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Relationship between the Corynebacterium pseudotuberculosis, phospholipase D inoculation and the fertility characteristics of crossbred Boer bucks
Livestock Science 191 (2016) 12–21
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Livestock Science journal homepage: www.elsevier.com/locate/livsci
Relationship between the Corynebacterium pseudotuberculosis, phospholipase D inoculation and the fertility characteristics of crossbred Boer bucks Zaid Khudhur Mahmood a, Zurin-Azlin Mohd Jin a, Faez Firduas Jesse a,n, Abdul Aziz Saharee a, Jasni Sabri b, Rosnina Yusoff a, Abd Wahid Haron a a b
Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, UPM, Serdang, Selangor, Malaysia Department of Clinical Studies, Faculty of Veterinary Medicine, UMK, Kelantan, Malaysia
art ic l e i nf o
a b s t r a c t
Article history: Received 28 August 2015 Received in revised form 22 June 2016 Accepted 27 June 2016
The aim of this study was to investigate the effects of Corynebacterium pseudotuberculosis and phospholipase D (PLD) on buck's fertility, specifically, testosterone concentration, scrotal circumference and semen quality. Crossbred Boer goats (n¼13) aged 12–14 months were divided into three groups. The first group (n¼ 3) was inoculated with one ml sterile phosphate buffer saline subcutaneously into subaxillary area as the control. The second group (n ¼5) was inoculated with live C. pseudotuberculosis 1 109 cfu subcutaneously into subaxillary area. The third group (n¼ 5) was inoculated with PLD 1 ml/20 kg BW intravenously into jugular vein. Blood collection was done twice a week over a period of three months for testosterone analyses. Semen was collected using electro-ejaculator once every two weeks whilst scrotal circumference was measured once every three weeks. Semen volume, semen pH and sperm progressive motility, concentration, live/dead percentage and morphology were evaluated. Bucks were euthanized three months' post inoculation and histopathological examination of the testicles and epididymis was performed. The results showed seven folds' significant decrease (P o0.05) in testosterone concentration in both C. pseudotuberculosis and PLD inoculated groups compared to the control. Semen volume, percentage of dead/live and abnormal sperm morphology showed significant increase (P o0.05) in both inoculated groups compared to the control. The following sperm defects were observed; tapered sperm, decapitated and knobbed head, thickened midpiece, distal and proximal droplet, sterilizing tail and folded tail. Scrotal circumference, semen pH, semen wave pattern, sperm motility and concentration showed significant decrease (Po 0.05) in both inoculated groups compared to the control. The testicles showed varied degrees of degeneration and necrosis with shrunken seminferous tubules. In conclusion, testosterone concentration, scrotal circumference and semen quality were negatively affected in both inoculated groups. Moreover, percentages of live/dead and abnormal sperm morphology were also increased. The results suggest that both C. pseudotuberculosis and PLD had detrimental effects on buck's fertility. & 2016 Elsevier B.V. All rights reserved.
Keywords: Boer Buck Goat Corynebacterium pseudotuberculosis Phospholipase D Fertility
1. Introduction Reproduction is the ability of an individual to create a new individual to maintain its kind. The natural process of reproduction in the animal kingdom is called breeding. Normally, mammalian offspring are born juveniles with complete developed sex organs, yet not reproductively functional. Later on, after a certain period of time, could be months to years, the sex organs develop
n
Corresponding author. E-mail addresses: [email protected], [email protected] (F.F. Jesse).
http://dx.doi.org/10.1016/j.livsci.2016.06.015 1871-1413/& 2016 Elsevier B.V. All rights reserved.
further toward maturity and the animal become sexually mature (Klein, 2007). The insidious nature of some infectious conditions is associated with the pathology of the reproductive system and evidence suggests that infectious disease-related reproductive abnormality reduces the reproductive efficiency in farm animals (Grooms, 2004; Robert and Walter, 2007). Chronic diseases such as Johne's disease, caprine arthritis-encephalitis (CAE) and caseous lymphadenitis have serious effects on reproductive health (Peterhans et al., 2004). First, and foremost effects are fever, reduction of appetite, loss of weight and reduced ambulation. A sick animal is less likely to breed (Robert and Walter, 2007).
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Goat CLA is a chronic disease caused by C. pseudotuberculosis a gram positive, facultative anaerobic and pyogenic organism belong to the family Actinomycetaceae. Caseous lymphadenitis has a long incubation period ranging between 3 and 20 weeks. It has distinct clinical manifestation when the lesions become progressive. Most common manifestations are abscesses in the superficial lymph nodes of the body and less often internally (Brown and Olander, 1987; Valli and Parry, 1993; Fontaine and Baird, 2008; Paton, 2010). C. pseudotuberculosis has a potent exotoxin, so-called, phospholipase D, which is a key virulence factor in the development of CLA (Baird and Fontaine, 2007). The notion that PLD is a significant virulence factor has been demonstrated with some isolates of C. pseudotuberculosis that had their pld genes encoding PLD removed, were not capable of causing abscess formation in the lymph nodes of CLA infected sheep (McNamara et al., 1994). PLD is a sphingomyelin-specific phospholipase that can dissociate sphingomyelin into phosphate and choline (Pepin et al., 1994). It has many biological activities such as dermonecrosis (Muckle and Gyles, 1986), lethality (Brogden and Engen, 1990) and destruction of macrophages during infection in the goats (Tashjian and Campbell, 1983), interferes with ovine neutrophil chemotaxis process and is lethal to the cells themselves (Yozwiak and Songer, 1993). This contributes to the dissemination of the pathogen from the primary infection location to other parts of the animal's body (Guimarães et al., 2011). Infectious reproductive diseases are common problem in male sheep and goats and often manifested as infertility. Infectious diseases such as Brucellosis, Actinobacillus spp and Histophilus spp have destructive nature on the reproductive performance in male and female which can lead to infertility (Robert and Walter, 2007). Interestingly, Othman et al. (2014) concluded that C. pseudotuberculosis infection in non-pregnant goats could be incriminated for hormonal imbalance resulting in infertility. Bacterial epididymitis is a significant cause of subfertility in rams. However, the pathogen type, the age and the sexual experience of the affected animal play an important role in the disease. Brucella melitensis, Histophilus somni and Streptococcus spp were isolated from clinical cases of epididymitis in rams. Often, these cases are accompanied with various degrees of testicular degeneration and to a lesser extent testicular atrophy with impaired spermatogenesis in nearly all cases (Quartuccio et al., 2009). Microscopic examinations revealed necrotic reaction of the entire parenchyma of the testicles and intratubular and interstitial orchitis were also observed. Histopathology of the epididymitis was of necrotic, sclerosing and suppurative form (Marino et al., 2009). Semen quality and fertility of rams can be affected by many infectious pathogens such as C. pseudotuberculosis, Brucella bovis, Actinobacilus spp and Histophilus spp. Looking from a scientific point of view, a large percentage of abnormal morphology of sperm is proof of the damage imposed on the reproductive tract and it is tricky to determine whether these sperm deformities are transient or permanent in nature (Robert and Walter, 2007). Goat's caseous lymphadenitis is gaining foothold in Malaysia with no proper vaccination or control programs. There is scanty information on how caseous lymphadenitis in general, C. pseudotuberculosis and/or PLD in particular affect fertility characteristics in male goat. Therefore, the objectives of this study were to estimate the testosterone concentration, evaluate semen quality and to observe the cellular changes in the testicles and the epididymis, post inoculation with C. pseudotuberculosis and PLD.
