Trichomonas fetus, Campylobacter fetus, and BoHV-1. Buffalo bulls that are positive for any of the above mentioned diseases should be removed from breeding/semen collection due to potential dangers of disease transmission. Before semen collection, the prepuce of buffalo bulls should be washed and disinfected and any bull with indications of inflammatory changes in the external genitals should be examined and evaluated. Likewise, bulls with poor semen characteristics should be carefully evaluated and semen from affected buffalo bulls should not be used for preservation or insemination.

Inherited Sperm Defects

Inherited sperm defects are uncommon in buffalo. Dag defect [183] and midpiece reflex defect [184] have been recorded in buffalo bulls.

Dag and Tail Stump Defect

These abnormalities are characterized by presence of non-specific sperm defects with low or absent sperm motility [185] and are also reported in Zebu and buffalo bulls [186]. All affected bulls had poor initial sperm motility (10 to 15%) and also very poor fertilizing ability. Most of these bulls had ∼40 to 50% of their sperm with strongly coiled, folded and/or split tails. The axial filament fibers in sperm were normal in the testis, but were abnormal when the cells reached the cauda epididymis. These defects were mainly due to an autosomal recessive factor/ gene [185]. Similarly, in sperm affected by the Dag defect, the axial fibers become abnormal and were translocated or missing and the coiled tail was enclosed in a common membrane. Consequently, both motility and fertilizing ability were limited. The principle piece is affected more frequently than the mid-piece [187]. Some of these sperm morphological abnormalities were reported in Murrah buffalo males and attributed to uncontrolled inbreeding in some herds [183].

Chromosomal Aberrations

River buffalo have 50 (2n) chromosomes; the first five pairs of autosomes are sub-metacentric, whereas all other chromosomes are acrocentric, including sex chromosomes [188]. Moreover, the X chromosome is the largest acrocentric chromosome and it could be studied without GTG-banding. The Y-chromosome, one of the smaller acrocentric chromosomes [188], could be observed by conventional C-banding, one of the simplest and best methods to assess sex chromosomes [188,189]).

Predominant reproductive problems in buffalo bulls are late maturity, subfertility, infertility and/or sterility [186]. Chromosomal aberrations/defects associated with infertility and reduced fertility in river buffalo bulls have been summarized [48].

During breeding soundness examination of buffalo bulls, special importance should be attached towards hereditary diseases and chromosomal aberrations, which can cause many morpho-functional disturbance related to the genital system, including gonadal hypoplasia, arrested development of mesonephric ducts, epididymal dysfunctions, poor or no libido, and inbreeding [186]. Various chromosomal abnormalities associated with infertility and reduced fertility, sub fertility and sterility have been reported in cattle [22,190,191], and have also been documented in riverine buffaloes [51,192,193]. Most chromosomal anomalies in buffaloes involving autosomes are translocations [194], inversions [195], chromosomal polymorphism [196,197], fragile sites [198] and secondary constrictions [199,200] or sex chromosome aneuploidy, including XO syndrome [201-203], XXX syndrome [199,204,205] and XX/XY chimerism [206,207]. Gonadal dysgenesis [192] and sex reversal syndrome [204] are also reported in buffalo. Klinefelter’s syndrome (XXY) is more common in humans [208] than in domestic species, especially cattle [209]. A male river buffalo calf (Mehsana buffalo) with an XXY-syndrome (Klinefelter’s syndrome) with an XX translocation with diploid number of chromosome (2n=50) was reported [210]. The buffalo bull was phenotypically normal with normal reproductive behavior at maturity; however, he was azoospermic and thus infertile [211]. Patel et al. [196] reported that higher frequency of aneuploidy, premature centromeric division (PCD) chromosomal displacement and other mitotic disturbances were associated with aspermia and increased sperm abnormalities in affected buffalo bulls.

Impotentia generandi with Abnormal Semen

This type of infertility is caused by defects in testes, duct system and accessory sex glands [52].

Testicular Abnormalities

Testicular dimensions and daily sperm output were both lower in buffalo bulls than European breeds of bulls [45]. Pathologies described in abattoir-derived buffalo testes include testicular hypoplasia and degeneration, orchitis and tumors, with testicular degeneration and hypoplasia being the most and second most common testicular abnormality, respectively (Table 2).

