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Mid-Piece Abnormalities in Bovine Semen Following Experimental and Natural Cases of Bovine Ephemeral Fever
Br. vet. ]. ( 1975), 131, 536
MID-PIECE ABNORMALITIES IN BOVINE SEMEN FOLLOWING EXPERIMENTAL AND NATURAL CASES OF BOVINE EPHEMERAL FEVER By
G. W.
BURGESS· AND
P.
J.
CHENOWETHt
Veterinary School, University of Queensland, St Lucia, Brisbane 4067 SUMMARY
A study of the effects of bovine ephemeral fever on the semen quality of bulls was carried out using two experimental and four natural cases of the disease. A consistent, dramatic rise in the percentage of abnormal mid-pieces was seen. Peak levels were recorded at approximately 35 days after the peak fever. After 155 days, the semen of one experimental case had returned to normal and similar trends were seen with the semen collected from the other bulls. A decrease in motility associated with the increase in abnormal mid-pieces also occurred. This finding supports field reports of a decrease in fertility of bulls following bovine ephemeral fever. It was not possible to show whether these changes are the result of viral growth in the testis or the pyrexia which usually occurs. INTRODUCTION
Bovine ephemeral fever is a viral disease of cattle, usually observed clinically as an acute febrile reaction which may be accompanied by lameness or paresis. The disease occurs mainly in Mrica, Asia and Australia (Burgess, 197 I). In Australia, there have been four epizootics (1936-37, 1955-56, 1967-68 and 1970-7 I) as well as numerous isolated outbreaks. The disease is now probably endemic in some northern areas of Australia (St George et al., 1973). The economic importance may not be readily apparent. Many cases are never seen by the owners and those which are seen are often accepted and soon forgotten. Losses have been reported to be due to a reduction in milk production, disruption of markets, reduction in condition of prime animals, individual deaths and reduced performance as a result of permanent lameness. A preliminary report of this study has been made (Chenoweth & Burgess, 1972). During the 1970-71 epizootic it was suggested that some bulls suffered a reduction in reproductive performance following this disease. In order to establish a better understanding of this situation, two experimental cases were observed in detail and semen samples collected from four natural cases were also studied. • Present address : Department of Veterinary Pathology and Public Health, Massey University, Palmerston North, New Zealand. t Present address: 52 Green Street, North Mackay, Queensland, Australia, 4740.
ABNORMALITIES IN BOVINE SEMEN
537
MATERIALS AND METHODS
Experimental animals Two bulls approximately 18 months old were used. One (No. 415) was a Friesian/Hereford weighing 209 kg, and the other (No. 418) was a Guernsey weighing 289 kg. Natural cases During the 1970-7 I epizootic several Droughtmaster bulls on the University Farm at Moggill showed clinical signs of Bovine Ephemeral Fever (BEF). Semen samples were subsequently collected from four of these animals. Virus inoculum The two experimental bulls were inoculated intravenously with 20 ml of infective blood which represented the third bovine passage of virus from the 1967-68 epizootic (Doherty, Standfast & Clark, 1969). This virus was generously supplied at the second passage level by Mr I. Clark, Animal Research Institute, Yeerongpilly. Semen collection technique Semen was collected by electro-ejaculation using a Watson Transtimulator Mk. I I, which was under manual control. Techniques for confirmation oj BEF infection Observations of clinical signs were made twice daily during the period of clinical reaction. Daily serum and whole blood samples were collected from the two experimental animals. Whole blood samples in ACD were used for virus isolation while EDT A samples were used for haematological studies. Serum samples were tested for neutralizing antibodies using infant mice, tube cultures of Vero (African green monkey kidney cell line) cells and the microtitre serum neutralization test (Burgess, 1974). Whole blood samples for virus isolation and acute and convalescent serum samples were collected from the four natural cases. Frequency of collection Three collections were made from each of the two experimental animals within eight days prior to inoculation. Six collections were made within 30 days post-pyrexia. Five samples were collected in the next 33-day period and a further four collections were made in the period 63-155 days post-pyrexia. Fewer collections were made from the natural cases. Collections from bull No. 622 were made at 5, 12, 19, 50 and 80 days post-pyrexia, and from bull No. 618 at 10, 17,52, 78, 95 and 115 days. A single collection was made from bull No. 580 at 34 days post-pyrexia. Bull No. 589 did not completely recover from the infection and had a hindlimb ataxia which prevented him from successfully mounting to serve. Two collections were made from this animal at 49 and 76 days post-pyrexia.