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2. Methods and methods 2.1. Animals and experimental design Thirteen buck aged between 12 and 14 months with no history of vaccination against CLA was screened seronegative toward CLA before the commencement of the experiment. The experiment was conducted according to the regulations of Institutional Animal Care and Use Committee (IACUC) of UPM (UPM/FPV/PS/3.2.1.551/ AUP-R119). The bucks were divided randomly into three groups. The first group consisted of three bucks as the control (we choose three bucks after the IACUC committee suggestions to reduce the number of animals used, especially it is control not treatment group as well as cost effective), inoculated with one ml sterile phosphate buffer saline (PBS) subcutaneously into subaxillary area. The second group consisted of five bucks were inoculated with live C. pseudotuberculosis 1 109 cfu subcutaneously into subaxillary area. The third group also consisted of five bucks inoculated intravenously with PLD one ml/20 kg BW into jugular vein. Post inoculation, blood was collected twice a week (starting week one through week 12) from the bucks via jugular vein puncture using hypodermic syringe and heparin tubes. The plasma was separated and stored at 20 °C until the analysis was done. The experiment lasted for three months (12 weeks, post inoculation) then the bucks were euthanized. 2.2. Isolation and identification of C. pseudotuberculosis C. pseudotuberculosis was isolated from clinical cases of goat's caseous lymphadenitis (seven cases of superficial lymph nodes and visceral organs abscessation). Isolates were sent to the Veterinary Laboratory Service Unit (VLSU), Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia for identification and confirmation of the bacteria according to principles and methods described in the microbiological diagnostic laboratory at the Veterinary Medical Teaching Hospital, University of California, Davis, Revised Edition 2008. 2.3. Extraction of phospholipase D Phospholipase D was extracted following the method described by Zaki (1968). Briefly, two or three loops of a 48 h pure culture of C. pseudotuberculosis were inoculated in flask of freshly prepared bovine heart-liver medium. The flask was incubated anaerobically for seven days at 37 °C in slanting position of 15–20°. The culture that developed a pellicle was used. Phospholipase D separation started with centrifugation of the culture medium at 8000 rpm/ 15 min in refrigerated centrifuge. The supernatant was collected and passed via sterile cellulose membrane filter (0.2 mm) and stored at 4 C° then used in the experiment. 2.4. Hormone analysis Radioimmunoassy kit (RIA) were used to analyze the goat's plasma concentration of testosterone (RIA, REF: IM 1119) from IMMUNOTECH, Beckman Coulter Company. The samples and calibrators were incubated using competitive assay kit with 125I-labeled tracer in antibodies-coated tubes. After a certain period of incubation, the content of the tubes was decanted and the bounded radioactivity was measured. Briefly, the calibrators (0 6) were provided by the manufacturer as a ready to use reagent. The calibrators were added to the calibrator's tubes at 50 ml, the samples and the control were added to the sample's tubes at 50 ml. Then, the tracer (125I-labeled testosterone) was added at 500 ml to all tubes. The tubes were then
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incubated on a vortex shaker for three hours at 37 °C to mix all the contents inside the tubes. Finally, the contents of the tubes were decanted and the radioactivity of the tubes was measured using a gamma counter machine (Wallac WIZARD 1470, Perkin Elmer, life sciences). The testosterone RIA sensitivity was 0.00065 ng/ml, inter-assay CV was 0.88 ng/ml and the intra-assay CV was 0.88 ng/ ml. The cross reactivity is o1.0 10 2% toward testosterone glucuronide, cortisol, corticosterone, cholesterol, deoxycorticosterone, 17-epitestosterone, testosterone sulfate, androsterone, 3 betaoxyandrostan, 5 beta, 17 one, progesterone, estrone, estradiol, ethynylestradiol, estriol and dexamethasone (RIA, REF: IM 1119). 2.5. Scrotal circumference (SC) Three weeks post inoculation SC measurement was started through week 12. The measurements were taken once every three weeks. The scrotal neck was held with one hand and then the testicles were gently forced down into the scrotum. A measuring tape was placed around the greatest circumference. Several measurements (three) were taken and their average was calculated to increase the accuracy of SC. 2.6. Semen collection and evaluation Semen was collected from the bucks once every two weeks (starting week two, post inoculation through week 12) using an electro-ejaculator (Electro-Ejaculator, P-T Electronics, Oregon, USA). The buck was held against the wall and a lubricated electrical probe with electrodes was introduced around 10–12 cm into the rectum adjacent to the prostate gland. An intermittent stimulation of 3–10 V was applied in cycles of 8–10 s. The semen was collected using a collecting funnel with a graded sterile glass tube covered with aluminum foil to avoid light shock. Evaluation was done on the semen for volume, pH, wave pattern, progressive motility, sperm concentration, live/dead percentage and abnormal morphology.
2.6.6. Sperm morphology Sperm morphology was determined using the Nigrosin-Eosin staining method and the smears were examined using a light microscope under high magnification (1000x) where 200 sperm were counted and abnormal morphology was determined within these 200 sperm according to Hafez (1993). 2.7. Histopathology Twelve weeks, post inoculation, the bucks were euthanized and reproductive organs (testicles and epididymis) were harvested and placed in 10% buffered formalin for histopathological examination. After fixation, all samples were processed using an automatic tissue processor, embedded in paraffin wax and cut at 4 mm. The tissue sections were placed on glass slides, stained with hematoxylin and eosin and covered with a drop of DPX and cover slip (Luna, 1968). 2.8. Statistical analysis Data were analyzed using SPSS version 19.0. Repeated measures ANOVA was used since all parameters were repeatedly measured over time (12 weeks). All values were reported as mean 7SE at Po0.05.
3. Results Generally, all bucks' developed fever, post inoculation in both treated groups. The C. pseudotuberculosis inoculated bucks (all five bucks) showed abscess formation at the site of inoculation as well as enlargement of their superficial lymph nodes two weeks post inoculation. In contrast, PLD inoculated bucks showed no lesions at the site of inoculation. There was no enlargement in any of their lymph nodes. 3.1. Testosterone concentration
2.6.1. Semen volume Semen volume was measured directly after collection and it was expressed in milliliter per animal. The semen pH also was estimated using pH strips (Whatman™). 2.6.2. Wave pattern A drop of raw semen was placed on a warmed glass slide and was examined using light microscope under low magnification (100x) to estimate the wave pattern of the sperm. The wave pattern was given grades between 0 and 5 depending on the aggressiveness of the waves made by sperm movement (Hafez, 1993). 2.6.3. Sperm motility A drop of raw semen and a drop of warmed saline were placed on warmed glass slide and were mixed together using the corner of cover slip and were examined using a light microscope under low magnification (100x) to view the individual motion of the sperm. If the individual sperm could not be identified due to concentrated semen, a new preparation was made with less semen. The number of motile sperm was expressed as a percentage.
Corynebacterium pseudotuberculosis inoculated group showed a significant decrease (p o0.05) in plasma testosterone concentration between week 2 to week 12 compared to the control. The PLD group plasma testosterone concentration was also significantly decreased (p o0.05) in week two and between week 5 to week 12 compared to the control. Both treated groups showed striking seven folds’ significant decrease (p o0.05) between week 5 to week 12 compared to the control (Fig. 1). 3.2. Scrotal circumference (cm) Inoculation with the C. pseudotuberculosis showed a significant decrease (p o0.05) in scrotal circumference within week 6, week 9 and week 12 as compared to the control. The PLD inoculated group showed a significant decrease (po 0.05) in week 9 and week 12 as compared to the control (Figs. 2 and 3). 3.3. Semen evaluation
2.6.4. Sperm concentration Sperm concentration was measured and calculated as previously described by Hafez (1993).
3.3.1. Semen volume The volume of the semen showed a significant increase (p o0.05) in both C. pseudotuberculosis and the PLD inoculated animals in week eight as compared to the control (Fig. 4).
2.6.5. Sperm live/dead percentage A semen film was made carefully using Nigrosin-Eosin stain mixture, and the average percentage was obtained after counting 200 sperm (Hafez, 1993).
3.3.2. Semen pH Corynebacterium pseudotuberculosis group showed a significant decrease (p o0.05) in pH of the semen in week four and week six, post inoculation as compared to the control. In the PLD group,
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Fig. 1. Testosterone concentration of the bucks’ post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼13.