Testicular Hypoplasia

Testicular hypoplasia is a congenital and hereditary condition caused by a single recessive autosomal gene with incomplete penetrance and characterized by marked reduction of spermatogonia in one or both testes during fetal life [216]. The condition has been attributed to inbreeding [186]. Testicular hypoplasia is often detected only at puberty or later due to reduced fertility or sterility [96,214]. Testicular hypoplasia may be unilateral or bilateral and may be total or partial, with the unilateral condition being more common [186]. Affected testes are considerably smaller (approximately 25 to 70%) than normal (Fig. 2 and Fig. 3) and usually do not reach the bottom of the scrotal pouch. The right testis (12.0 x 8.8 x 6.5 cm) is more affected than left testis (14.6 x 8.7 x 6.0 cm) [97]. Maurya and Bhalla [217] and Kerur et al., [218] reported testicular hypoplasia in Indian buffalo bulls. Furthermore, various authors have reported varying degrees of testicular hypoplasia/ aplasia in buffalo bulls [1,22,97,212,214,217,219]. Kodagali et al. [1] reported that testicular hypoplasia was the main reproductive abnormality for culling of Surti buffalo (7.1% of bulls were culled for this defect). Bulls with testicular hypoplasia had smaller testes (Fig. 2 and Fig. 3), poor quality semen, a lack of libido, under-developed seminiferous tubules and various forms of germinal tissue hypoplasia [22]. The incidence of unilateral hypoplasia was 3.9% in a recent study [26] and in another study [215] the incidence was 8.9% in 123 buffalo bulls (and was attributed to inbreeding) [28,215].

Figure 2. Unilateral testicular hypoplasia in a buffalo bull (before and after castration; photos courtesy Prof. William Vale).

Figure 3. Unilateral testicular hypoplasia in a buffalo bull. (Photos courtesy Prof. William Vale).

In testicular hypoplasia, the sperm producing seminiferous tubules are poorly developed (Fig. 4), although the remaining reproductive organs develop normally and the animal expresses normal libido and mounting behavior, as Leydig cells and testosterone concentrations are usually unaffected [216]. Gonadal aplasia in a Berari bull was reported by Kaikini and Patil [97]. Similarly, Chaudhuri et al. [212] also observed 4.0% cases of absence of testis for 197 buffaloes examined. Out of these, 8 cases were bilateral, 3 cases of right and 2 cases of left testicular aplasia. Buffalo bulls with testicular aplasia have a rudimentary wrinkled testis [49,214]. Moreover, the incidence of testicular hypoplasia in bubaline bulls has been reported as 4-6% [212,219].

Figure 4. Histopathology of a hypoplastic testis from a buffalo bull. Arrows show seminiferous tubules with partially lacking spermatogenic epithelium lined with empty Sertoli cells. (Photo courtesy Prof. William Vale).

However, Arangasamy et al. [220] reported that the right testes and the vas deferens together weighed 87.9 g and left testes and vas deferens weighed ∼61.0 g. The length and thickness of right and left epididymis were 35.2 x 2.7 cm and 25.7 cm x 2.0 cm, respectively as compared to abnormal epididymis (Rt. 14.1 x 0.9 cm; Lt. 13.0 x 0.7 cm). The right and left vas deferens were 8.9 x 1.6 cm and 8.7 x 1.0 cm, respectively. Histologically, when there was a hypoplastic testis and epididymis, the lumen of the epididymal head, body and tail were lined with low columnar to cuboidal cells, with no perceptible microvilli, micropinocytic invagination and eosinophilic bodies [220] and a pseudostratified epithelium with moderately tall columnar epithelial cells. There was no sperm within the lumen and limited fluid was present [220], or there was a greater quantity of immature sperm (9.3%) and low sperm concentration (650 x 106/ml) [220] and thick layering of smooth muscle cells and connective tissue at around the ducts. The interstitial connective tissue was thickened and loosely arranged. In cases of severe hypoplasia, diagnosis was based on testis size and scrotal circumference. Ohashi et al. [221] reported a case of bilateral partial hypoplasia in a Mediterranean buffalo bull, aged 3 y, with symmetrical testes (7.5 x 3.5 cm) and scrotal circumference less than normal. Semen characteristics in buffalo bulls with testicular hypoplasia depend on degree of involvement. In unilateral cases, there is reduced sperm concentration and low motility, whereas in bilateral condition, the semen is watery with few or no sperm [216]. Often the condition becomes evident at physical examination when the male is 2 y old and has sub-fertility [28].

The prognosis is very poor and due to hereditary origin [28,214] therapy is not suggested and affected buffalo bulls should be culled.