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BRITISH VETERINARY JOURNAL,
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Semen examination procedure Field examination. Immediately after collection, volume, colour and density were recorded. Mass activity was scored (0 to + + + +) and the percentage of actively motile spermatozoa was estimated. The percentage of sperm alive was estimated by using nigrosin-eosin stain, read within 24 h (Hancock, 1951). Laboratory examination. Head and mid-piece morphology of the spermatozoa was studied using Williams's stained preparations (Lagerlof, 1934) and wet preparations in buffered formol saline were examined under phase contrast microscopy after the method of Hancock (Chenoweth et al., 1970) for tail morphology, loose heads and cytoplastic droplets. Two hundred sperm were counted in each instance. The following classifications were employed, using Williams's stain: for sperm heads; normal, narrow at base (pyriform), large, pear-shaped, small, round and broad. For mid-pieces; thick, knobbed, filiform, "moth-eaten", corkscrew, dissociated, split, defective, broken, abaxial and mid-piece coiled around the head (Fig. 1). Using wet preparations; loose heads, proximal droplets, single bent tails, double bent tails and coiled tails.
RESULTS
There was a sharp rise in rectal temperature which coincided with a neutrophilia with a left shift occurring on days five and eight post inoculation for animals 415 and 418 (the experimental animals) respectively. A rise in serum antibody titres occurred shortly after the clinical signs were seen. Virus was isolated from blood samples collected on the day of peak fever. Virus was also isolated from a blood sample taken from animal No. 580. A rise in serum neutralizing antibody levels was demonstrated for all four Droughtmasters which had clinical signs of the disease.
Semen changes The most consistent and dramatic change was a rise in the percentage of abnormal mid-pieces as shown in Fig. 2. This reached a peak at approximately 35 days after the peak temperature rise. A further small rise was noted approximately 60 days post-pyrexia. Mid-piece abnormalities of most types were encountered at all tests conducted during the post inoculation period (Figs 4-7). However, during the period prior to the sharp rise at 27 days post-pyrexia, the main abnormalities encountered were in the "offshoot defect", "dissociated" and "filiform" categories. From the period of dramatic rise at 27 days postpyrexia, the "kinked mid-piece" abnormality assumed major importance. The pre-inoculation levels of mid-piece abnormalities were consistently low and the highest percentage recorded at this time was 2 per cent, whereas the peak values exceeded 70 per cent. There appeared to be a corresponding drop in motility. The number of animals involved in the experiment was such that a statistical confirmation of this observation was not possible. Changes in other parameters were not consistent and were not considered to be related to the
ABNORMALITIES IN BOVINE SEMEN
539
CLASSIFICATION OF MIDDLE-PIECE MORPHOLOGY OF BULL SPERM --
I
IlQrmal
,I t, r thick
knob
I t
filiform
'motheaten '
abaxial
! , t I J
dissociated kink / corkscrew
double
split
defective attachment
o
.1
coiled around . head Fig. I . Classification of mid-piece morphology of bull spermatozoa. The illustrations are a diagramatic representation of the mid-piece abnormalities observed using Williams's stained preparations. offshoot defecf
broken
infection. The percentage of abnormal mid-pieces in semen samples collected from the natural cases tends to confirm these observations and the results are shown in Table I. The percentage of mid-piece abnormalities d eclined in both experimental cases after 80 days. By 155 days the level for No. 418 had reached the pre-inoculation figure . Number 415 had dropped to 17 per cent by 88 days. However, an increase then occurred and by 152 days a level of 45 per cent had been reached. The significance of this rise is not clear. DISCUSSION
The most important of the semen results was considered to be the dramatic rise in mid-piece abnormalities which occurred in both experimental bulls and in the naturally infected bulls. There has been little work done on the effects
BRITISH VET ERI NARY JOURNAL, 13 1, 5
540
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• _ ___ _
2
. ___
20
4050
8 0 70
90
•
100
110
TIME POST l'Y'EXIA {rlA.ys 1
;.;f'
150
Fig. 2. Semen changes in two animals following experimental bovine ephemeral fever. The values at d ay 0 represent the m ean of three results of samples collected within 8 days prior to inoculation.
of specific pathogenic agents on spermatogenesis. Macleod (1964) found that abnormal spermatozoa increase significantly in the semen of men who had suffered viral infections. Previous workers have shown that illness associated with pyrexia can lead to increased numbers of abnormal spermatozoa (Gunn, Sanders & Granger, 1942; Macleod, 1964; Watson, 1964; Singleton, 1966; Rathore, 1969; Cameron, 1970). This increase can be detected as early as the fourth day following the pyrexia and is reflected at this stage in increased numbers of loose sperm heads and abnormal sperm tails (McKenzie & Berliner, 1937; Gunn et al., 1942). Cameron (1970) working with rams, found that nuclear changes in sperm first became apparent at 17- 21 days post-pyrexia and that there was a second rise in abnormalities between 30 days and 56 days post-pyrexia. Rathore (1969), TABLE I P E RCENTA G E OF MID-PIECE ABNORMALITIES IN BOVINE S E MEN FOLLOWIN G NATURAL CASES OF BOVINE EPHEMERAL FEVER
Animal N umber
0-10
10- 20
days
days
622
15
27
580 618
58 9
*
30-40
days
70 -80
27
days ,
gO-1 00
100-120
days
days
16
10
9
33 8
* Epithelial cells were the only cells present in -
50 - 60 days
Represents no observation.