Fig. 4. Semen volume of the bucks' post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼13.
Fig. 2. Scrotal circumference of the bucks’ post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼ 13.
Fig. 5. Semen pH of the bucks' post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼ 13.
semen pH showed a significant decrease (po 0.05) in semen pH in week four only compared to the control (Fig. 5). 3.3.3. Semen wave pattern The semen wave pattern showed a significant decrease (p o0.05) for C. pseudotuberculosis and the PLD inoculated groups in week four and week six compared to the control (Fig. 6). 3.3.4. Sperm motility Sperm motility showed a significant decrease (p o0.05) in both, C. pseudotuberculosis and the PLD groups in week four and week six compared to the control (Fig. 7). 3.3.5. Sperm concentration Corynebacterium pseudotuberculosis and PLD groups showed a significant decrease (p o0.05) in sperm concentration in week four and week six compared to the control (Fig. 8). 3.3.6. Dead/ live sperm The percentage of dead sperms showed a significant increase
Fig. 6. Semen wave pattern of the bucks' post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼ 13.
(p o0.05) in both C. pseudotuberculosis and the PLD groups in week four, week six and week eight compared to the control (Fig. 9). 3.3.7. Abnormal sperm morphology Corynebacterium pseudotuberculosis
and
the
PLD
groups
Fig. 3. Testes of the bucks showing their sizes at the end of experiment period (week 12). Corynebacterium pseudotuberculosis inoculated animals (A), phospholipase D inoculated animals (B) and the control (C).
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Fig. 7. Sperm motility of the bucks’ post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼ 13.
Fig. 10. Percentage of abnormal sperm morphology of the bucks' post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼ 13.
and necrosis of the lining epithelium of the epididymal tubules. There were morphologically abnormal sperms inside the lumen of the epididymal tubules (Figs. 15 and 16).
4. Discussion
Fig. 8. Sperm concentration of the bucks' post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼ 13.
Fig. 9. Percentage of dead/live sperm of the bucks' post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼13.
showed a significant increase (po 0.05) of the percentage of abnormal sperm morphology in week four, week six and week eight compared to the control (Figs. 10–12). 3.4. Histopathology of the reproductive organs Corynebacterium pseudotuberculosis inoculated group showed significant cellular changes in the testicles represented by irregular seminiferous tubules, shrinkage of seminiferous tubules, edema, degeneration and necrosis of some spermatogonia cells, there were spermatides inside the lumen of the seminiferous tubules, mild necrosis of Leydig cells and testicular atrophy, whilst in the PLD inoculated group testicles showed edema, severe congestion, irregular and shrinkage of the seminiferous tubules, less spermatides were seen in the lumen of the seminiferous tubules, severe degeneration and necrosis of the spermatogonia cells (Figs. 13 and 14). Moreover, the epididymis in both, the Corynebacterium pseudotuberculosis and the PLD groups showed edema, degeneration
To the authors’ knowledge, this is the first detailed study to evaluate the effects of the C. pseudotuberculosis and the PLD on testosterone level, semen quality and testicular lesions of male goat. Determination of infertility in animals caused by a specific pathogen requires the knowledge of the infectivity, the incubation period, prevalence and the virulence factors of that pathogen as well as the susceptibility of the animals (Givens and Marley, 2008). In males, the invasion of microbes into the genital tract is often associated with impaired sperm function and infertility (Sanocka et al., 2004). In mice, the C. pseudotuberculosis and the PLD inoculation showed a significant decrease in plasma testosterone level, where C. pseudotuberculosis inoculated mice showed 5 times lower compared to the control group (Khuder et al., 2012). This is in agreement with the current study which showed that the testosterone concentration was significantly decreased (seven folds) in both treated groups C. pseudotuberculosis and PLD. Leydig cells are testosterone producing cells; the low-level of the testosterone could be due to the direct effect of PLD on these cells in rams (Ibtisam, 2008). These accords with our findings when the testicles showed necrosis of the Leydig cells and similar signs of atrophy, post inoculation with both the C. pseudotuberculosis and the PLD. The testicles also showed irregular shape and shrinkage of some seminiferous tubules with interstitial edema. The results of this study is in agreement with Ibtisam (2008) who stated that rams naturally infected with C. pseudotuberculosis showed significant decreased of serum testosterone level (2.11 70.63 ng/ml) compared to non-infected rams (3.42 70.82 ng/ml). Scrotal circumference is an inherited trait that is related to reproductive performance and is a good indicator of puberty in bulls when it reaches certain size (25–27 cm) instead of the body weight and the age (Coulter, 1986). Semen quality is also associated with scrotal circumference at the age of the puberty in beef bulls (Coulter and Kastelic, 1999). Moreover, scrotal circumference is highly correlated with sperm production, total sperm per ejaculate and sperm concentration in adult rams and bucks (Robert and Walter, 2007). In the present study scrotal circumference was significantly decreased in both the C. pseudotuberculosis and the PLD groups and negatively reflected on other fertility attributes such as semen wave pattern, sperm motility, sperm concentration, percentage of dead sperm and abnormal sperm morphology. Hence, all the latter parameters showed a significant decrease in their level. These findings are in strong agreement with Schoenian
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Fig. 11. Different types of abnormal sperm morphology from the Corynebacterium pseudotuberculosis inoculated bucks; Nigrosin-Eosin stain 1000x. Spiral head (A), tapered sperm (B), coiled tail and decapitated heads (C), double forms (D), broken neck (E) and distal protoplasmic droplet (F).
Fig. 12. Different types of abnormal sperm morphology from the PLD inoculated bucks; Nigrosin-Eosin stain 1000x. Detached knobbed head and bent tail sperm (A), distal protoplasmic droplet with bent tail (B), sterilizing tail stump (C), thickened midpiece (D), proximal protoplasmic droplet with coiled tail, (E) and distal protoplasmic droplet with bent tail (F).
(2005) and Gimenez (2007) stated that semen quality, semen quantity and fertility rates are sturdily correlated with scrotal circumference in rams and bucks. There is a high positive correlation between semen volume along with the number of sperms per ejaculate and sperm concentration with the age in bucks. However, some diseases are well known to have a negative impact on semen quality such as the epididymitis (Robert and Walter, 2007). In the current study semen volume showed a significant increase in the C. pseudotuberculosis inoculated group in week eight of the experiment. It is hypothesized that the C. pseudotuberculosis have reached the epididymis and accessory sex glands leading to inflammatory
reaction increasing their secretions and adding additional volume to the ejaculated semen; hence, the increased volume was noticed two months, post inoculation. Additionally, C. pseudotuberculosis was isolated and identified from the buck's semen in this treated group (unpublished data). The latter hypothesis also agreed with Radostits et al. (2007), Fontaine and Baird (2008) and Paton (2010) who stated that the C. pseudotuberculosis was recovered from the accessory sex organs, epididymis and preputial cavity in apparently healthy rams. The consequences of bacterial presence in the male genital tract can affect fertility (Sanocka et al., 2004). Feasible measures should be taken prior to implicate C. pseudotuberculosis as sexually transmitted bacteria in small ruminants.
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Fig. 13. Testes shows irregular and shrinkage of the seminiferous tubules (red arrow), edema (orange arrow), degeneration and necrosis (yellow arrow), fewer spermatides inside the lumen of the seminiferous tubules (black arrow) and necrosis of Leydig cells (white arrow) 12 weeks post inoculation with the Corynebacterium pseudotuberculosis; H&E 400X. (For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.)
Fig. 14. Testes shows irregular and shrinkage of the seminiferous tubules (red arrow), severe congestion (white arrow), edema (orange arrow), degeneration and necrosis (yellow arrow), and fewer spermatides inside the lumen of the seminiferous tubules (black arrow) 12 weeks post inoculation with the phospholipase D; H&E 400X. (For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.)