Testicular Degeneration

Testicular degeneration appears to be the most frequent testicular pathology in male buffaloes [51]. This condition leads to production of poor semen quality in buffalo bulls [52]. Most reports describing testicular degeneration evaluated abattoir-derived genitalia [1,22,25,212,222]. The incidence varied from 1.0 [212] to 4.8% [1]. In buffalo bulls with poor semen quality, testicular degeneration should be considered as a possible cause. Raja [223] reported azoospermia in an 8-y-old Murrah buffalo. Exposure to toxins and high temperatures can cause testicular degeneration [50,51]. Experimental exposure of Brazilian buffalo bulls to temperatures of 39°C for 54 d resulted only in change in the testicular consistency but did not result in degeneration, nor did it alter blood testosterone concentrations [18,65]. However, scrotal insulation of buffalo bulls for 20-60 d resulted in transient to prolonged testicular degeneration, with a decline in sperm motility and sperm concentration and increases in sperm morphological abnormalities [224,225]. Testicular degeneration can also occur as a result of unilateral castration, febrile and toxic conditions, advancing age, vaccination and vitamin A deficiency [216]. Testicular degeneration was noted in buffalo bulls with tuberculosis in Brazil [169]. Tripathi and Saxena [226] recorded seminal disturbances in Murrah buffalo bulls subsequent to FMD vaccination and similarly Murugavel et al., [123] reported significant increases in sperm morphologic abnormalities and decreased sperm concentrations following FMD vaccination in Murrah buffalo bulls. In addition, enrofloxacin (5 mg/kg IM) for 7 d resulted in similar changes in seminal quality in buffalo bulls [227]. Buffaloes with poor semen quality and semen freezability often had testicular pathologies, frequently testicular degeneration [23]. Ultrasonography of testes and epididymis of infertile buffalo bulls revealed numerous hyperechoic areas and acoustic shadowing, consistent with testicular degeneration [24].

Buffalo bulls with decreased fertility should be suspected of having undergone testicular degeneration. Testis size and consistency are not usually altered, except in bulls with chronic degeneration and resultant fibrosis [216]. Semen concentration varies from normal to low, with varying degrees of decreased motility, depending upon the degree and duration of the condition [216]. An increased proportion of detached sperm heads is the first morphologic sign of degeneration [216] and there is increase in percentage of sperm heads with morphologic abnormalities (∼30 to 50%). However, libido is usually normal.

Diagnosis of testicular degeneration is usually based on poor seminal quality and decreased fertility. Ultrasonographic evaluation of testicular tissue reveals hyperechoic areas and acoustic shadowing [24]. Histologic examination of testicular biopsy specimens can confirm the diagnosis [228]. Therapy involves removal of the cause and administration of Vitamins A and E. Sexual rest for 3-5 mo is suggested [216].

Orchitis

Orchitis can be of hematogenous origin, from wounds penetrating the scrotum, or retrograde progression through the vas deferens from infected accessory sex glands [229]. Traumatic scrotal injuries due to fights or lacerated wounds are uncommon causes of orchitis [28]. Orchitis in buffaloes has been sporadically detected in abattoir specimens [212,222]. Orchitis in buffalo due to Brucella has been reported [145,230,231]. In two recent abattoir studies, the incidence of orchitis was relatively high (3.3% [26] and 9.7% [25]). In a clinical report of orchitis caused by Brucella in a buffalo bull, the scrotum and testes were enlarged and semen quality was exceptionally poor [230]. Testicular tuberculosis in buffalo bulls in Brazil caused asymmetry (Fig. 5), lobulation and increased scrotal volume with fibrotic lesions visible when the testis was cut [169]. Buffalo bulls with orchitis had enlarged testes (Fig. 5), with ultrasonographic examination helpful to make a diagnosis [24]. Febrile changes may be evident. Adhesion of testes to adjacent structures (Fig. 6) and peri-orchitis have been reported. Therapy for severe orchitis is seldom successful and affected buffalo bulls are simply culled. In contrast, minor traumatic injuries of the testes of valuable buffalo bulls need immediate attention and should be treated with local and parenteral antibiotics and anti-inflammatory compounds [28].

Figure 5. Left: Unilateral testicular enlargement in a buffalo bull with orchitis. Right: Longitudinal section of the right testis. Note the diffuse creamy white nodules of varying sizes with fibrous consistency (Photos courtesy Prof. William Vale).

Figure 6. Buffalo testis with adhesions between the parietal, vaginal and albuginea tunics, but no evidence of active inflammation.(Photo courtesy Prof. William Vale).