25 24'5
this sample.
31 20 ' 5
Fig. 3. Abnormal mid-pieces in a typical field in semen from bull 415 following bovine ephemeral fever. This illustrates the high incidence of mid-piece abnormalities encountered (Williams's stain). X 400. Fig. 4. Dissociated mid-piece (Williams's stain). X 1000.
Fig. 6 Fig. 5. Split mid-piece (Williams's stain). X 1000. Fig. 6. Thickened mid-pieces (note: in our Williams's stains, cytoplasmic droplets stain much more lightly). X 1000.
Fig. 7 Fig. 7. "Moth-eaten" mid-pieces (Williams's stain).
X 1500.
542
BRITISH VETERINARY JOURNAL, 13 1, 5
again working with rams, specifically mentioned mid-piece abnormalities which were first detected in the ejaculate at 15 days post-heating and reached a peak at 24 days post-heating. In a study of tick-borne fever in cattle, R etief, Neitz & MacFarlane (1971) found that a high percentage of head abnormalities of sperm could be produced in the absence of a systemic reaction. McKenzie & Phillips (1934) stated that abnormalities involving the midpiece were in evidence in most cases of lowered fertility in the ram. Williams (1950) working with human semen stated that "abnormalities of the mid-piece are frequent in infertile men and may be of considerable clinical significance". There is no lack of published work concerning the apparent relationship between specific mid-piece abnormalities and infertility (Blom, 1950, 1959, 1968; Savage & Isa, 1963; Coubrough & Barker, 1966; Rathore, 1969 and Chenoweth et at., 1970). Rao (197 I) reported that the incidence of mid-piece abnormalities was higher in bulls with poor quality semen than in normal bulls, indicating that such defects are in some way associated with pathological changes in the testis. The persistence of high incidences of abnormal mid-pieces in the semen of these bulls following a relatively mild and transient illness is perhaps surprising. Watson (1964) found that changes in the morphology of spermatozoa of rams following pyrexia caused by tick-borne fever persisted for 4 months after th e infection. If the average time lapse between division of the A-type spermatogonium and the appearance of mature spermatozoa in the ejaculate of the bull is accepted as about 60 days (Kofoed-Johnson, 1960; Orgebin-Christ, 1962), then the continued appearance of morphologically abnormal sperm in excess of 60 days after the illness, as described here, must suggest damage to the spermatogenic process. At this stage it is not completely clear whether these changes are a result of virus growth in the testis or merely the pyrexia which is usually present. It is hoped that further work will clarify this point. ACKN O WLEDGEMENTS
The authors gratefully acknowledge the helpful advice received from Dr P. B. Spradbrow and Mr M. G. Osborne of the University of Queensland, and we \vish to thank Professor J. Francis for access to bulls and facilities. This work ".-as supported by the Australian Meat Research Committee. REFERENCES
BLOM, E. A. (1950). J . Fert. Steril. I, 233. BLOM, E. A. (1959). Nature, Lond. 183, 1280. BLOM, E. A. (1968). Nord. Vet. Med. 20, 279. BURGESS, G. W. (1971) . Vet. Bull., WlJlbridge, 41, 887. Burgess, G. W. (1974). Aust. J. expo BioI. m£d. Sci. 52, 851. CAMERON, R. A. (1970) . M.V.Sc. Thesis, University of Queensland. CHENOWETH, P. j. & BURGESS, G. W. (1972). Aust. vet. J. 48, 37. CHENOWETH, P. j., PASCOE, R. R. R., McDOUGALL, HEATHER & MCCOSKER, P. j. (1970).
Br. vet. J. 126, 476.
COUBROUGH, R. I. & BARKER, C. A. V . (1964). Proc. Vth Int. Gongr. Anim. Reprod. 5, 219. DOHERTY, R. L., STANDFAST, H. A. & CLARK, I. (1969)' Aust. J. Sci. 31, 365.
