ABSTRACT
Tolerance to African animal trypanosomosis (AAT) among several animal species involves a wide milieu of factors which modulate the animal’s response to the disease and is considered a breed attribute. To investigate the effect of breed on tolerance/resilience to trypanosome infection on pubertal boars, nine (9) Nigerian Native and nine (9) Large-White x Landrace crossbreed boars were experimentally inoculated with laboratory samples of Trypanosoma brucei brucei. Their comparative responses with regard to clinical symptoms, growth parameters, histopathological and histometrical features of the testis, Sertoli and germ cell numbers and spermatogenic output including cell ratios and daily sperm production were studied over two study periods- 63 days post infection (63d p.i.) and 98 days post infection (98d p.i.). Results obtained indicated that infected boars of both breeds were clearly parasitaemic in the first study period, with a significant (P<0.05) reduction observed in the native boars by 98d p.i. The general trend in the results obtained showed significant (P<0.05) differences in the various parameters, with the Nigerian Native boars exhibiting strong marginal gains by the second study period. This was not the case with the exotic Large-White x Landrace boars and suggested that the native boars possessed a superior ability to mitigate the more severe effects of the pathology and a tendency to return to normal. With respect to the clinical features investigated, the Nigerian Native boars presented significantly (P<0.05) higher values with respect to parasitaemia log values, rectal temperatures, as well as packed cell volume. Histopathological findings revealed that lesions, including tubular distortion, denudation of basement membrane, seminiferous epithelial damage led to the distortion of the architecture of the seminiferous epithelium as well as degradation of the inter-tubular compartment and values were significantly (P<0.05) lower among the native boars. The parameters on growth showed the nutrient-parasite interaction was influenced by breed attributes. Biometrical and linear body measurements were affected significantly (P<0.05) less in the native boars than in the exotic boars. Weight loss was minimized among the native boars with a tendency to significantly (P<0.05) increase growth rate as during the second study period, whereas this trend was not clearly observed among the exotic boars. The effect of the infection on clinical and histopathological features, as well as growth responses and especially in relation to the testes’ capacity for spermatogenesis was studied. We observed significant (P<0.05) reductions in testes weight, somatic and germ cell populations and also significant (P<0.05) reductions in the overall kinetics of spermatogenesis and daily sperm production. The mechanisms of action implicated in breed responses to the pathology appeared to relate to phenotypic characteristics as well as innate mechanisms which are known to modulate the pathogenesis of trypanosomosis. Equally the lowered parasitaemia observed among the native boars suggested that toxicological effects of trypanosomes on this breed of boars were limited. It was concluded that the Nigerian Native boars possessed an attribute that could reverse the adverse patho-physiological effects of T. b. brucei infection and were therefore more resilient to T. b. brucei infection than the exotic Large-White x Landrace boars.
TABLE OF CONTENTS
Page
Title page … … … … … … … … … i
Certification … … … … … … … … … ii
Dedication … … … … … … … … … iii
Acknowledgement … … … … … … … … iv
Abstract … … … … … … … … vi
Table of Contents … … … … … … … … … vii
List of Tables … … … … … … … … … xiii
List of Figures … … … … … … … … … xiv
List of Plates … … … … … … … … … xv
Chapter 1: Introduction
1.1 Background … … … … … … … 1
1.2 Objectives of the study … … … … … … 5
1.3 Justification … … … … … … 6
Chapter 2: Literature Review
2.1 African Animal Trypanosomosis … … … … … 7
2.2 Economic Importance of AAT … … … … … 7
2.3 Epidemiology of AAT … … … … … … 9
2.3.1 Occurrence … … … … … … … 10
2.3.2 Host range … … … … … … … … 12
2.3.3 Trypanosomes … … … … … … … 12