Testicular Fibrosis and Calcification

Prolonged testicular degeneration or testicular inflammation can result in testicular fibrosis [216]. Ribeiro et al. [169] observed fibrotic changes in testes of Brazilian buffalo bulls (the testes were hard on palpation) affected with tuberculosis; furthermore, similar fibrotic changes with degeneration have been reported [22]. Testicular calcification was detected (incidence, 5.5 to 31.0% [97,212]) in abattoir-derived buffalo bull testes [29,97,212,232]. Calcified masses in seminiferous tubules were apparent on histologic examination (Fig. 7) and were either unilateral or bilateral [97,212]. The lack of reports on the clinical diagnosis of these 2 conditions suggests that most affected buffalo bulls are excluded from breeding.

Figure 7. Photomicrograph of epididymal tubules filled with cheese-like exudate, with central calcification (H&E:10x).(Photo courtesy Prof. William Vale).

Testicular Tumors and Other Conditions

Testicular tumors appear to be uncommon in buffalo bulls [216]. Zanwar et al., [213] examined 97 abattoir-derived buffalo bull genitalia and identified 3 cases of unilateral testicular neoplasm (all were Sertoli cell tumors). Furthermore, 1 buffalo bull in this study with bilateral neoplasia; that buffalo was a male pseudohermaphrodite and its testes were located inguinally and there was a uterus masculinus, vestibule and mammary glands. A male pseudohermaphrodite buffalo has been reported [93,233] however; no tumor was noted. In 1 study of abattoir-derived genitalia [234], 36 buffaloes were affected with various types of lymphosarcoma of which 8 were in the testes and 4, 1, 2, 4, and 7 were in the seminal vesicles, prostrate, bulbourethral gland, pelvic urethra, and epididymis, respectively. Testicular cysts were detected in buffalo bulls [29,235]. Although diagnosis of a testicular tumor in live animals can be confirmed with ultrasonography [236] and testicular biopsy, therapy is seldom suggested.

Congenital Defects of Testes

The common congenital defects of testes are testicular aplasia, hypoplasia, absence of testes [212], and adhesions of tunica vaginalis, underdeveloped testes, bifid scrotum [237,238], uni- or bilateral misplaced testes [222], elongated testes, rounded testes, rotated testes [26], distinctively cleaved testes, scrotal hernia, and incomplete descent of testes. Bulls with these testicular abnormalities should not be used for breeding, as most of these defects are considered heritable [28].

Cryptorchidism

Cryptorchidism refers to retention of one or both testes in the abdomen (Fig. 8). The incidence of cryptorchidism in buffalo is rare [222], but increased with inbreeding [239]. A Pandharpuri buffalo calf had both testes under the skin of the ventral abdomen (alongside the penis), and an empty scrotum [240]. In a study of 97 abattoir-derived buffalo genital tracts [241], 6 bulls had cryptorchid testicles (4 bilateral and 2 unilateral right-sided cryptorchid testes). However, Osman and Sherry [222] reported only 4 cases of unilateral cryptorchidism from 1000 abattoir-derived buffalo bull genitalia. Cryptorchid testes are usually small, soft and flaccid, and fail to produce sperm. Affected bulls should be culled.

Figure 8. A unilateral cryptorchid buffalo bull. (Photo courtesy Prof. William Vale).

Scrotal Abnormalities

In Murrah buffalo bulls, there are various scrotal shapes, including oblong, square and overlapping types. However, these are all considered normal variants, as semen characteristics are not affected by scrotal shape [242].

Scrotal abnormalities that have been reported include scrotal hernia, morphologic abnormalities, bifid scrotum, hydrocele, and varicocele .The scrotum has an important role in testicular thermoregulation [49]; consequently, scrotal abnormalities can affect spermatogenesis. Furthermore, painful scrotal lesions can reduce libido.

Scrotal enlargement can accompany abnormalities of the testes and surrounding structures (Fig. 9). For example, scrotal hernia results in scrotal enlargement; the accompanying interference with scrotal/testicular thermoregulation can dramatically decrease fertility. Furthermore, scrotal hernias in buffalo bulls may interfere with serving ability [216]. Scrotal hernias are due to a double recessive autosomal gene/factor [48]. They predominate on the left side [243] and are usually first apparent between birth and 30 d of age.

Morphologic abnormalities of the scrotum (including scrotal torsion) were recorded in 25.2% of 123 Murrah buffalo males subjected to breeding soundness evaluation in Brazil [244]; defects included scrotal torsion, horizontal displacement, horizontal-ventral localization of scrotum and asymmetric volume reduction and were attributed to inbreeding. The incidence of testicular torsion in another study was 10.2% and in 1 male buffalo, the testes were horizontal [26]. Many of other testicular displacements, including testicular torsion and horizontal and vertical placement of testicles (Fig. 10) were reported in another study in Brazil [28].