ABNORMALITIES IN BOVINE SEMEN
543
GUNN, R. M. C., SANDERSR. N. & GRANGER, W. (1942). Bull. Coun. sci. indo Res., Melb. No. 148. HANCOCK, j. L. (1951). Nature, Lond. 16,323. KOFOED-JOHNSON, H. H. (1960). Nature, Lond. 185,49. LAGERLOF, N. (1934). Acta. path. microbiol. scand., Supp!. 19, I. MACLEOD, j. (1964). J. Fert. Steril. I, 199. McKENZIE, F. F. & BERLINER, V. (1937). Mo. agric. expo Sta. Res. Bull. 265, I. McKENZIE, F. F. & PHILLIPS, R. W. (1934). J. Am. vet. med. Ass. 84, 189. ORGEBIN-CHRIST, M. C. (1962). Ann. Bioi. Animale Biochem. Biophys. 2, 51. RAO, A. R. (1971). Ph.D. Thesis, Royal Veterinary College, Stockholm. RATHORE, K. A. (1969). Br. vet. J. 125,534. RETIEF, G. P., NEITZ, W. O. & McFARLANE, I. S. (1971). Jl. S. 4fr. vet. med. Ass. 42,321. SAVAGE, A. & ISA, j. M. (1963). Cornell Vet. 53, 338. SINGLETON, E. F. (1966). M.V.Sc. Thesis, University of Queensland. ST GEORGE, T. D., STANDFAST, H. A., ARMSTRONG, j. M., CHRISTIE, D. G., IRVING, M. R., KNOTT, S. G. & RIDEOUT, B. L. (1973). Aust. vet. J. 49, 441. WATSON, W. A. (1964). Vet. Rec. 76, 1131. WILLIAMS, W. W. (1950). J. Fert. Steril. I, 199. (Acceptedfor publication 15 October 1974) ADloDlalies des corps intermediaires au niveau du sperme bovin a la suite de cas experimentaux et naturels de fievre bovine ep heDlere (Burgess et Chenoweth) ResuDle. On mit en evidence une etude concernant les dfets de la fievre bovine ephemere sur la qualite du sperme de tau reaux, en utilisant deux cas experimentaux et quatre cas naturels de la maladie. Une augmentation importante, dramatique du pourcentage de corps intermediaires anormaux fut notee. On enregistra les niveaux des pics 35 hours approximativement apres Ie pic thermique. Au bout de 155 jours, Ie sperme d'un cas experimental etait redevenu normal et I'on nota des tendances similaires avec Ie sperme recueilli chez les autres taureaux. Une diminution de la motilite associee a une augmentation des corps intermediaires anormaux survenaient aussi. Cette decouverte confirme les rapports efi'ectues dans les champs decrivant une dimunition de la fertilite des taureaux consecutive a la fievre bovine ephemere. line fut pas possible de montrer si ces modifications sont Ie resultat de la croissance virale dans les testicules, de la pyrexie qui survient habituellement. AnoDlalien des Mittelstiicks von SperDlatozoen von Rindern nach experiDlenteller oder natiirlicher Erkrankung nUt ephemoralem Fieber (Burgess und Chenoweth) ZusaDlmenfassung. Die Wirkung von ephemoralem Fieber auf die Qualitat des Semen bei Rindern wurde untersucht bei zwei experimentell infizierten Tieren und bei vier, bei denen es sich urn eine nattirliche Infektion handelte. Man sah eine bestandige dramatische Zunahme des Prozentsatzes abnormaler Spermatozoen. Diese erreichte ihren Hohepunkt etwa 35 Tage nach der hochsten Temperatur. Nach 155 Tagen war der Semen in einem experimentellen Fall wieder normal und der von den anderen Stieren gewonnene Semen verhielt sich ahnlich. Eine Herabsetzung der Motilitat trat ebenfalls auf, wenn die Mittelstticke der Spermatozoen Anomalien zeigten. Dies bestatigt die Beobachtung, dass die Fertilitat von Stieren nach ephemeral em Fieber vermindert ist. Es liess sich nicht zeigen, ob diese Veranderungen das Resultat von Viren in den Testes oder von der gewohnlich auftretenden hohen Temperatur sind. Anomalias de la pieza Dledia en el semen bovino tras casos experiDlentales y naturales de fiebre efemera bovina (Burgess y Chenoweth) ResUDlen. Se llevo a cabo un estudio de los efectos de la fiebre efemera bovina sobre la calidad del semen de los toros, empleandose dos casos experimentales y cuatro naturales de
544
BRITISH VETERINARY JOURNAL,
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la enfermedad. Se observ6 un aumento consistente y dramatico del porcentaje de piezas medias anormales. Se registraron niveles cumbre aproximadamente 35 dias despues de que la fiebre alcanzara su cumbre. Despues de 155 dias, el semen de uno de los casos experimentales se habia normalizado y tendencias similares se observaron en el semen obtenido de los otros toros. Tambien se dio una disminuci6n en la motiIidad que estuvo asociada con el aumento de piezas medias anormales. Este hallazgo apoya los informes de campo de que ocurre una disminucion en la fertilidad de los toros despues de la fiebre efemera bovina. No rue posible demostrar si estos cambios son resllitado del crecimiento viral en los testiculos 0 de la pirexia que normalmente tiene lugar.