Routinely, semen pH is evaluated as one of the environmental parameters in human and animal semen, as it can affect the sperm characteristics, function and motility (De Pauw et al., 2003; Shum et al., 2011; Contri et al., 2013). However, a decreased pH of bull's sperm led to reduced sperm motility and increase in sperm longevity. Thus, lifespans of the sperms were increased (Carr and Acott, 1989; Jones and Bavister, 2000). The present study showed that semen pH was significantly low, post inoculation with both the C. pseudotuberculosis and the PLD. These findings agreed with Contri et al. (2013) who reported that a low pH (5.5) compromises
the sperm membrane integrity and motility in bulls whilst a high pH (8.5) led to a drop in sperm mitochondrial activity producing immobile sperm. The low semen pH in this experiment further explains the low semen quality in both inoculated groups. The wave pattern of the semen is dependent on many factors such as sperm concentration, percentage of motile sperm and the progressive motion of the sperm. Semen waves can be seen as rapid swirling which can be depressed if sperm concentration or motility is reduced (Robert and Walter, 2007). Semen wave patterns showed a significant decrease in the current study in both
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Fig. 15. Epididymis shows edema (white arrow), degeneration and necrosis of the lining epithelia of the epididymal tubules (red arrow), and morphologically abnormal sperms (orange arrow) inside the epididymal tubules 12 weeks post inoculation with the Corynebacterium pseudotuberculosis; H&E 400X. (For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.)
Fig. 16. Epididymis shows degeneration and necrosis (red arrow) of the lining epithelia of the epididymal tubules, morphologically abnormal sperms (orange arrow) inside the epididymal tubules and edema (white arrow) 12 weeks post inoculation with the phospholipase D; H&E 400X. (For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.)
the C. pseudotuberculosis and the PLD inoculated groups. Moreover, in the present study plasma testosterone concentration and semen pH were significantly low decreasing the semen wave pattern hence all of the semen quality characteristic are stimulated and controlled by the androgens, especially testosterone. In general male reproductive function could be affected by pathogenic bacterial strains especially when presenting in the semen where it can affect the sperm motility directly via its agglutination effect on the motile sperm rendering it immotile and also disrupting the acrosome integrity (Monga and Roberts, 1994;
Sanocka et al., 2004). The current study showed that the sperm motility and sperm concentration were significantly decreased four weeks post inoculation in both the C. pseudotuberculosis and the PLD groups. These findings are supported by the significant histopathological lesions that have been found in the testicular tissue such as degeneration and necrosis of some of the spermatogonia cells and the reduced number of spermatids that was observed in the lumen of the seminiferous tubules. Spermatozoa morphology is one of the semen quality characteristics and of great value in fertility assessment of animals
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especially in extensive rearing farms in the tropics (Chacón, 2001). Abnormal spermatozoa morphology has immediate effect on the animal's fertility and the proportion of those abnormalities in the semen is negatively correlates with fertility (Shamsuddin et al., 1993). In the current study both the C. pseudotuberculosis and the PLD inoculated groups showed a significant increase in the percentage of dead sperm and the percentage of abnormal morphology of the sperm. In addition, some morphologically abnormal sperm were observed in the lumen of epididymal tubules. These findings are agreed with Chandler et al. (1988) and Barth and Oko (1989) who reported that spermatozoa morphological abnormalities can be influenced by diseases. It also indicates the poor semen quality and reduced fertility of many animals’ species including goat. We hypothesized that if CLA lesions (of chronic nature) develop in the scrotum or the inguinal lymph nodes, the testicles within the affected side may become smaller than normal due to atrophy. In addition, affected testicles may produce low concentration of spermatozoa with high percentage of abnormal sperm morphology. Moreover, “heat” produced by the local caseous inflammatory process may have adverse effect on spermatogenesis.
5. Conclusion In conclusion, the present findings suggest that CLA, in general, C. pseudotuberculosis and the PLD in particular have had direct detrimental effects on testosterone concentration. As a result, toll reflected and decreased the scrotal circumference, lowered semen pH and semen quality. All that have been mentioned above could potentially implicate and affect the reproduction and fertility of male goat. However, the main influence of CLA on buck's fertility may be due to the general effect of this chronic disease (wasting and debilitating nature of CLA) on the body systems resulting in collateral damages of reproductive system.
Competing interests The authors declare that they have no competing interests.
Authors contributions FF Jesse and ZKH Mahmood contributed to the design of the field trial. ZKH Mahmood and Z Mohd Jin ran the experiment and collect the samples. ZKH Mahmood and FF Jesse analyzed the results and drafted the paper. FF Jesse, AA Saharee, J Sabri, R Yusoff and H Wahid have contributed to the design of the study, writing the manuscript and coordination of the study. All authors have read and approved the manuscript.
Acknowledgements The authors are grateful to Mr. Yap Keng Chee, Mr. Mohd Fahmi Mashuri and Mr. Mohd Jefri Norsidin for their assistance. This work was funded by the Research University Grant Scheme (RUGS), Universiti Putra Malaysia.
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Coulter, G.H., Kastelic, J.P., 1999. Management programs for developing beef bulls. In: Robert, Y.S., Walter, T.R. (Eds.), Current Therapy in Large Animal Theriogenology., second ed., Saunders, United States of America, pp. 221–228. Coulter, G.H., 1986. Puberty and postpubertal development of beef bulls. In: Robert, Y.S., Walter, T.R. (Eds.), Current Therapy in Large Animal Theriogenology., second ed., Saunders, United States of America, pp. 221–228. De Pauw, I.M.C., Van Soom, A., Mintiens, K., Verberckmoes, S., de Kruif, A., 2003. In vitro survival of bovine spermatozoa stored at room temperature under epididymal conditions. Theriogenology 59 (5), 1093–1107. Fontaine, M.C., Baird, G.J., 2008. Caseous lymphadenitis. Small Rumin. Res. 76, 42–48. Gimenez, D., 2007. Reproductive management of goats and sheep. Alabama cooperative extension system. Bull. Anr, 1316. Givens, M.D., Marley, M.S.D., 2008. Pathogens that cause infertility of bulls or transmission via semen. Theriogenology 70 (3), 504–507. Grooms, D.L., 2004. Reproductive consequences of infection with bovine viral diarrhea virus. Vet. Clin. N. Am.: Food Anim. Pract. 20 (1), 5–19. Guimarães, A., Carmo, F.B., Paulett, R.B., Seyffert, N., Ribeiro, D., Lage, A.P., Heinemann, M.B., Miyoshi, M., Azevedo, V., Gouveia, A.M.G., 2011. Caseous lymphadenitis: epidemiology, diagnosis, and control. IIOAB. Journal (2) 2, 33–43. Hafez, E.S.E., 1993. Reproduction in Farm Animals, sixth edition.,. Lea And Febiger,, Philadelphia, PA, p. 571. Ibtisam, M.A., 2008. Some clinicopathological and pathological studies of C. ovis infection in sheep. Egypt. J. Comp. Pathol. Clin. Pathol. 21 (1), 327–343. Jones, J.M., Bavister, B.D., 2000. Acidification of intracellular pH in bovine spermatozoa suppresses motility and extends viable life. J. Androl. 