Figure 9. A buffalo bull (10-y old) with chronic testicular degeneration (small, flaccid testes) and a distended scrotum. (Photo courtesy Prof. William Vale).

Figure 10. Left: horizontal testicular displacement in a buffalo bull. Middle: Murrah buffalo (8 y) with both testes located vertical to the scrotum. Right: Bifid scrotum in a buffalo bull. (All photos courtesy Prof. William Vale).

Bifid scrotum has been reported for buffalo bulls [26,237,238] and considered to be congenital. In Brazil, a high incidence of bifid scrotum (7.9%) was attributed to inbreeding [26]. Bifid scrotum can be easily recognized by clear division of scrotal segments (Fig. 10) [28]. Higher sperm morphological abnormalities in buffalo bulls with bifid scrotum [245,246] warrant their removal from breeding.

Scrotal hernia can be verified by palpation and ultrasonography [236]. Surgical repair of scrotal hernia in the bull has been reported [247]; however, bulls with inguinal hernia should not be used for breeding.

Other scrotal affections described for bulls include uni- or bilateral hydrocele (accumulation of fluid in the tunica vaginalis) manifested as fluctuating scrotal swelling without pain [236,248,249] and varicocele which is a dilatation and tortuosity of the pampiniform plexus and cremasteric veins [250]. Varicocele is characterized by painful nodular enlargement of the spermatic cord and an enlarged pendulous scrotum [250]. Hydrocele developed secondary to ascites in 28 bulls in 1 study [248]. Scrotal tumors are rare in bulls [251]. None of the scrotal affections except bilateral hydrocele have been mentioned for buffalo bulls. The hydrocele developed subsequent to castration by burdizzo [252].

Abnormalities of the Duct System

Infections of the epididymis (epididymitis) and vas deferens are less frequent in buffalo bulls than in domestic bulls, and are usually associated with testicular infections [26,216]. Bilateral epididymitis was noted in 1 abattoir-derived buffalo specimen, whereas 6 other specimens had both orchitis and epididymitis [145]. Similarly, there were lobular focal lesion involving epididymis and testes in Brazilian buffalo bulls with tuberculosis [169]. Varying degrees of fibrous adhesions in buffalo testis, epididymis and tunica vaginalis have been reported [232] The incidence of epididymitis/spermiostasis in buffalo bulls varied from 3.6 to 18.0% (Table 2). When detected clinically, buffalo bulls with epididymitis had enlarged epididymis (Fig. 11), pain on palpation and ejaculates with numerous leucocytes and decapitated sperm heads [28]. Dissected specimens had pus and caseous material (Fig. 11).

Figure 11. Left: Enlarged epididymis tail in a buffalo bull. Right: Dissected longitudinal section of bilateral epididymis tail, with pasty white contents. (Photos courtesy Prof. William Vale).

Malformation or congenital defects of the duct system include aplasia or hypoplasia of epididymis, epididymal or ampullary cysts, spermiostasis and spermatocele [48]. This may be unilateral (with bulls being fertile to infertile) or bilateral (sterile). Segmental aplasia of epididymis and ampullae has been reported as the most common abnormality of the Wolffian duct system [96,253]. The condition is characterized by partial or total absence of 1 or both epididymis [253]. Three cases of cysts in caput epididymis and 1 case of epididymitis in buffalo bulls were reported [222]. In an investigation of 39 male genitalia of mature Berari buffalo bulls, there was bilateral spermiostasis and fusion of ampulla near the urethral end in 8 cases [254]. Similarly, fusion of ampulla was the most common abnormality in the buffalo bull and there were abnormalities of ampulla and vas deferens in 30 and 32 cases, in 197 abattoir-derived genital organs examined [255]. Fusion of ampullae was also reported in other studies [232,254]. Other abnormalities were unilateral segmental constriction of right ampulla [254], atrophy and displacement of vas deferens [232], bilateral spermiostasis, fusion of the ampulla near the urethral ends [254], fusion of ampulla and remnants of Mullerian duct [222,255], fused ampulla (44.4%) and spermiostasis [254].