MID-PIECE ABNORMALITIES IN BOVINE SEMEN FOLLOWING EXPERIMENTAL AND NATURAL CASES OF BOVINE EPHEMERAL FEVER By
G. W.
BURGESS· AND
P.
J.
CHENOWETHt
Veterinary School, University of Queensland, St Lucia, Brisbane 4067 SUMMARY
A study of the effects of bovine ephemeral fever on the semen quality of bulls was carried out using two experimental and four natural cases of the disease. A consistent, dramatic rise in the percentage of abnormal mid-pieces was seen. Peak levels were recorded at approximately 35 days after the peak fever. After 155 days, the semen of one experimental case had returned to normal and similar trends were seen with the semen collected from the other bulls. A decrease in motility associated with the increase in abnormal mid-pieces also occurred. This finding supports field reports of a decrease in fertility of bulls following bovine ephemeral fever. It was not possible to show whether these changes are the result of viral growth in the testis or the pyrexia which usually occurs. INTRODUCTION
Bovine ephemeral fever is a viral disease of cattle, usually observed clinically as an acute febrile reaction which may be accompanied by lameness or paresis. The disease occurs mainly in Mrica, Asia and Australia (Burgess, 197 I). In Australia, there have been four epizootics (1936-37, 1955-56, 1967-68 and 1970-7 I) as well as numerous isolated outbreaks. The disease is now probably endemic in some northern areas of Australia (St George et al., 1973). The economic importance may not be readily apparent. Many cases are never seen by the owners and those which are seen are often accepted and soon forgotten. Losses have been reported to be due to a reduction in milk production, disruption of markets, reduction in condition of prime animals, individual deaths and reduced performance as a result of permanent lameness. A preliminary report of this study has been made (Chenoweth & Burgess, 1972). During the 1970-71 epizootic it was suggested that some bulls suffered a reduction in reproductive performance following this disease. In order to establish a better understanding of this situation, two experimental cases were observed in detail and semen samples collected from four natural cases were also studied. • Present address : Department of Veterinary Pathology and Public Health, Massey University, Palmerston North, New Zealand. t Present address: 52 Green Street, North Mackay, Queensland, Australia, 4740.
ABNORMALITIES IN BOVINE SEMEN
537
MATERIALS AND METHODS
Experimental animals Two bulls approximately 18 months old were used. One (No. 415) was a Friesian/Hereford weighing 209 kg, and the other (No. 418) was a Guernsey weighing 289 kg. Natural cases During the 1970-7 I epizootic several Droughtmaster bulls on the University Farm at Moggill showed clinical signs of Bovine Ephemeral Fever (BEF). Semen samples were subsequently collected from four of these animals. Virus inoculum The two experimental bulls were inoculated intravenously with 20 ml of infective blood which represented the third bovine passage of virus from the 1967-68 epizootic (Doherty, Standfast & Clark, 1969). This virus was generously supplied at the second passage level by Mr I. Clark, Animal Research Institute, Yeerongpilly. Semen collection technique Semen was collected by electro-ejaculation using a Watson Transtimulator Mk. I I, which was under manual control. Techniques for confirmation oj BEF infection Observations of clinical signs were made twice daily during the period of clinical reaction. Daily serum and whole blood samples were collected from the two experimental animals. Whole blood samples in ACD were used for virus isolation while EDT A samples were used for haematological studies. Serum samples were tested for neutralizing antibodies using infant mice, tube cultures of Vero (African green monkey kidney cell line) cells and the microtitre serum neutralization test (Burgess, 1974). Whole blood samples for virus isolation and acute and convalescent serum samples were collected from the four natural cases. Frequency of collection Three collections were made from each of the two experimental animals within eight days prior to inoculation. Six collections were made within 30 days post-pyrexia. Five samples were collected in the next 33-day period and a further four collections were made in the period 63-155 days post-pyrexia. Fewer collections were made from the natural cases. Collections from bull No. 622 were made at 5, 12, 19, 50 and 80 days post-pyrexia, and from bull No. 618 at 10, 17,52, 78, 95 and 115 days. A single collection was made from bull No. 580 at 34 days post-pyrexia. Bull No. 589 did not completely recover from the infection and had a hindlimb ataxia which prevented him from successfully mounting to serve. Two collections were made from this animal at 49 and 76 days post-pyrexia.