21 (5), 616–624. Khuder, Z., Osman, A.Y., Jesse, F.F., Wahid, A., Saharee, A.A., Jasni, S., Yusoff, R., Rasedee, A., 2012. Sex hormone profiles and cellular changes of reproductive organs of mice experimentally infected with C. pseudotuberculosis and its exotoxin phospholipase D (PLD). J. Agric. Veter-.-. Sci. (IOSR-JAVS). (1) 3, 24–29. Klein, B.G. (2007). Cunningham’s Textbook of Veterinary Physiology. Elsevier Health Sciences. Marino, G., Catone, G., Barna, A., Russo, M., Zanghì, A., 2009. Testicular inflammatory diseases in the buck. J. Comp. Pathol. 141 (4), 297. Luna, L.G., 1968. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, Blakiston Division, McGraw-Hill, third ed. University Of Michigan. McNamara, P.J., Bradley, G.A., Songer, J.G., 1994. Targeted mutagenesis of the phospholipase D gene results in decreased virulence of Corynebacterium pseudotuberculosis. Mol. Microbiol. 12, 921–930. Monga, M., Roberts, J.A., 1994. Spermagglutination by bacteria: receptor-specific interactions. J. Androl. 15 (2), 151–156. Muckle, C.A., Gyles, C.L., 1986. Exotoxic activities of Corynebacterium pseudotuberculosis. Curr. Microbiol. 13, 57–60. Othman, A.M., Jesse, F.F.A., Adamu, L., Abba, Y., Adza Rina, M.N., Saharee, A.A., Wahid, A.H., Zamri-Saad, M., 2014. Changes in serum progesterone and estrogen concentrations in non-pregnant boer does following experimental infection with corynebacterium pseudotuberculosis. J. Vet. Adv. 4 (5), 524–528. Paton, M.W., 2010. The epidemiology and control of caseous lymphadenitis in australian sheep flocks. Murdoch University. Pepin, M., Paton, M., Hodgson, A.L., 1994. Pathogenesis and epidemiology of Corynebacterium pseudotuberculosis infection in sheep. Curr. Top. Vet. Res. 1, 63–82. Peterhans, E., Greenland, T., Badiola, J., Harkiss, G., Bertoni, G., Amorena, B., Eliaszewicz, M., Juste, R.A., Kraßnig, R., Lafont, J.P., Lenihan, P., Pétursson, G., Pritchard, G., Thorley, J., Vitu, C., Mornex, J.F., Pépin, M., 2004. Routes of transmission and consequences of small ruminant lentiviruses (SRLVs) infection and eradication schemes. Vet. Res. 35 (3), 257–274. Quartuccio, M., Marino, G., Cristarella, S., Zanghì, A., 2009. Ram epididymitis: case reports. J. Comp. Pathol. 141 (4), 297. Radostits, O.M., Gay, C.C., Hinchcliff, K.W., Constable, P.D., 2007. A textbook of the diseases of cattle, horses, sheep, pigs and goats. Veter-. Med. 10, 2045–2050. Robert, Y.S., Walter, T.R., 2007. Current Therapy in Large Animal Theriogenology, second ed. Saunders,, United States of America. Sanocka, D., Fra̧czek, M., Jȩ drzejczak, P., Szumała-Ka̧kol, A., Kurpisz, M., 2004. Male genital tract infection: an influence of leukocytes and bacteria on semen. J. Reprod. Immunol. 62 (1), 111–124. Schoenian, 2005. Reproduction in the Ram. In: Getting Started in the Meat Goat Business. McKenzie-Jakes, Angela. Shamsuddin, M., Rodriguez-Martinez, H., Larsson, B., 1993. Fertilizing capacity of bovine spermatozoa selected after swim up in hyaluronic acid containing medium. Reprod. Fert. Dev. 5, 7–15.
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Shum, W.W., Ruan, Y.C., Silva, N., Breton, S., 2011. Establishment of cell-cell cross talk in the epididymis: control of luminal acidification. J. Androl. 32 (6), 576–586. Tashjian, J.J., Campbell, S.G., 1983. Interaction between caprine macrophages and Corynebacterium pseudotuberculosis: an electron microscopic study. Am. J. Vet. Res. 44, 690–693.
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Relationship between the Corynebacterium pseudotuberculosis, phospholipase D inoculation and the fertility characteristics of crossbred Boer bucks Zaid Khudhur Mahmood a, Zurin-Azlin Mohd Jin a, Faez Firduas Jesse a,n, Abdul Aziz Saharee a, Jasni Sabri b, Rosnina Yusoff a, Abd Wahid Haron a a b
Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, UPM, Serdang, Selangor, Malaysia Department of Clinical Studies, Faculty of Veterinary Medicine, UMK, Kelantan, Malaysia
art ic l e i nf o
a b s t r a c t
Article history: Received 28 August 2015 Received in revised form 22 June 2016 Accepted 27 June 2016
The aim of this study was to investigate the effects of Corynebacterium pseudotuberculosis and phospholipase D (PLD) on buck's fertility, specifically, testosterone concentration, scrotal circumference and semen quality. Crossbred Boer goats (n¼13) aged 12–14 months were divided into three groups. The first group (n¼ 3) was inoculated with one ml sterile phosphate buffer saline subcutaneously into subaxillary area as the control. The second group (n ¼5) was inoculated with live C. pseudotuberculosis 1 109 cfu subcutaneously into subaxillary area. The third group (n¼ 5) was inoculated with PLD 1 ml/20 kg BW intravenously into jugular vein. Blood collection was done twice a week over a period of three months for testosterone analyses. Semen was collected using electro-ejaculator once every two weeks whilst scrotal circumference was measured once every three weeks. Semen volume, semen pH and sperm progressive motility, concentration, live/dead percentage and morphology were evaluated. Bucks were euthanized three months' post inoculation and histopathological examination of the testicles and epididymis was performed. The results showed seven folds' significant decrease (P o0.05) in testosterone concentration in both C. pseudotuberculosis and PLD inoculated groups compared to the control. Semen volume, percentage of dead/live and abnormal sperm morphology showed significant increase (P o0.05) in both inoculated groups compared to the control. The following sperm defects were observed; tapered sperm, decapitated and knobbed head, thickened midpiece, distal and proximal droplet, sterilizing tail and folded tail. Scrotal circumference, semen pH, semen wave pattern, sperm motility and concentration showed significant decrease (Po 0.05) in both inoculated groups compared to the control. The testicles showed varied degrees of degeneration and necrosis with shrunken seminferous tubules. In conclusion, testosterone concentration, scrotal circumference and semen quality were negatively affected in both inoculated groups. Moreover, percentages of live/dead and abnormal sperm morphology were also increased. The results suggest that both C. pseudotuberculosis and PLD had detrimental effects on buck's fertility. & 2016 Elsevier B.V. All rights reserved.
Keywords: Boer Buck Goat Corynebacterium pseudotuberculosis Phospholipase D Fertility
1. Introduction Reproduction is the ability of an individual to create a new individual to maintain its kind. The natural process of reproduction in the animal kingdom is called breeding. Normally, mammalian offspring are born juveniles with complete developed sex organs, yet not reproductively functional. Later on, after a certain period of time, could be months to years, the sex organs develop
n
Corresponding author. E-mail addresses: [email protected], [email protected] (F.F. Jesse).
http://dx.doi.org/10.1016/j.livsci.2016.06.015 1871-1413/& 2016 Elsevier B.V. All rights reserved.
further toward maturity and the animal become sexually mature (Klein, 2007). The insidious nature of some infectious conditions is associated with the pathology of the reproductive system and evidence suggests that infectious disease-related reproductive abnormality reduces the reproductive efficiency in farm animals (Grooms, 2004; Robert and Walter, 2007). Chronic diseases such as Johne's disease, caprine arthritis-encephalitis (CAE) and caseous lymphadenitis have serious effects on reproductive health (Peterhans et al., 2004). First, and foremost effects are fever, reduction of appetite, loss of weight and reduced ambulation. A sick animal is less likely to breed (Robert and Walter, 2007).