2.3.3.1 Sub-genus: Nannomonas … … … … … … 15
2.3.3.2 Sub-genus: Duttonella … … … … … … 16
2.3.3.3 Sub-genus: Trypanozoon … … … … … … 17
2.3.4 The Tsetse fly (Glossina spp) … … … … … … 19
2.4 Pathogenesis of AAT … … … … … 21
2.5 Detection and diagnosis of AAT … … … … … 28
2.6 Trypanotolerance as a control measure of AAT … … … 31
2.7 Growth and development of pigs in the humid tropics … … 34
2.7.1 Characteristics of the humid tropics in relation to pig production … 34
2.7.2 Phenotypic characteristics of native and exotic pigs … … … 35
2.7.3 Factors affecting pig growth … … … … … … 37
2.8 Testis characteristics and function in the boar … … … 39
2.8.1 Morphology, growth and development of boar testis … … … 39
2.8.2 Spermatogenesis and the cycle of the seminiferous epithelium in the boar…43
2.8.3 The cycle of the seminiferous epithelium … … … … 45
2.8.4 Regulation of testis function … … … … … … 49
2.8.5 Measurement of testicular function … … … … … 52
Chapter 3: Materials and Methods
3.1 Experimental Site … … … … … … … 55
3.2 Experimental Animals … … … … … … 55
3.3 Trypanosomes … … … … … … … … 57
3.4 Experimental Procedure … … … … … … 57
3.4.1 Harvesting of blood trypanosomes … … … … … 57
3.4.2 Inoculation of experimental animals … … … … … 57
3.4.3 Blood collection … … … … … … … 58
3.4.4 Clinical observations and data collection … … … … 58
3.4.4.1 Rectal Temperature (oC) … … … … … … 58
3.4.4.2 Parasitaemia (log) … … … … … … … 58
3.4.4.3 Packed cell volume (%) … … … … … … 59
3.4.4.4 Live-weight (kg) … … … … … … … 59
3.4.4.5 Daily weight gains … … … … … … … 59
3.4.4.6 Scrotal width … … … … … … … … 59
3.4.4.7 Increases in scrotal width … … … … … 60
3.4.4.8 Live-weight: scrotal width … … … … … 60
3.4.4.9 Final body weight … … … … … … … 60
3.4.4.10 Gonadosomatic index … … … … … … 60
3.4.5 Tissue processing … … … … … … … 61
3.4.5.1 Castration … … … … … … … … 61
3.4.5.2 Testis Sectioning … … … … … … … 62
3.4.6 Photomicrography … … … … … … 62
3.4.7 Light Microscopy … … … … … … 63
3.4.7.1 Histopathology … … … … … … … 63
3.4.7.2 Testis Morphometry … … … … … … … 63
3.4.7.2.1 Diameter of Seminiferous Tubule … … … … … 63
3.4.7.2.2 Volume Density of Testis Cmponents … … … … 64
3.4.7.2.3 Volume of Testis Components … … … … … 64
3.4.7.2.4 Length of Seminiferous Tubule … … … … … 64
3.4.7.2.5 Nuclear Diameter of Sertoli and Germ Cells … … … … 65
3.4.7.2.6 Nuclear volume of Sertoli and Germ Cells … … … … 65
3.4.7.3 Cell Counts … … … … … … … … 65
3.4.7.3.1 Sertoli Cell Nuclei Enumeration … … … … … 65
3.4.7.3.2 Germ Cell Enumeration … … … … … … 66
3.4.7.3.3 Leydig Cell Enumeration … … … … … … 67
3.4.8 Cell Ratios … … … … … … … … 68
3.4.9 Sperm Production Rates … … … … … … 69
3.4.9.1 Daily Sperm Production/testis … … … … … 69
3.4.9.2 Daily Sperm Production/gram/testis … … … … … 69
3.5 Statistical Analysis … … … … … … 69
Chapter 4: Results and Discussion
4.1 Results … … … … … … … … … 71
4.1.1 Rectal Temperature, Packed cell volume and Parasitaemia … … 71
4.1.1 Rectal Temperature … … … … … … … 71
4.1.1.1 Packed Cell Volume … … … … … … … 72
4.1.1.3 Parasitaemia … … … … … … … 72
4.1.2 Live-weights and Linear Testicular Measurements … … … 73
4.1.2.1 Live-weight … … … … … … … … 74
4.1.2.2 Daily Weight Gains … … … … … … … 74
4.1.2.3 Scrotal Width … … … … … … … … 75
4.1.2.4 Increase in Scrotal Width … … … … … 75
4.1.2.4 Liveweight: Scrotal Width … … … … … … 76
4.1.3 Biometric Measurements … … … … … … 77
4.1.3.1 Age at castration … … … … … … … 78
4.1.3.2 Final Body Weight … … … … … … 78
4.1.3.3 Paired testis weight … … … … … … … 79
4.1.3.4 Left/Right Testis Weight … … … … … … 79
4.1.3.5 Gonadosomatic Index … … … … … … 80
4.1.4 Histopathological observations … … … … … 81
4.1.4.1 Testis smear … … … … … … … 93
4.1.4.2 Tubular distortion … … … … … … … 93
4.1.4.3 Tunica Propria … … … … … … … … 94
4.1.4.4 Spermatogenic Profile … … … … … … … 94
4.1.4.5 Interstitial region … … … … … … … 95
4.1.5 Tubular Measurements … … … … … … 96
4.1.5.1 Tubular Diameter … … … … … … 96
4.1.5.2 Tubular Length/testis … … … … … … 97
4.1.5.2 Tubular Length/g/testis … … … … … 97
4.1.6 Volume density of testis components … … … … 98
4.1.6.1 Seminiferous tubule … … … … … … … 98
4.1.6.2 Tunica propria … … … … … … … 99