Spermiostasis (accumulation of sperm in the caput epididymis) is a condition caused by blind, rudimentary mesonephric tubules or ducti aberrantis and is common in bucks and rams and less common in bulls and buffalo bulls [216]. This condition is probably of genetic origin [256] and unilateral as well as bilateral spermiostasis of cauda epididymis was reported by Kaikini and Patil [97] in Berrari buffalo bulls. Maurya et al. [232] observed that there are varying degrees of fibrous adhesion in testis, epididymis and tunica vaginalis and it is unilateral or bilateral. Similarly, they have reported 4.9% of spermatocele (bilateral enlargement of vas deferens), of which 2.9% was unilateral and 1.3% was bilateral. Spermatocele of cauda epididymis was also reported [97]. Other pathological conditions of epididymis such as cyst in caput epididymis and epididymitis [222] were also reported.

Clinical diagnoses of affections of the duct system are not mentioned and their therapy is not advocated.

Abnormalities of Accessory Sex Glands

Seminal vesiculitis is the most common abnormality of the accessory sex glands in cattle and buffalo [216]. The condition is often unilateral and has been associated with brucellosis, tuberculosis and mycoplasmosis [216]. In addition, Tuerpella pyogenes and various gram negative anaerobic bacteria have often been associated with seminal vesiculitis in bulls [257]. A hematogenous source of infection has often been favored, as high-energy diets predispose to vesiculitis [257]. In domestic breed, the condition has been detected in 0.9-10.0% of yearling bulls during routine breeding soundness examination [257], although the incidence of clinical or subclinical vesiculitis was much higher (49%) in abattoir-derived specimens [258]. In one report regarding Murrah buffalo bulls with seminal vesiculitis, the semen had a high leucocyte concentration [259]. Diagnosis of seminal vesiculitis is based on transrectal palpation, ultrasonography, and presence of leucocytes in semen. Antibiotics such as tilmicosin (1 mL/30 kg SC every 48 h for 3 treatments) were effective for treating vesiculitis in bulls [260]. Chronic cases (refractory to treatment) may best be managed by surgical removal of the affected glands [216].

Seminal vesicles are the accessory sex glands most commonly affected with congenital defects. These developmental abnormalities in the Wolffian duct system occur during fetal development, with indications of a hereditary predisposition [261].

The most common congenital defects of accessory glands in buffalo bulls are fusion of vesicular glands and unilateral segmental aplasia of seminal vesicles [97], under-development of the seminal vesicle [255], under-development of seminal vesicle or a cystic vesicle [261], seminal vesiculitis [259], abnormal fusion of vesicular gland and unilateral segmental aplasia of vesicular gland [254], pouch formation, non-patent lumen, ampulitis, concretion and gross abnormalities of seminal vesicle [255]. The fused ampulla is also associated with other genital malformations such as spermiostasis and pigmentation melanosis of the pelvic urethra [220]. Congenital defects are seldom detected in live animals and are not amenable to treatment.

Seasonal Effects on Buffalo Bull Semen Quality and Fertility

Buffalo bulls are comparatively sluggish and more susceptible to heat stress due to their poor thermoregulatory abilities compared to females [262]. Testes are extremely sensitive to high ambient temperature, resulting in degenerative changes characterized by reduction in testicular size and change in its structure, consistency and functions [263]. The extent to which seasonal breeding in buffalo is a genetic characteristic or due to environmental factors is not well known. Regardless, based on recent research, endocrine and reproductive systems of buffaloes are strongly affected by biometeorological factors, including photoperiod, ambient environmental temperature, relative humidity, precipitation, etc. [264]. Buffalo are considered poor breeders with low reproductive efficiency due to late attainment of puberty and sexual maturity, and frequently poor fertility [264]. Buffalo bulls are capable of breeding throughout the year, but due to some seasonal influences, endocrinological and reproductive functions of buffalo are depressed [16,59]. Reduced sexual activity, especially reaction time and libido score in buffalo bulls coincided with both an increase in ambient temperature [265-268] and increasing day length (photo period) [269]. During hot summer months, thyroid is depressed, resulting in low serum concentrations of thyroxin and testosterone, which probably increase reaction time and decrease sexual libido in buffalo bulls [60,62,94]. Scrotal circumference, libido and testicular parameters such as testicular volume, width and length are higher during peak breeding season (winter) than during low breeding season (summer) [57,270,271], with better quality semen during winter [272,273]. Diameters of seminiferous tubules and height of epithelial cells in corpus and cauda epididymis are significantly higher in breeding than in non-breeding seasons [9].

High ambient temperatures during summer with resultant thermal degeneration of testes [274] contribute to poor reproductive performance. Furthermore, tunica albuginea adhesions may occur in dry hot season, causing testicular degeneration that reduces SC [263]. Similarly, testicular weight, length and breadth in buffalo bulls were lesser during the late dry season than during other seasons of the year [275].