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Goat CLA is a chronic disease caused by C. pseudotuberculosis a gram positive, facultative anaerobic and pyogenic organism belong to the family Actinomycetaceae. Caseous lymphadenitis has a long incubation period ranging between 3 and 20 weeks. It has distinct clinical manifestation when the lesions become progressive. Most common manifestations are abscesses in the superficial lymph nodes of the body and less often internally (Brown and Olander, 1987; Valli and Parry, 1993; Fontaine and Baird, 2008; Paton, 2010). C. pseudotuberculosis has a potent exotoxin, so-called, phospholipase D, which is a key virulence factor in the development of CLA (Baird and Fontaine, 2007). The notion that PLD is a significant virulence factor has been demonstrated with some isolates of C. pseudotuberculosis that had their pld genes encoding PLD removed, were not capable of causing abscess formation in the lymph nodes of CLA infected sheep (McNamara et al., 1994). PLD is a sphingomyelin-specific phospholipase that can dissociate sphingomyelin into phosphate and choline (Pepin et al., 1994). It has many biological activities such as dermonecrosis (Muckle and Gyles, 1986), lethality (Brogden and Engen, 1990) and destruction of macrophages during infection in the goats (Tashjian and Campbell, 1983), interferes with ovine neutrophil chemotaxis process and is lethal to the cells themselves (Yozwiak and Songer, 1993). This contributes to the dissemination of the pathogen from the primary infection location to other parts of the animal's body (Guimarães et al., 2011). Infectious reproductive diseases are common problem in male sheep and goats and often manifested as infertility. Infectious diseases such as Brucellosis, Actinobacillus spp and Histophilus spp have destructive nature on the reproductive performance in male and female which can lead to infertility (Robert and Walter, 2007). Interestingly, Othman et al. (2014) concluded that C. pseudotuberculosis infection in non-pregnant goats could be incriminated for hormonal imbalance resulting in infertility. Bacterial epididymitis is a significant cause of subfertility in rams. However, the pathogen type, the age and the sexual experience of the affected animal play an important role in the disease. Brucella melitensis, Histophilus somni and Streptococcus spp were isolated from clinical cases of epididymitis in rams. Often, these cases are accompanied with various degrees of testicular degeneration and to a lesser extent testicular atrophy with impaired spermatogenesis in nearly all cases (Quartuccio et al., 2009). Microscopic examinations revealed necrotic reaction of the entire parenchyma of the testicles and intratubular and interstitial orchitis were also observed. Histopathology of the epididymitis was of necrotic, sclerosing and suppurative form (Marino et al., 2009). Semen quality and fertility of rams can be affected by many infectious pathogens such as C. pseudotuberculosis, Brucella bovis, Actinobacilus spp and Histophilus spp. Looking from a scientific point of view, a large percentage of abnormal morphology of sperm is proof of the damage imposed on the reproductive tract and it is tricky to determine whether these sperm deformities are transient or permanent in nature (Robert and Walter, 2007). Goat's caseous lymphadenitis is gaining foothold in Malaysia with no proper vaccination or control programs. There is scanty information on how caseous lymphadenitis in general, C. pseudotuberculosis and/or PLD in particular affect fertility characteristics in male goat. Therefore, the objectives of this study were to estimate the testosterone concentration, evaluate semen quality and to observe the cellular changes in the testicles and the epididymis, post inoculation with C. pseudotuberculosis and PLD.
13
2. Methods and methods 2.1. Animals and experimental design Thirteen buck aged between 12 and 14 months with no history of vaccination against CLA was screened seronegative toward CLA before the commencement of the experiment. The experiment was conducted according to the regulations of Institutional Animal Care and Use Committee (IACUC) of UPM (UPM/FPV/PS/3.2.1.551/ AUP-R119). The bucks were divided randomly into three groups. The first group consisted of three bucks as the control (we choose three bucks after the IACUC committee suggestions to reduce the number of animals used, especially it is control not treatment group as well as cost effective), inoculated with one ml sterile phosphate buffer saline (PBS) subcutaneously into subaxillary area. The second group consisted of five bucks were inoculated with live C. pseudotuberculosis 1 109 cfu subcutaneously into subaxillary area. The third group also consisted of five bucks inoculated intravenously with PLD one ml/20 kg BW into jugular vein. Post inoculation, blood was collected twice a week (starting week one through week 12) from the bucks via jugular vein puncture using hypodermic syringe and heparin tubes. The plasma was separated and stored at 20 °C until the analysis was done. The experiment lasted for three months (12 weeks, post inoculation) then the bucks were euthanized. 2.2. Isolation and identification of C. pseudotuberculosis C. pseudotuberculosis was isolated from clinical cases of goat's caseous lymphadenitis (seven cases of superficial lymph nodes and visceral organs abscessation). Isolates were sent to the Veterinary Laboratory Service Unit (VLSU), Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia for identification and confirmation of the bacteria according to principles and methods described in the microbiological diagnostic laboratory at the Veterinary Medical Teaching Hospital, University of California, Davis, Revised Edition 2008. 2.3. Extraction of phospholipase D Phospholipase D was extracted following the method described by Zaki (1968). Briefly, two or three loops of a 48 h pure culture of C. pseudotuberculosis were inoculated in flask of freshly prepared bovine heart-liver medium. The flask was incubated anaerobically for seven days at 37 °C in slanting position of 15–20°. The culture that developed a pellicle was used. Phospholipase D separation started with centrifugation of the culture medium at 8000 rpm/ 15 min in refrigerated centrifuge. The supernatant was collected and passed via sterile cellulose membrane filter (0.2 mm) and stored at 4 C° then used in the experiment. 2.4. Hormone analysis Radioimmunoassy kit (RIA) were used to analyze the goat's plasma concentration of testosterone (RIA, REF: IM 1119) from IMMUNOTECH, Beckman Coulter Company. The samples and calibrators were incubated using competitive assay kit with 125I-labeled tracer in antibodies-coated tubes. After a certain period of incubation, the content of the tubes was decanted and the bounded radioactivity was measured. Briefly, the calibrators (0 6) were provided by the manufacturer as a ready to use reagent. The calibrators were added to the calibrator's tubes at 50 ml, the samples and the control were added to the sample's tubes at 50 ml. Then, the tracer (125I-labeled testosterone) was added at 500 ml to all tubes. The tubes were then
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incubated on a vortex shaker for three hours at 37 °C to mix all the contents inside the tubes. Finally, the contents of the tubes were decanted and the radioactivity of the tubes was measured using a gamma counter machine (Wallac WIZARD 1470, Perkin Elmer, life sciences). The testosterone RIA sensitivity was 0.00065 ng/ml, inter-assay CV was 0.88 ng/ml and the intra-assay CV was 0.88 ng/ ml. The cross reactivity is o1.0 10 2% toward testosterone glucuronide, cortisol, corticosterone, cholesterol, deoxycorticosterone, 17-epitestosterone, testosterone sulfate, androsterone, 3 betaoxyandrostan, 5 beta, 17 one, progesterone, estrone, estradiol, ethynylestradiol, estriol and dexamethasone (RIA, REF: IM 1119). 2.5. Scrotal circumference (SC) Three weeks post inoculation SC measurement was started through week 12. The measurements were taken once every three weeks. The scrotal neck was held with one hand and then the testicles were gently forced down into the scrotum. A measuring tape was placed around the greatest circumference. Several measurements (three) were taken and their average was calculated to increase the accuracy of SC. 2.6. Semen collection and evaluation Semen was collected from the bucks once every two weeks (starting week two, post inoculation through week 12) using an electro-ejaculator (Electro-Ejaculator, P-T Electronics, Oregon, USA). The buck was held against the wall and a lubricated electrical probe with electrodes was introduced around 10–12 cm into the rectum adjacent to the prostate gland. An intermittent stimulation of 3–10 V was applied in cycles of 8–10 s. The semen was collected using a collecting funnel with a graded sterile glass tube covered with aluminum foil to avoid light shock. Evaluation was done on the semen for volume, pH, wave pattern, progressive motility, sperm concentration, live/dead percentage and abnormal morphology.
2.6.6. Sperm morphology Sperm morphology was determined using the Nigrosin-Eosin staining method and the smears were examined using a light microscope under high magnification (1000x) where 200 sperm were counted and abnormal morphology was determined within these 200 sperm according to Hafez (1993). 2.7. Histopathology Twelve weeks, post inoculation, the bucks were euthanized and reproductive organs (testicles and epididymis) were harvested and placed in 10% buffered formalin for histopathological examination. After fixation, all samples were processed using an automatic tissue processor, embedded in paraffin wax and cut at 4 mm. The tissue sections were placed on glass slides, stained with hematoxylin and eosin and covered with a drop of DPX and cover slip (Luna, 1968). 2.8. Statistical analysis Data were analyzed using SPSS version 19.0. Repeated measures ANOVA was used since all parameters were repeatedly measured over time (12 weeks). All values were reported as mean 7SE at Po0.05.