4.1.6.3 Seminiferous epithelium … … … … … 100
4.1.6.4 Lumen … … … … … … … … … 100
4.1.7 Volumes of testis components … … … … … 101
4.1.7.1 Seminiferous tubule … … … … … … … 101
4.1.7.2 Tunica propria … … … … … … … 102
4.1.7.2 Seminiferous epithelium … … … … … … 102
4.1.7.3 Lumen … … … … … … … … … 103
4.1.8 Nuclear diameter of Sertoli and Germ Cells … … … … 104
4.1.8.1 Sertoli cell … … … … … … … … 104
4.1.8.2 Type-A spermatogonia … … … … … … 105
4.1.8.3 Pachytene primary spermatocyte … … … … … 105
4.1.8.4 Round spermatid … … … … … … 106
4.1.9 Nuclear volume of Sertoli and Germ cells … … … … 107
4.1.9.1 Sertoli cell … … … … … … … 107
4.1.9.2 Type-A spermatogonia … … … … … … 108
4.1.9.3 Pachytene primary spermatocyte … … … … … 108
4.1.9.4 Round spermatid … … … … … … 109
4.1.10 Sertoli and germ cells/seminiferous tubule … … … … 110
4.1.10.1 Sertoli cell/tubular profile … … … … … … 110
4.1.10.2 Type A-spermatogonia/tubular profile … … … … 111
4.1.10.3 Pachytene primary spermatocyte/tubular profile… … … … 111
4.1.10.4 Round spermatid/tubular profile … … … … … 112
4.1.11 Sertoli and germ cells/testis … … … … … … 113
4.1.11.1 Sertoli cells/testis … … … … … … … 113
4.1.11.2 Type A-spermatogonia/testis … … … … … … 114
4.1.11.3 Pachytene primary spermatocytes/testis … … … … 114
4.1.11.4 Round spermatids/testis … … … … … … 115
4.1.12 Sertoli and germ cell ratios and daily sperm production… … … 116
4.1.12.1 Sertoli cell efficiency … … … … … … … 117
4.1.12.2 Overall rate of spermatogenesis … … … … … 117
4.1.12.3 Meiotic index … … … … … … … 118
4.1.12.4 Measure of germ cell degeneration … … … … … 118
4.1.12.5 Coefficient of the efficiency of spermatogonial mitosis… … … 119
4.1.12.6 Efficiency of mitotic division … … … … … … 119
4.1.12.7 Daily sperm production/testis … … … … … … 120
4.1.12.8 Spermatogenic efficiency … … … … … … 120
4.2 Discussion … … … … … … … 121
Chapter 5: Conclusion … … … … … … … 162
References … … … … … … … … … … 164
Appendix … … … … … … … … … … 206
CHAPTER ONE
Introduction
1.1 Background
Trypanosome species cause a wide range of damage to their livestock hosts, resulting in a disease condition known as trypanosomosis. The trypanosomes, which are largely implicated in this disease condition, are the pathogenic African trypanosomes. Thus, the disease is aptly called African Animal Trypanosomosis (AAT). AAT constitutes a significant impediment to animal husbandry in sub-Saharan Africa, where it is known to limit the full potential, not only of the livestock industry but also the human population (Onyiah, 1997; Swallow, 2000; WHO, 2004). In this regard, the disease has been implicated as a major cause of livestock under-development in the countries where it is endemic (FAO, 1983). Hursey (2000), describes AAT as the single most devastating disease in Africa in terms of its contribution to poverty and loss of agricultural production. In fact, it is estimated that in the absence of trypanosomosis, there could be a three-fold increase in livestock production in areas currently classified as endemic to AAT. In Nigeria, trypanosomosis is endemic to the rain forest region up to the derived savannah belt, an area noted for extensive production of exotic and indigenous breeds of livestock including swine (NITR, 1985; Kalu et al, 1991).
Trypanosomes have been detected in practically all classes of livestock (Ige and Amodu, 1975; Ikede and Losos, 1975; Murray et al., 1979; Agu and Bajeh, 1987; Luckins, 1995; Budd, 1999; Hursey, 2000). Infections by trypanosomes may occur singly, by one species of the parasite or be mixed, by simultaneous infection by more than one trypanosome species (Logan-Henfrey et al., 1992). Cyclical transmission, i.e. infections arising following a bite from an infected tsetse fly (Glossina spp), in which the parasite undergoes a series of cyclical development, is the usual mode of infection in susceptible mammalian hosts. However, infections may also occur mechanically by means of biting insect flies other than Glossina spp, in which the parasite undergoes no further development, or iatrogenically through sharp objects such as syringes and needles contaminated with infected blood (Jones and Davila, 2001).