In temperate regions, semen quality of buffalo is better in winter and spring than during summer and autumn [276,277], whereas in tropical regions, semen quality was better during the rainy season. Furthermore, buffalo bulls had their best semen quality during monsoon [13]. Season has much influence on production and quality of semen as there was poor quality and quantity of semen (and poor freezability) during summer months than in winter months [61]. Presumably, sperm collected during summer might be more fragile and less able to withstand freezing stress and thus semen should be frozen only during winter and spring season. However, in some studies, semen quantity in buffalo bulls was better during the hot and humid summer than in colder months [57,278]. Higher semen volume in autumn and low volume in humid summer were reported in Murrah buffalo [279], Italian [280], Iraqi [281] and in Surti buffalo bulls [12]. Similarly, higher semen volume during summer was reported in Surti and Murrah [15], Egyptian [282], Nili-Ravi [283] and in Italian buffalo bulls [284]. In Murrah buffalo, semen volume was higher in autumn season and lower in hot humid summer season [6].

Variation in different reports with regard to semen volume in different seasons might be due to differences in genetics, number of observations and study interval. The color of semen during autumn is between milky to creamy and indicates good sperm concentration [6]. The pH is low in autumn collection as compared to winter season [283,285], although it was reported that semen pH was relatively higher in winter than in spring and summer [284], with some of the difference due to sperm concentration. Furthermore, there was greater mass activity in autumn and spring than in humid summer and winter seasons [276,283,286] and higher motility was reported in winter season in Murrah buffaloes [276,287]. Overall, fertility of buffalo bulls is compromised during hot summer months.

Breeding Soundness Evaluation (BSE) of Buffalo Bulls

Potential requirements for selection of buffalo bulls for natural mating and as donors of semen warrant that buffalo bulls be evaluated for breeding soundness. Breeding soundness evaluation includes assessment of physical capability of the bull, SC and sperm morphology that is highly correlated with fertility [36] and most easily repeated. Whereas BSE of domestic bulls has been developed by the Society for Theriogenology and described in detail [288-290] descriptions for buffalo bulls are few [28,44,291]. Four components of BSE appear important; physical examination, SC, sperm motility and sperm morphology [290,292]. A bull is classified as satisfactory potential breeder if it has passed a physical examination, meet the requirements for scrotal circumference, has at least 30% progressive sperm motility and produces at least 70% sperm with normal morphology [292]. Similar descriptions are not mentioned for buffalo bulls, yet presumably they should be similar.

General and Reproductive Physical Examination

The requirements for body conformation include a bull being bright and alert and having a normal body conformation. The head of the bull should be high and shoulders and chest should be normal. Hind leg conformation is important; bulls with abnormal conformation of hind legs (as viewed from the side and back) such as sickle hock, post-legged or bow legged (Fig. 12) should not be used for breeding [290]. Descriptions on such leg abnormalities are not available for buffalo bulls, yet such abnormalities are undesirable. Overgrown hooves may also be a reason for disqualification. Bulls should be examined for abnormalities in conformation such as hernias, hock joint injuries and testicular, epididymal and scrotal abnormalities such as hernias, enlargements, and congenital malformations [290], any of which render the buffalo bull unfit for breeding. The penis and prepuce should be examined for lacerations, adhesions, or warts which may interfere with extension of the penis during coitus [290]. The preputial sheath in Murrah buffalo bulls was described as pendulous, medium or and tight (Fig. 13), with the median type being common [293]. Abnormalities of penis and prepuce are rare in buffalo bulls. Only 2 of 305 buffalo bulls in a Brazilian report had abnormalities of the penis or prepuce [26]. In another report from Brazil, penile dorsal curvature was present in 1 buffalo bull that was able to mount but unable to achieve intromission or ejaculation [294]. Furthermore, transrectal ultrasonography was recently described for evaluation of seminal vesicles [32], testes, epididymis and the scrotum [24,295].

Figure 12. Appropriate and undesirable leg confirmation in domestic bulls.

Figure 13. Surti buffalo bulls. Note the very tight prepuce (left) and a prepuce with median laxity (right).

