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INSTITUTE OF INSPECTORS OF STOCK OF N.S.W. YEAR BOOK.
INFERTILITY NOTES
H. E. R. BEATTIE, B.V.Sc., H.D.A., Special Veterinary Research Officer, Veterinary Research Station, Glenfield, N.S.W.
OVINE BRUCELLOSIS:
The presence of this disease in New South Wales has been confirmed, but the proportion of epididymitis cases with which it is associated, remains a matter for conjecture. Possibly the infection is fairly widespread, though less likely to make its presence felt where 2 to 3 per cent, rams are used in mass mating than under stud conditions.
The rams we are working on at present came from two connected properties on which infertility was causing concern. Of 12 Merinos and 5 Crossbreds showing marked epididymitis lesions, positive complement-fixation tests were obtained from five and a suspicious reaction from one. One of the positive serum samples was from a Crossbred ram; the remaining reactors being Merinos.
It would be difficult to identify the reactors in this group on palpation alone. The ovine brucellosis lesion is essentially a spermatocoele, commonly causing a firm enlargement of the tail of the epididymis. In most of the serum-negative rams slaughtered here to date old abscesses have been found but the scrotal contents in some of these cases had felt very similar to those of some of the positive rams.
Some abscess lesions lay between the epididymis and the tunic, making the former seem larger and harder than it was. Marked thickening of the tunic and extensive adhesions in some instances had made the various lines of demarcation difficult to feel.
Organisms resembling Brucella ovis have been recovered on cultural examination of semen samples obtained by electro-ejaculation, but sub-cultures were unsatisfactory. Further sampling will be undertaken in the near future. It is hoped also to mate each ram with experimental ewes.
Sometimes the organism can be demonstrated in direct smears of semen. It is a small, Gram-negative cocco-bacillus, and apparently is weakly acidfast. Cultural examination is preferable. Contamination is hard to avoid however, and may cause difficulties.
BOVINE TRICHOMONIASIS:
The story of the so-called "trichomonad cyst" presented for consideration this year has been submitted for publication in The Australian Veterinary Journal. The presence of a Gartnerian cyst had been noted shortly after this Cow was served by a trichomoniasis-positive bull. On being opened nearly 5 months later the cyst was found to contain numerous viable T. foetus organisms. No protozoa had been recovered from the vagina during the preceding 11 weeks, and a mucus agglutination test (carried out in Melbourne by Dr. Murnane) had proved negative.
Retention cysts of Gartner's ducts are not rare, but usually they contain only clear fluid. No previous report of the recovery of T. foetus from such a site has been found. Possibly such an occurrence could cause a "mysterious" break-down in a careful control programme.
Previously this cow had conceived to third service by another infected bull, calving normally at full term. She was then rested for 9 months, served by a non-tricho, bull, calved at full term, and rested for 14 months before being re-infected as above. Theoretically it seems she should have been quite susceptible again by then; and trichomonads were recovered from her vagina continuously for 7 weeks, from the 10th day post-coitus. A right-horn pregnancy was diagnosed early, but there was some doubt as to the contents of the uterus at the time of the incision of the cyst. At the time of writing, however-approximately 7 months after service-the pregnancy appears to be proceeding normally. Two further samples have been secured carefully from the cyst, which had reappeared within a few days. These and all additional vaginal "exudate" specimens have proved negative for T. foetus.
It would be interesting to know how long the organisms would have persisted had the cyst not been drained (after irrigating the vagina with dettol). The latest stage of pregnancy at which trichomonads apparently have been present, in our experience, is "between 7 and 8 months". Dr. Seddon submitted a specimen from a cow which had aborted approximately 3 weeks previously at that stage (in the owner's words). T. foetus organisms we detected in direct smears and grew satisfactorily in culture media. A blood serum sample secured at the same time returned a strongly positive reaction to the agglutination test for brucellosis. One wonders whether the trichomonads might have been carried through to a full-term parturition in this case but for the latter infection. (Some workers are not satisfied that any instance of T. foetus remaining throughout normal pregnancy has ever been proven).
EFFECTS OF "DEEP-FREEZING"—AND GLYCEROL:
Sub-cultures from both the above cases were among the sources of T. foetus used in a special series of experiments carried out by Mr. A. W. Blackshaw and the writer. This work indicated that T. foetus is more sensitive to glycerol than are bovine spermatozoa. Glycerol in concentrations of approximately 10 per cent, is capable both of protecting the trichomonads against the otherwise fatal effects of freezing to minus 79 deg, C., and of itself destroying them in the unfrozen state.