Infections by trypanosomes in livestock may result in sub-clinical, acute or chronic forms of the disease, characterized primarily by anaemia (Kobayashi et al., 1976; Suliman and Feldman, 1989; Omeke and Odo, 1999). Further symptoms of AAT include febrile conditions as well as the rapid loss of body condition often resulting in death (Losos and Ikede, 1972). Of much greater importance are the inflammation, necrosis and degeneration of several tissues and organs, arising from invasion by trypanosome species such as Trypanosoma brucei (Mare, 1994). These changes, which apparently reflect the hosts’ immunologic responses to the infection, result in emaciation and general infertility among others (Omeke and Onuora, 1992b; Walgren et al., 1993; Mare, 1994; Sekoni et al., 2004).
Among porcine breeds, the incidence of AAT has of recent been well documented. Initial assumptions held that only Trypanosoma simiae was pathogenic to swine. However, it is now well established that trypanosome species other than T. simiae are equally pathogenic to pigs (Mally, 1982; Agu and Bajeh, 1987; Kageruka 1987; Onah and Uzoukwu, 1991; Onah, 1991; Omeke and Onuora, 1992; Omotainse et al., 1993; N’gayo et al., 2004). Symptoms common to all infected animals include a detectable parasitemia, lowered packed cell volume, fluctuating pyrexia as well as significant loss of body weight (Anosa, 1988). However, it is not often that trypanosomosis is fatal to pigs except when infected animals are left untreated for an extended period. Therefore the occurrence of fatality among trypanosome-infected animals depends to a large extent on several factors including plane of nutrition, environmental stressors, concurrent infections as well as breed characteristics (Murray et al., 1981; Dolan, 1987; Fagbemi et al., 1990; Otesile et al., 1991; d’Ieteren et al., 1998; Seck et al., 2000; Naessens et al., 2002). With regard to breed characteristics, anecdotal reports suggest that indigenous swine breeds are more resilient to infections by trypanosomes than exotic breeds. Pioneer works by Birkett (1958) and Stephen (1960) showed that although native pig breeds succumb to T. simiae infection, they remain relatively productive and suffer minimal mortality. Also, studies by Onah (1991) have shown that local pig breeds exhibit reasonable levels of trypanotolerance.
Trypanosome-induced infertility in boars appears to be largely due to the parasites’ effects on testis structure and function. These effects, which could be direct or indirect, are principally mediated through several processes including damage to testicular parenchyma, destruction of the interstitial tissues (including connective tissues, blood and lymphatic vessels) and probable effects on the reproductive endocrine system often resulting in the disruption of spermatogenesis (Isoun et al., 1975; Omeke and Onuora, 1992; Sekoni, 1994; Omeke and Igboeli, 2000; Adamu et al., 2006). Sertoli cells as well as other spermatogenic cell, including spermatogonia, early and late spermatocytes and spermatids are known to be adversely affected at varying degrees by trypanosome infections.
The critical functions of these cells in male reproduction have been fully elucidated (Amann and Schanbacher, 1983; Berndston et al., 1987; Hochereau-de-Reviers et al., 1987; Orth, 1988; Johnson et al., 2007). Equally, the close morphological associations existing between Sertoli cells and between Sertoli and germ cells, as well as interactions between germ cells within the seminiferous epithelium mean that a disruptive effect on any of these cell types would likely impact adversely on the other cell types with consequent implications on the kinetics of spermatogenesis (Robaire et al., 1995; Mruk and Cheng, 2004).
Sertoli cells support the process of spermatogenesis through a variety of specialized functions including the regulation of the mileux within which germ cells develop (Lunstra et al., 2003). This is achieved principally through the physiological arrangement of the tight junctional complexes between adjacent Sertoli cells and the basal lamina, known as the blood-testis barrier (Dym and Fawcett, 1970; Setchell, 1980; Russell and Peterson, 1985; Mruk and Cheng, 2004). Sertoli cells are also implicated in the maintenance of high intra-testicular concentration of testoterone, which is requisite for spermatogenesis (Means et al., 1976). Germ cells rely on Sertoli cells for nutritional and structural support (Courot et al., 1970; Mita et al., 1982; Russell et al., 1990). It is clear therefore that a close morphological and functional association exists between Sertoli cells and germ cells throughout the spermatogenic process which results in extensive interactions and communications at the cellular and molecular levels (Skinner, 1991; Jegou, 1993; Wright, 1993; Griswold, 1995).