3. Results Generally, all bucks' developed fever, post inoculation in both treated groups. The C. pseudotuberculosis inoculated bucks (all five bucks) showed abscess formation at the site of inoculation as well as enlargement of their superficial lymph nodes two weeks post inoculation. In contrast, PLD inoculated bucks showed no lesions at the site of inoculation. There was no enlargement in any of their lymph nodes. 3.1. Testosterone concentration
2.6.1. Semen volume Semen volume was measured directly after collection and it was expressed in milliliter per animal. The semen pH also was estimated using pH strips (Whatman™). 2.6.2. Wave pattern A drop of raw semen was placed on a warmed glass slide and was examined using light microscope under low magnification (100x) to estimate the wave pattern of the sperm. The wave pattern was given grades between 0 and 5 depending on the aggressiveness of the waves made by sperm movement (Hafez, 1993). 2.6.3. Sperm motility A drop of raw semen and a drop of warmed saline were placed on warmed glass slide and were mixed together using the corner of cover slip and were examined using a light microscope under low magnification (100x) to view the individual motion of the sperm. If the individual sperm could not be identified due to concentrated semen, a new preparation was made with less semen. The number of motile sperm was expressed as a percentage.
Corynebacterium pseudotuberculosis inoculated group showed a significant decrease (p o0.05) in plasma testosterone concentration between week 2 to week 12 compared to the control. The PLD group plasma testosterone concentration was also significantly decreased (p o0.05) in week two and between week 5 to week 12 compared to the control. Both treated groups showed striking seven folds’ significant decrease (p o0.05) between week 5 to week 12 compared to the control (Fig. 1). 3.2. Scrotal circumference (cm) Inoculation with the C. pseudotuberculosis showed a significant decrease (p o0.05) in scrotal circumference within week 6, week 9 and week 12 as compared to the control. The PLD inoculated group showed a significant decrease (po 0.05) in week 9 and week 12 as compared to the control (Figs. 2 and 3). 3.3. Semen evaluation
2.6.4. Sperm concentration Sperm concentration was measured and calculated as previously described by Hafez (1993).
3.3.1. Semen volume The volume of the semen showed a significant increase (p o0.05) in both C. pseudotuberculosis and the PLD inoculated animals in week eight as compared to the control (Fig. 4).
2.6.5. Sperm live/dead percentage A semen film was made carefully using Nigrosin-Eosin stain mixture, and the average percentage was obtained after counting 200 sperm (Hafez, 1993).
3.3.2. Semen pH Corynebacterium pseudotuberculosis group showed a significant decrease (p o0.05) in pH of the semen in week four and week six, post inoculation as compared to the control. In the PLD group,
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Fig. 1. Testosterone concentration of the bucks’ post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼13.
Fig. 4. Semen volume of the bucks' post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼13.
Fig. 2. Scrotal circumference of the bucks’ post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼ 13.
Fig. 5. Semen pH of the bucks' post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼ 13.
semen pH showed a significant decrease (po 0.05) in semen pH in week four only compared to the control (Fig. 5). 3.3.3. Semen wave pattern The semen wave pattern showed a significant decrease (p o0.05) for C. pseudotuberculosis and the PLD inoculated groups in week four and week six compared to the control (Fig. 6). 3.3.4. Sperm motility Sperm motility showed a significant decrease (p o0.05) in both, C. pseudotuberculosis and the PLD groups in week four and week six compared to the control (Fig. 7). 3.3.5. Sperm concentration Corynebacterium pseudotuberculosis and PLD groups showed a significant decrease (p o0.05) in sperm concentration in week four and week six compared to the control (Fig. 8). 3.3.6. Dead/ live sperm The percentage of dead sperms showed a significant increase
Fig. 6. Semen wave pattern of the bucks' post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼ 13.
(p o0.05) in both C. pseudotuberculosis and the PLD groups in week four, week six and week eight compared to the control (Fig. 9). 3.3.7. Abnormal sperm morphology Corynebacterium pseudotuberculosis
and
the
PLD
groups
Fig. 3. Testes of the bucks showing their sizes at the end of experiment period (week 12). Corynebacterium pseudotuberculosis inoculated animals (A), phospholipase D inoculated animals (B) and the control (C).
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Fig. 7. Sperm motility of the bucks’ post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼ 13.
Fig. 10. Percentage of abnormal sperm morphology of the bucks' post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼ 13.
and necrosis of the lining epithelium of the epididymal tubules. There were morphologically abnormal sperms inside the lumen of the epididymal tubules (Figs. 15 and 16).
4. Discussion
Fig. 8. Sperm concentration of the bucks' post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼ 13.
Fig. 9. Percentage of dead/live sperm of the bucks' post inoculated with Corynebacterium pseudotuberculosis and phospholipase D; n ¼13.
showed a significant increase (po 0.05) of the percentage of abnormal sperm morphology in week four, week six and week eight compared to the control (Figs. 10–12). 3.4. Histopathology of the reproductive organs Corynebacterium pseudotuberculosis inoculated group showed significant cellular changes in the testicles represented by irregular seminiferous tubules, shrinkage of seminiferous tubules, edema, degeneration and necrosis of some spermatogonia cells, there were spermatides inside the lumen of the seminiferous tubules, mild necrosis of Leydig cells and testicular atrophy, whilst in the PLD inoculated group testicles showed edema, severe congestion, irregular and shrinkage of the seminiferous tubules, less spermatides were seen in the lumen of the seminiferous tubules, severe degeneration and necrosis of the spermatogonia cells (Figs. 13 and 14). Moreover, the epididymis in both, the Corynebacterium pseudotuberculosis and the PLD groups showed edema, degeneration
To the authors’ knowledge, this is the first detailed study to evaluate the effects of the C. pseudotuberculosis and the PLD on testosterone level, semen quality and testicular lesions of male goat. Determination of infertility in animals caused by a specific pathogen requires the knowledge of the infectivity, the incubation period, prevalence and the virulence factors of that pathogen as well as the susceptibility of the animals (Givens and Marley, 2008). In males, the invasion of microbes into the genital tract is often associated with impaired sperm function and infertility (Sanocka et al., 2004). In mice, the C. pseudotuberculosis and the PLD inoculation showed a significant decrease in plasma testosterone level, where C. pseudotuberculosis inoculated mice showed 5 times lower compared to the control group (Khuder et al., 2012). This is in agreement with the current study which showed that the testosterone concentration was significantly decreased (seven folds) in both treated groups C. pseudotuberculosis and PLD. Leydig cells are testosterone producing cells; the low-level of the testosterone could be due to the direct effect of PLD on these cells in rams (Ibtisam, 2008). These accords with our findings when the testicles showed necrosis of the Leydig cells and similar signs of atrophy, post inoculation with both the C. pseudotuberculosis and the PLD. The testicles also showed irregular shape and shrinkage of some seminiferous tubules with interstitial edema. The results of this study is in agreement with Ibtisam (2008) who stated that rams naturally infected with C. pseudotuberculosis showed significant decreased of serum testosterone level (2.11 70.63 ng/ml) compared to non-infected rams (3.42 70.82 ng/ml). Scrotal circumference is an inherited trait that is related to reproductive performance and is a good indicator of puberty in bulls when it reaches certain size (25–27 cm) instead of the body weight and the age (Coulter, 1986). Semen quality is also associated with scrotal circumference at the age of the puberty in beef bulls (Coulter and Kastelic, 1999). Moreover, scrotal circumference is highly correlated with sperm production, total sperm per ejaculate and sperm concentration in adult rams and bucks (Robert and Walter, 2007). In the present study scrotal circumference was significantly decreased in both the C. pseudotuberculosis and the PLD groups and negatively reflected on other fertility attributes such as semen wave pattern, sperm motility, sperm concentration, percentage of dead sperm and abnormal sperm morphology. Hence, all the latter parameters showed a significant decrease in their level. These findings are in strong agreement with Schoenian
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Fig. 11. Different types of abnormal sperm morphology from the Corynebacterium pseudotuberculosis inoculated bucks; Nigrosin-Eosin stain 1000x. Spiral head (A), tapered sperm (B), coiled tail and decapitated heads (C), double forms (D), broken neck (E) and distal protoplasmic droplet (F).