53 8
BRITISH VETERINARY JOURNAL,
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Semen examination procedure Field examination. Immediately after collection, volume, colour and density were recorded. Mass activity was scored (0 to + + + +) and the percentage of actively motile spermatozoa was estimated. The percentage of sperm alive was estimated by using nigrosin-eosin stain, read within 24 h (Hancock, 1951). Laboratory examination. Head and mid-piece morphology of the spermatozoa was studied using Williams's stained preparations (Lagerlof, 1934) and wet preparations in buffered formol saline were examined under phase contrast microscopy after the method of Hancock (Chenoweth et al., 1970) for tail morphology, loose heads and cytoplastic droplets. Two hundred sperm were counted in each instance. The following classifications were employed, using Williams's stain: for sperm heads; normal, narrow at base (pyriform), large, pear-shaped, small, round and broad. For mid-pieces; thick, knobbed, filiform, "moth-eaten", corkscrew, dissociated, split, defective, broken, abaxial and mid-piece coiled around the head (Fig. 1). Using wet preparations; loose heads, proximal droplets, single bent tails, double bent tails and coiled tails.
RESULTS
There was a sharp rise in rectal temperature which coincided with a neutrophilia with a left shift occurring on days five and eight post inoculation for animals 415 and 418 (the experimental animals) respectively. A rise in serum antibody titres occurred shortly after the clinical signs were seen. Virus was isolated from blood samples collected on the day of peak fever. Virus was also isolated from a blood sample taken from animal No. 580. A rise in serum neutralizing antibody levels was demonstrated for all four Droughtmasters which had clinical signs of the disease.
Semen changes The most consistent and dramatic change was a rise in the percentage of abnormal mid-pieces as shown in Fig. 2. This reached a peak at approximately 35 days after the peak temperature rise. A further small rise was noted approximately 60 days post-pyrexia. Mid-piece abnormalities of most types were encountered at all tests conducted during the post inoculation period (Figs 4-7). However, during the period prior to the sharp rise at 27 days post-pyrexia, the main abnormalities encountered were in the "offshoot defect", "dissociated" and "filiform" categories. From the period of dramatic rise at 27 days postpyrexia, the "kinked mid-piece" abnormality assumed major importance. The pre-inoculation levels of mid-piece abnormalities were consistently low and the highest percentage recorded at this time was 2 per cent, whereas the peak values exceeded 70 per cent. There appeared to be a corresponding drop in motility. The number of animals involved in the experiment was such that a statistical confirmation of this observation was not possible. Changes in other parameters were not consistent and were not considered to be related to the
ABNORMALITIES IN BOVINE SEMEN
539
CLASSIFICATION OF MIDDLE-PIECE MORPHOLOGY OF BULL SPERM --
I
IlQrmal
,I t, r thick
knob
I t
filiform
'motheaten '
abaxial
! , t I J
dissociated kink / corkscrew
double
split
defective attachment
o
.1
coiled around . head Fig. I . Classification of mid-piece morphology of bull spermatozoa. The illustrations are a diagramatic representation of the mid-piece abnormalities observed using Williams's stained preparations. offshoot defecf
broken
infection. The percentage of abnormal mid-pieces in semen samples collected from the natural cases tends to confirm these observations and the results are shown in Table I. The percentage of mid-piece abnormalities d eclined in both experimental cases after 80 days. By 155 days the level for No. 418 had reached the pre-inoculation figure . Number 415 had dropped to 17 per cent by 88 days. However, an increase then occurred and by 152 days a level of 45 per cent had been reached. The significance of this rise is not clear. DISCUSSION
The most important of the semen results was considered to be the dramatic rise in mid-piece abnormalities which occurred in both experimental bulls and in the naturally infected bulls. There has been little work done on the effects
BRITISH VET ERI NARY JOURNAL, 13 1, 5
540
~': ~\'-'---- \ . ~
/''>
50
c:!
'- .
\ f~·,t;/-- -. -~<· --
"
' __
~ 2S
.
\/ "
-.......
"
.
- .
•
~~ ' :~O <;\----·' ·'/:~-~·:=:~· ~-~·--- ---·-------'~------' .~ - --_ --~ .'
~
\ _.
"/
- '
• _ ___ _
2
. ___
20
4050
8 0 70
90
•
100
110
TIME POST l'Y'EXIA {rlA.ys 1
;.;f'
150
Fig. 2. Semen changes in two animals following experimental bovine ephemeral fever. The values at d ay 0 represent the m ean of three results of samples collected within 8 days prior to inoculation.
of specific pathogenic agents on spermatogenesis. Macleod (1964) found that abnormal spermatozoa increase significantly in the semen of men who had suffered viral infections. Previous workers have shown that illness associated with pyrexia can lead to increased numbers of abnormal spermatozoa (Gunn, Sanders & Granger, 1942; Macleod, 1964; Watson, 1964; Singleton, 1966; Rathore, 1969; Cameron, 1970). This increase can be detected as early as the fourth day following the pyrexia and is reflected at this stage in increased numbers of loose sperm heads and abnormal sperm tails (McKenzie & Berliner, 1937; Gunn et al., 1942). Cameron (1970) working with rams, found that nuclear changes in sperm first became apparent at 17- 21 days post-pyrexia and that there was a second rise in abnormalities between 30 days and 56 days post-pyrexia. Rathore (1969), TABLE I P E RCENTA G E OF MID-PIECE ABNORMALITIES IN BOVINE S E MEN FOLLOWIN G NATURAL CASES OF BOVINE EPHEMERAL FEVER
Animal N umber
0-10
10- 20
days
days
622
15
27
580 618
58 9
*
30-40
days
70 -80
27
days ,
gO-1 00
100-120
days
days
16
10
9
33 8
* Epithelial cells were the only cells present in -
50 - 60 days
Represents no observation.