Scrotal Circumference

Scrotal circumference (SC) is an indicator of testis size and therefore potential for semen production, as well as having some value to predict semen quality and fertility. The SC is measured using a scrotal tape. Due to the presence of numerous wrinkles on the surface of the buffalo scrotum (Fig. 14), prior to measurement of SC, the testes should be gently forced to the bottom of the scrotum (Fig. 14), thereby ensuring a smooth scrotal skin and accurate measurement. Care should be taken to avoid excessive downward force, or putting a thumb between the testes and separating them, both of which will generally artificially increase the SC measurement. Although standards for SC of bulls have been defined [290], there is a relative lack of similar standards for buffalo bulls [28]. Notwithstanding, there are many reports on buffalo bulls (various breeds and ages) describing SC and the number and morphology of sperm (Table 3). Although scrotal shapes in Murrah buffalo bulls were described as oblong (69.8%), square (20.9%) or overlapping (9.3%), semen quality did not differ among shapes [242].

Figure 14. Left: Scrotum of a Surti buffalo bull with dorsally displaced testes and wrinkled scrotal skin. Right: Scrotum of a buffalo bull after testes were moved ventrally. Note that the hand is on the anterior aspect of the scrotum, with care to avoid increasing apparent scrotal size by either using excessive ventral force on the testes or placing the thumb between the testes and forcing them apart. (Photo courtesy Prof. William Vale).

Scrotal circumference increases significantly in male buffalo from 1 to 24 mo of age [45,296,297]. Spermatocytes appeared at 12 mo of age in Nili-Ravi buffalo, but spermatogenesis with appearance of spermatids was evident at 18 mo [298].

Scrotal circumference increased with age and bodyweight and buffalo bulls with higher SC produced better quality semen [44,57,270,274,299]. The SC and testicular dimensions in buffalo bulls was significantly lower during summer compared to winter [57,270,271,300]. Testis weight and volume and diameter of seminiferous tubules in Iraqi buffalo were greater in winter compared to summer [271]. Therefore, SC evaluation in buffalo bulls should take into account effects of season. There were no significant effects of breed on SC in buffalo bulls at <7 mo or >60 mo of age [301,302]. Significant and positive correlations of SC with ejaculate volume, motility and sperm concentration were reported in buffalo bulls [45,270,303]. Sajjad et al. [304] reported positive correlations between testosterone concentrations and SC (r=0.414) and ejaculatory volume. Similarly positive correlations between SC with age, ejaculate volume and sperm concentration were reported for Murrah buffaloes [272]. However, sperm quality (as assessed by computer assisted semen motility analyzer) was lower in old buffalo bulls (84-107 mo) and those with SC >39.0 cm compared to mature (48-83 mo) buffalo bulls or those with SC of 36.0-39.0 cm [273]. First service conception rate (FSCR) was 48% in Murrah buffalo bulls that had SC of >44 cm, although there was no significant correlation between FSCR and SC and no consistent relation between sperm concentration and SC [305].

Selection of buffalo bulls on the basis of sexual behavior index and SC has been recommended [274]. Reaction time and aggressiveness were deemed important in 2 studies that evaluated mating ability on the basis of dominance [294,295].

Sperm Motility and Morphology

Standards for minimum progressive sperm motility and morphology have not been standardized for buffalo bulls, although 1 study [23] reported semen quality from normal and abnormal buffalo bulls. In a recent study [314], post-thaw motility of buffalo semen was 31.8+1.75%. In one study evaluating buffalo bull selection criteria, it was noted that evaluation of sexual behavior was important in selection of breeding bulls [44], although it was also noted that semen evaluation was also required.

Morphologic abnormalities involving various parts of buffalo sperm have been mentioned [315,316] and varied from 7.5 to 18.5%. Buffalo bulls with testicular degeneration had a higher percentage of morphologically abnormal sperm [317]. There is a need to standardize sperm motility and morphology parameters for buffalo bulls for BSE. Karyotyping of buffalo bulls [318] is suggested to cull buffalo bulls with chromosomal abnormalities [48].

Ranges for semen parameters for various breeds of buffalo bulls have been recently summarized [319]. There are few studies describing semen quality problems in buffalo bulls [317]. Jafarabadi buffalo bulls producing poor semen quality had poor initial sperm motility (48-50%), low sperm concentration (842x106/mL), numerous morphologically abnormal sperm (17-60%) and poor pre-freeze motility (23-34%) [23]. Similarly, buffalo bulls with very poor libido had poor individual sperm motility (42%) and increased morphologically abnormal sperm (35%) [21].

Acknowledgements: We thank Prof. William Vale (Programa Nacional de Professor Visitante Sênior, Universidade Federal do Oeste Do Para A, Instituto de Biodiversidade e Florestas – IBEF Rua Vera Paz s/n - Bairro do Salé, CEP 68035, Para, Brazil) for kindly providing many original photographs.