It was found possible to store T. foetus successfully at the temperature mentioned, with the aid of glycerol concentrations ranging from 7½ to 15 per cent., when freezing was commenced very soon after mixing and subculturing was carried out immediately after rapid thawing.
From 4 per cent. to about 8 per cent, glycerol has failed to prevent growth in some ordinary T. foetus cultures, but 10 per cent, usually does so. (In one trial, conducted here by student David Jones under supervision, two series of sub-cultures in 3 different media were set up and examined after 16 hours incubation at 37 deg. C. Glycerol to 10 per cent. had been added to one tube of each pair. These were all negative at 16 hours, while the untreated controls were positive). In the early semen-freezing work in England, 15 per cent. glycerol was used, while Professor Emmens and Mr. Blackshaw of Sydney University whom we first started supplying with bovine semen for this purpose in 1949-reported best results with 74 per cent. glycerol (plus a pentose sugar). Concentrations of the latter order are now widely used. There is a tendency also to reduce, or even omit, the 16 hours "equilibration" period at 5 deg. C. prior to freezing.
Where there is any question of suspect or known-positive bulls being used for artificial insemination it would be wise to use not less than 10 per cent. glycerol in the case of frozen semen, and to allow a reasonable equilibration period so that any trichomonads present could be acted upon prior to freezing. Slow thawing, now favoured by some workers in place of rapid, would decrease the chances of survival of such organisms, as would any delay between thawing and insemination-accepted in the past as being contra-indicated.
Higher concentrations of glycerol have been suggested in the case of suspect semen stored at 5 deg. C. Too much, however, might "protect the Cows as effectively against conception as against infection. Following th A.V.A. Conference discussion, Mr. Hewetson carried out some trials with semen from non-infected bulls at the Artificial Stock-breeding Station at Berry. Observing no deleterious effect on chilled samples to which glycerol had been added to a final concentration of 10 per cent. he inseminated about 20 cows with similarly treated semen. The conception rate proved better than average, especially in the case of samples used on the 3rd day after collection. In a more extensive subsequent trial, however, the over-all conception rate was slightly depressed, though the figures varied with different bulls.
The possible value of glycerol preparations in treatment and prophylaxis is among matters discussed in a letter published in the March, 1955, issue of the Australian Veterinary Journal
(Before leaving the subject of frozen semen it might be mentioned that this development, through increasing the facility with which samples can be transported over long distances and reducing wastage in storage, could lead to an undesirable increase in the amount of newly-imported "blood" herds. The attention of interested breeders might be drawn to a joint statement by Professor McClymont and Dr. Morley, which appeared in the "Sydney Morning Herald" on 22/4/55).
VIBRIOSIS,etc.:
This disease might prove to be at least as widespread as trichomoniasis in N.S.W. cattle. Diagnosis is the difficulty. Some people regard a good response to the intra-uterine administration of streptomycin-penicillin preparations shortly after or within a few days before service or artificial insemination, where there has been marked infertility of uncertain aetiology, as circumstantial evidence of the presence of vibriosis.
A freshly aborted foetus, or a pipette of its stomach contents, is the best specimen to submit; but abortions may be few-and the chances of securing suitable specimens fewer.
Little significance can be attached to the finding of a few small, curved, Gram-negative rods in smears of, say, vaginal exudate. These are not necessarily vibrios. Further, there may be vibrios other than V. fetus. Again, it is not yet definitely established that the latter alone will cause trouble always. Another question not yet answered satisfactorily is the period for which "recovered" cows may remain carriers. Four months sexual rest is recommended, but further knowledge is needed.
There is a general similarity between this disease and trichomoniasis,e.g. venereal transmission, tendency to spontaneous recovery in females and development of temporary immunity. In both instances trouble occurs mostly in heifers and introduced cows after a time; infected bulls serving as reservoirs of infection
Organisms occasionally associated with sporadic abortion or dead calves include Listeria monocytogenes and Leptospira pomona. Either might occur in the vibriosis-positive herd. The former is stated to affect heifers more commonly than cows. In one instance a foot infection was suspected to have been the source. In the latter (Leptospirosis) the cow's urine may be dark just before abortion occurs, and she may show a thickened, corn-cob-like vaginal mucosa due to the development of longitudinal and transverse (circular) striations. The maternal vagina and the foetal skin and cotyledons may be leathery, but the membranes are said not to become opaque and leathery as they often do in brucellosis.