25 24'5
this sample.
31 20 ' 5
Fig. 3. Abnormal mid-pieces in a typical field in semen from bull 415 following bovine ephemeral fever. This illustrates the high incidence of mid-piece abnormalities encountered (Williams's stain). X 400. Fig. 4. Dissociated mid-piece (Williams's stain). X 1000.
Fig. 6 Fig. 5. Split mid-piece (Williams's stain). X 1000. Fig. 6. Thickened mid-pieces (note: in our Williams's stains, cytoplasmic droplets stain much more lightly). X 1000.
Fig. 7 Fig. 7. "Moth-eaten" mid-pieces (Williams's stain).
X 1500.
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again working with rams, specifically mentioned mid-piece abnormalities which were first detected in the ejaculate at 15 days post-heating and reached a peak at 24 days post-heating. In a study of tick-borne fever in cattle, R etief, Neitz & MacFarlane (1971) found that a high percentage of head abnormalities of sperm could be produced in the absence of a systemic reaction. McKenzie & Phillips (1934) stated that abnormalities involving the midpiece were in evidence in most cases of lowered fertility in the ram. Williams (1950) working with human semen stated that "abnormalities of the mid-piece are frequent in infertile men and may be of considerable clinical significance". There is no lack of published work concerning the apparent relationship between specific mid-piece abnormalities and infertility (Blom, 1950, 1959, 1968; Savage & Isa, 1963; Coubrough & Barker, 1966; Rathore, 1969 and Chenoweth et at., 1970). Rao (197 I) reported that the incidence of mid-piece abnormalities was higher in bulls with poor quality semen than in normal bulls, indicating that such defects are in some way associated with pathological changes in the testis. The persistence of high incidences of abnormal mid-pieces in the semen of these bulls following a relatively mild and transient illness is perhaps surprising. Watson (1964) found that changes in the morphology of spermatozoa of rams following pyrexia caused by tick-borne fever persisted for 4 months after th e infection. If the average time lapse between division of the A-type spermatogonium and the appearance of mature spermatozoa in the ejaculate of the bull is accepted as about 60 days (Kofoed-Johnson, 1960; Orgebin-Christ, 1962), then the continued appearance of morphologically abnormal sperm in excess of 60 days after the illness, as described here, must suggest damage to the spermatogenic process. At this stage it is not completely clear whether these changes are a result of virus growth in the testis or merely the pyrexia which is usually present. It is hoped that further work will clarify this point. ACKN O WLEDGEMENTS
The authors gratefully acknowledge the helpful advice received from Dr P. B. Spradbrow and Mr M. G. Osborne of the University of Queensland, and we \vish to thank Professor J. Francis for access to bulls and facilities. This work ".-as supported by the Australian Meat Research Committee. REFERENCES
BLOM, E. A. (1950). J . Fert. Steril. I, 233. BLOM, E. A. (1959). Nature, Lond. 183, 1280. BLOM, E. A. (1968). Nord. Vet. Med. 20, 279. BURGESS, G. W. (1971) . Vet. Bull., WlJlbridge, 41, 887. Burgess, G. W. (1974). Aust. J. expo BioI. m£d. Sci. 52, 851. CAMERON, R. A. (1970) . M.V.Sc. Thesis, University of Queensland. CHENOWETH, P. j. & BURGESS, G. W. (1972). Aust. vet. J. 48, 37. CHENOWETH, P. j., PASCOE, R. R. R., McDOUGALL, HEATHER & MCCOSKER, P. j. (1970).
Br. vet. J. 126, 476.
COUBROUGH, R. I. & BARKER, C. A. V . (1964). Proc. Vth Int. Gongr. Anim. Reprod. 5, 219. DOHERTY, R. L., STANDFAST, H. A. & CLARK, I. (1969)' Aust. J. Sci. 31, 365.