Fig. 12. Different types of abnormal sperm morphology from the PLD inoculated bucks; Nigrosin-Eosin stain 1000x. Detached knobbed head and bent tail sperm (A), distal protoplasmic droplet with bent tail (B), sterilizing tail stump (C), thickened midpiece (D), proximal protoplasmic droplet with coiled tail, (E) and distal protoplasmic droplet with bent tail (F).
(2005) and Gimenez (2007) stated that semen quality, semen quantity and fertility rates are sturdily correlated with scrotal circumference in rams and bucks. There is a high positive correlation between semen volume along with the number of sperms per ejaculate and sperm concentration with the age in bucks. However, some diseases are well known to have a negative impact on semen quality such as the epididymitis (Robert and Walter, 2007). In the current study semen volume showed a significant increase in the C. pseudotuberculosis inoculated group in week eight of the experiment. It is hypothesized that the C. pseudotuberculosis have reached the epididymis and accessory sex glands leading to inflammatory
reaction increasing their secretions and adding additional volume to the ejaculated semen; hence, the increased volume was noticed two months, post inoculation. Additionally, C. pseudotuberculosis was isolated and identified from the buck's semen in this treated group (unpublished data). The latter hypothesis also agreed with Radostits et al. (2007), Fontaine and Baird (2008) and Paton (2010) who stated that the C. pseudotuberculosis was recovered from the accessory sex organs, epididymis and preputial cavity in apparently healthy rams. The consequences of bacterial presence in the male genital tract can affect fertility (Sanocka et al., 2004). Feasible measures should be taken prior to implicate C. pseudotuberculosis as sexually transmitted bacteria in small ruminants.
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Fig. 13. Testes shows irregular and shrinkage of the seminiferous tubules (red arrow), edema (orange arrow), degeneration and necrosis (yellow arrow), fewer spermatides inside the lumen of the seminiferous tubules (black arrow) and necrosis of Leydig cells (white arrow) 12 weeks post inoculation with the Corynebacterium pseudotuberculosis; H&E 400X. (For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.)
Fig. 14. Testes shows irregular and shrinkage of the seminiferous tubules (red arrow), severe congestion (white arrow), edema (orange arrow), degeneration and necrosis (yellow arrow), and fewer spermatides inside the lumen of the seminiferous tubules (black arrow) 12 weeks post inoculation with the phospholipase D; H&E 400X. (For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.)
Routinely, semen pH is evaluated as one of the environmental parameters in human and animal semen, as it can affect the sperm characteristics, function and motility (De Pauw et al., 2003; Shum et al., 2011; Contri et al., 2013). However, a decreased pH of bull's sperm led to reduced sperm motility and increase in sperm longevity. Thus, lifespans of the sperms were increased (Carr and Acott, 1989; Jones and Bavister, 2000). The present study showed that semen pH was significantly low, post inoculation with both the C. pseudotuberculosis and the PLD. These findings agreed with Contri et al. (2013) who reported that a low pH (5.5) compromises
the sperm membrane integrity and motility in bulls whilst a high pH (8.5) led to a drop in sperm mitochondrial activity producing immobile sperm. The low semen pH in this experiment further explains the low semen quality in both inoculated groups. The wave pattern of the semen is dependent on many factors such as sperm concentration, percentage of motile sperm and the progressive motion of the sperm. Semen waves can be seen as rapid swirling which can be depressed if sperm concentration or motility is reduced (Robert and Walter, 2007). Semen wave patterns showed a significant decrease in the current study in both
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Fig. 15. Epididymis shows edema (white arrow), degeneration and necrosis of the lining epithelia of the epididymal tubules (red arrow), and morphologically abnormal sperms (orange arrow) inside the epididymal tubules 12 weeks post inoculation with the Corynebacterium pseudotuberculosis; H&E 400X. (For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.)
Fig. 16. Epididymis shows degeneration and necrosis (red arrow) of the lining epithelia of the epididymal tubules, morphologically abnormal sperms (orange arrow) inside the epididymal tubules and edema (white arrow) 12 weeks post inoculation with the phospholipase D; H&E 400X. (For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.)
the C. pseudotuberculosis and the PLD inoculated groups. Moreover, in the present study plasma testosterone concentration and semen pH were significantly low decreasing the semen wave pattern hence all of the semen quality characteristic are stimulated and controlled by the androgens, especially testosterone. In general male reproductive function could be affected by pathogenic bacterial strains especially when presenting in the semen where it can affect the sperm motility directly via its agglutination effect on the motile sperm rendering it immotile and also disrupting the acrosome integrity (Monga and Roberts, 1994;
Sanocka et al., 2004). The current study showed that the sperm motility and sperm concentration were significantly decreased four weeks post inoculation in both the C. pseudotuberculosis and the PLD groups. These findings are supported by the significant histopathological lesions that have been found in the testicular tissue such as degeneration and necrosis of some of the spermatogonia cells and the reduced number of spermatids that was observed in the lumen of the seminiferous tubules. Spermatozoa morphology is one of the semen quality characteristics and of great value in fertility assessment of animals
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especially in extensive rearing farms in the tropics (Chacón, 2001). Abnormal spermatozoa morphology has immediate effect on the animal's fertility and the proportion of those abnormalities in the semen is negatively correlates with fertility (Shamsuddin et al., 1993). In the current study both the C. pseudotuberculosis and the PLD inoculated groups showed a significant increase in the percentage of dead sperm and the percentage of abnormal morphology of the sperm. In addition, some morphologically abnormal sperm were observed in the lumen of epididymal tubules. These findings are agreed with Chandler et al. (1988) and Barth and Oko (1989) who reported that spermatozoa morphological abnormalities can be influenced by diseases. It also indicates the poor semen quality and reduced fertility of many animals’ species including goat. We hypothesized that if CLA lesions (of chronic nature) develop in the scrotum or the inguinal lymph nodes, the testicles within the affected side may become smaller than normal due to atrophy. In addition, affected testicles may produce low concentration of spermatozoa with high percentage of abnormal sperm morphology. Moreover, “heat” produced by the local caseous inflammatory process may have adverse effect on spermatogenesis.
5. Conclusion In conclusion, the present findings suggest that CLA, in general, C. pseudotuberculosis and the PLD in particular have had direct detrimental effects on testosterone concentration. As a result, toll reflected and decreased the scrotal circumference, lowered semen pH and semen quality. All that have been mentioned above could potentially implicate and affect the reproduction and fertility of male goat. However, the main influence of CLA on buck's fertility may be due to the general effect of this chronic disease (wasting and debilitating nature of CLA) on the body systems resulting in collateral damages of reproductive system.
Competing interests The authors declare that they have no competing interests.
Authors contributions FF Jesse and ZKH Mahmood contributed to the design of the field trial. ZKH Mahmood and Z Mohd Jin ran the experiment and collect the samples. ZKH Mahmood and FF Jesse analyzed the results and drafted the paper. FF Jesse, AA Saharee, J Sabri, R Yusoff and H Wahid have contributed to the design of the study, writing the manuscript and coordination of the study. All authors have read and approved the manuscript.
Acknowledgements The authors are grateful to Mr. Yap Keng Chee, Mr. Mohd Fahmi Mashuri and Mr. Mohd Jefri Norsidin for their assistance. This work was funded by the Research University Grant Scheme (RUGS), Universiti Putra Malaysia.
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