Leydig cells constitute the main components of the interstitial region along with myoid cells and blood vessels (Frandson et al., 2009). Their major function is the production and secretion of the male sex hormones. Androgen secretion by Leydig cells is critical to the initiation and maintenance of the reproductive process in the male as well as the elaboration of secondary sexual characteristics (Amann and Schanbacher, 1983). The interstitial region is also highly vascularized with blood vessels and capillaries which serve in the transport of metabolites and other chemicals within the testicular environment (Dietrichs et al., 1973; Payne et al., 1996). In view of the reported deleterious effects of trypanosomes on Sertoli and germ cells as well as on the cells and tissues of the interstinum, it is understandable as to how such damage could lead to derangement of spermatogenesis (Omeke and Igboeli, 2000; Aksglaede et al., 2006).
Testis morphometry is a valuable technique for assessing qualitative as well as estimating quantitative changes in testicular composition and spermatogenic function under various conditions (Swiestra, 1966; Johnson and Neaves, 1981; Jones and Berndston, 1986; Sinha-Hikim and Hoffer, 1987; Johnson et al, 1992). It has also been used to characterize the cycle of the seminiferous epithelium in various pig breeds (Swiestra, 1968; Lunstra et al., 1986; Mendis-Handagama et al., 1988; Almeida et al., 2006). These studies have consistently demonstrated a strong correlation between testis structure and function. Comparative studies using these stereological techniques would therefore be a useful method for estimating the responses of different pig breeds to the effect of pathological insult to the testis.
Much emphasis has been placed on the study of the effects of porcine trypanosomosis on growing and adult pigs of the exotic breed in the Nigerian environment (Fagbemi et al., 1990; Otesile et al., 1991; Omeke and Onuora, 1992a; b; Omeke and Igboeli, 2000). In contrast, there is a dearth of information on comparison between pig breeds which are native to Nigeria and the exotic breeds. In fact, to the best of our knowledge, there has been no morphometric study on the Nigerian native pig under a disease condition such as trypanosomosis. This study was therefore designed to determine how trypanosome infection would affect testis structure and function in the native boars in comparison to the exotic boar and to assess whether the Nigerian native boar would exhibit resistance to trypanosomes than the exotic (Large-White x Landrace) boar, when they are experimentally infected with Trypanosoma brucei brucei.
1.2 Objectives of the Study
The broad objective of this research is to determine the effects of Trypanosoma brucei brucei on growth indices and testis characteristics of two breeds of boars – the indigenous Nigerian native boar and the exotic Large-White x Landrace crossbreed boars.
Specific objectives included:
- To evaluate the responses of two breeds of boars infected with Trypanosoma brucei brucei, with particular reference to pyrexia, degree of parasitaemia and packed cell volume, (PCV);
- To determine the extent to which T. b. brucei infection would affect live-weight gains, scrotal width and the relationships between live-weight and reproductive organ size among the experimental boars;
- To determine the effect of T. b. brucei on the testis parenchyma and the interstitial compartments of the testis in the two breeds of boars;
- To asses through quantitative testicular histology, the effect of T. b. brucei infection on populations of Sertoli and germ cells including Type-A spermatogonia, pachytene primary spermatocytes and round spermatids in both breeds of boars;
- To estimate by quantitative testicular histology, spermatogenic function under trypanosome challenge in the testis of both breeds of boars by determining the germ cell ratios and sperm production rates.
1.3 Justification
Boars represent ideal models for the study of testis structure and function in male mammalian species (Russell and Griswold, 1999; Matzuk and Lamb 2002; Costa et al., 2010). However, few studies exists which detail testicular cell proliferation and enumeration in the boar under pathological conditions such as trypanosomosis. Equally, there does not appear to be any such study involving the presumed trypanotolerant Nigerian native boar. The implication of the presumed trypanotolerant attribute of the native pig could be far-reaching. The increasing awareness of the potential contributions of indigenous animal genetic resources, especially those assumed trypanotolerant and the possible exploitation of their traits, especially through cross-breeding protocols, in increasing animal productivity has been consistently emphasized (d’Ieteren, 1994). This makes a definitive statement on this issue very imperative. Therefore a clear understanding of the effect of trypanosomes on testicular characteristics of the Nigerian native pig is an ideal first step. Furthermore, this study would further contribute to the existing body of knowledge on the effects of trypanosome pathology on boar reproduction.
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