A condition which may or may not prove to be associated with vibriosis, and which is being reported from many districts now, is that in which a purulent vaginal discharge appears within a few days after service. Two suspected bulls have been investigated at Glenfield without any specific organism being incriminated. Heifers put to each conceived to first service, and the pregnancies proceeded normally, though slight abnormal discharges did occur for a time. It is said that when first service fails trouble follows.
A bacteria-free filtrate of semen from one of the bulls was placed in the vagina of a sexually immature heifer, and the unfiltered portion into that of her twin. The latter developed a transient vaginitis with shallow erosions and a slight, whitish discharge, while no effect was observed in the former.
TREATMENT :
Some authorities, including Lindley (J.A.V.M.A., March, 54), consider non-specific infections to be a major cause of infertility in dairy cattle. The mucus may be abundant and thin, showing perhaps clouding and flakes of pus, and there may be enlargement of the cervix, endometritis,etc. Lindley states that a cow becomes a candidate for intra-uterine therapy if bred unsuccessfully 2 or 3 times, and if examination of the genital tract indicates that an infection may be responsible.
His recommendations include infusion on first or second day post-service with 1 gramme of streptomycin sulphate and 200,000 to 500,000 units of penicillin (Na or K) in sterile saline, distilled water, or 12 per cent. sulphapyridine solution. Preferably the complete solution is placed in both uterine horns, which are then massaged. Suitable proprietary mixtures are now available.
Following treatment last year the conception rate in one herd in the Berry area increased from 40 per cent, to about 80 per cent. Mr. Hewetson speaks of good results from such treatment up to several days before service (or A.I.), as an alternative to a few hours or two days after.
Another preparation arousing interest at present is marketed in various forms, including pessaries, as "Hibitane". The makers claim high activity against both Gram-negative and Gram-positive organisms and Trichomonas foetus, low toxicity, excellent dispersion in the uterus and a good cleansing effect, among other features. Double the recommended dose has been suggested in the case of septic metritis.
Many will be familiar with "Penotrane", claimed to have "potent bacteriacidal and fungicidal properties which are not impaired by the presence of pus". We have found it useful,e.g., in malodorous pyometritis associated with Corynebacterium pyogenes; but we cannot agree that it is always non-irritant.
Various other preparations merit consideration also; and of course there are factors other than infection to be borne in mind.
Stilboestrol is used for various purposes-perhaps rather too freely. Some veterinarians use surprisingly large doses in septic metritis,etc. So far the writer prefers not to exceed 40 mgm. at one time. Incidentally he has administered such amounts experimentally to pregnant cows (2½ to 4m.) without inducing abortion. Some authorities contend that the use of stilboestrol while a sizeable corpus luteum is present may give rise to ovarian cysts.
An Italian veterinarian, by the way, claims to have cured nymphomania completely in 18 cows out of 25 by means of intramuscular injections of 10,000 I.U. of Gonadotrophin B. As it has been suggested that 43 per cent. of cases are inherited, however (see Durward Olds, J.A.V.M.A., April, '53), treatment may be considered contra-indicated.
EQUIPMENT : As an aid to pregnancy diagnosis during the difficult 4 to 6 months' period, vulsellum forceps may be found useful. The cervix can be pulled back carefully, lifting the uterus sufficiently to facilitate palpation (e.g., of cotyledons) per rectum. This instrument is employed more commonly to assist in passing a uterine catheter, especially where the rectal fixation technique has not been mastered.
A vaginal speculum is desirable for bovine infertility work if only to reduce the human Undulant Fever hazard. There is a good deal to be said for the familiar metal type with two blades of equal length. It permits ready examination of the vaginal wall and sub-urethal diverticulum,etc., as well as the cervix, but requires a separate source of light or a reflecting surface, and it may be too large for heifers. Tubular plastic types with in-built lights give an excellent view of the cervix and are easily introduced without damaging the mucosa. Leads to a separate battery may cause inconvenience, however, while sterilization presents difficulties. One metal cylindrical speculum, obtainable for about £15, has dry cells in the hand-grip and a bulb on the end of a rod within the cylinder. There are interchangeable cylinders of two sizes, for cows and heifers. The outfit is robust, convenient and easily sterilized. Care is called for in passing it through the vestibule, however. Further, it is not the best instrument for viewing the vaginal lining; and where exudation is profuse it may be difficult to see anything.
Perhaps P.P. Boards would be prepared to supply some such equipment in certain instances where it is considered necessary and not otherwise available.