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GUNN, R. M. C., SANDERSR. N. & GRANGER, W. (1942). Bull. Coun. sci. indo Res., Melb. No. 148. HANCOCK, j. L. (1951). Nature, Lond. 16,323. KOFOED-JOHNSON, H. H. (1960). Nature, Lond. 185,49. LAGERLOF, N. (1934). Acta. path. microbiol. scand., Supp!. 19, I. MACLEOD, j. (1964). J. Fert. Steril. I, 199. McKENZIE, F. F. & BERLINER, V. (1937). Mo. agric. expo Sta. Res. Bull. 265, I. McKENZIE, F. F. & PHILLIPS, R. W. (1934). J. Am. vet. med. Ass. 84, 189. ORGEBIN-CHRIST, M. C. (1962). Ann. Bioi. Animale Biochem. Biophys. 2, 51. RAO, A. R. (1971). Ph.D. Thesis, Royal Veterinary College, Stockholm. RATHORE, K. A. (1969). Br. vet. J. 125,534. RETIEF, G. P., NEITZ, W. O. & McFARLANE, I. S. (1971). Jl. S. 4fr. vet. med. Ass. 42,321. SAVAGE, A. & ISA, j. M. (1963). Cornell Vet. 53, 338. SINGLETON, E. F. (1966). M.V.Sc. Thesis, University of Queensland. ST GEORGE, T. D., STANDFAST, H. A., ARMSTRONG, j. M., CHRISTIE, D. G., IRVING, M. R., KNOTT, S. G. & RIDEOUT, B. L. (1973). Aust. vet. J. 49, 441. WATSON, W. A. (1964). Vet. Rec. 76, 1131. WILLIAMS, W. W. (1950). J. Fert. Steril. I, 199. (Acceptedfor publication 15 October 1974) ADloDlalies des corps intermediaires au niveau du sperme bovin a la suite de cas experimentaux et naturels de fievre bovine ep heDlere (Burgess et Chenoweth) ResuDle. On mit en evidence une etude concernant les dfets de la fievre bovine ephemere sur la qualite du sperme de tau reaux, en utilisant deux cas experimentaux et quatre cas naturels de la maladie. Une augmentation importante, dramatique du pourcentage de corps intermediaires anormaux fut notee. On enregistra les niveaux des pics 35 hours approximativement apres Ie pic thermique. Au bout de 155 jours, Ie sperme d'un cas experimental etait redevenu normal et I'on nota des tendances similaires avec Ie sperme recueilli chez les autres taureaux. Une diminution de la motilite associee a une augmentation des corps intermediaires anormaux survenaient aussi. Cette decouverte confirme les rapports efi'ectues dans les champs decrivant une dimunition de la fertilite des taureaux consecutive a la fievre bovine ephemere. line fut pas possible de montrer si ces modifications sont Ie resultat de la croissance virale dans les testicules, de la pyrexie qui survient habituellement. AnoDlalien des Mittelstiicks von SperDlatozoen von Rindern nach experiDlenteller oder natiirlicher Erkrankung nUt ephemoralem Fieber (Burgess und Chenoweth) ZusaDlmenfassung. Die Wirkung von ephemoralem Fieber auf die Qualitat des Semen bei Rindern wurde untersucht bei zwei experimentell infizierten Tieren und bei vier, bei denen es sich urn eine nattirliche Infektion handelte. Man sah eine bestandige dramatische Zunahme des Prozentsatzes abnormaler Spermatozoen. Diese erreichte ihren Hohepunkt etwa 35 Tage nach der hochsten Temperatur. Nach 155 Tagen war der Semen in einem experimentellen Fall wieder normal und der von den anderen Stieren gewonnene Semen verhielt sich ahnlich. Eine Herabsetzung der Motilitat trat ebenfalls auf, wenn die Mittelstticke der Spermatozoen Anomalien zeigten. Dies bestatigt die Beobachtung, dass die Fertilitat von Stieren nach ephemeral em Fieber vermindert ist. Es liess sich nicht zeigen, ob diese Veranderungen das Resultat von Viren in den Testes oder von der gewohnlich auftretenden hohen Temperatur sind. Anomalias de la pieza Dledia en el semen bovino tras casos experiDlentales y naturales de fiebre efemera bovina (Burgess y Chenoweth) ResUDlen. Se llevo a cabo un estudio de los efectos de la fiebre efemera bovina sobre la calidad del semen de los toros, empleandose dos casos experimentales y cuatro naturales de
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la enfermedad. Se observ6 un aumento consistente y dramatico del porcentaje de piezas medias anormales. Se registraron niveles cumbre aproximadamente 35 dias despues de que la fiebre alcanzara su cumbre. Despues de 155 dias, el semen de uno de los casos experimentales se habia normalizado y tendencias similares se observaron en el semen obtenido de los otros toros. Tambien se dio una disminuci6n en la motiIidad que estuvo asociada con el aumento de piezas medias anormales. Este hallazgo apoya los informes de campo de que ocurre una disminucion en la fertilidad de los toros despues de la fiebre efemera bovina. No rue posible demostrar si estos cambios son resllitado del crecimiento viral en los testiculos 0 de la pirexia que normalmente tiene lugar.