Cattle Practice 5.4

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CATTLE PRACTICE VOL 5 PART 4 A Survey of Lungworm Serology and Vaccination in First and Second Season Grazing Cattle Mawhinney I.C. Intervet UK Ltd, Science Park, Cambridge CB4 4FP. ABSTRACT The survey looked at the serological status of previously unvaccinated yearlings after one season on grass when treated with a long acting avermectin regime and the response to subsequent vaccination before their second season turnout. Exposure to lungworm in the first season was variable even when housed late. One farm had a significant exposure which may have lead to late season clinical disease in unvaccinated animals, and another farm had low titres suggesting only weak exposure and very low immunity prior to second season turnout. Vaccination prior to the second season produced a definite boosting effect. The fall off in titres during a short housing period was noticeable and the effect of treating such animals with a second season worming programme is suggested as being unwise with regard to Dictyocaulus immunity unless booster vaccination is considered. INTRODUCTION Husk incidence has increased in recent years (VIDA statistics) and is now particularly prevalent in adult cattle although there is still a significant proportion of outbreaks in first season grazers. Incidence of outbreaks correlates with periods of warm wet weather. There is circumstantial evidence to suggest that immunity in herds these days is lower than it has been in the past (G David 1996). This is undoubtedly related to a reduced usage of vaccine in recent years ( vaccine usage is only about one third of what it was 10 years ago) and presumably also a reduction in the lungworm challenge on farms which results in less opportunity for immunity to develop, or once developed, to be boosted. Immunity boosting by natural pasture exposure is important each year to maintain immunity to husk since immunity is relatively short-lived. Trials suggest that immunity is significantly reduced after 6 months without exposure, and little other than immune memory probably exists 12 months after a challenge or vaccination (Michel and others 1966, and Taylor and others 1990). The purpose of this survey on 4 farms was to monitor variation in the immunity level, as indicated by serology, at the end of the first grazing season and during housing, and examine the effect of vaccination in pre-grazed yearling animals. Serology is not necessarily indicative of presence or lack of protective immunity but it is the only available indicator of exposure and changes in immune stimulation. MATERIALS AND METHODS 4 Farms were identified in a practice on the Devon Cornwall border where long grazing seasons of warm wet weather are common and where therefore opportunity for exposure to lungworm in late season is probably the highest in the UK. The farms had all given up vaccination some years previously in favour of long-acting avermectin treatments in the first season. They had now become concerned about their herd protection status and were interested to discover their status and the effect of vaccination. Calves were blood sampled at housing at the end of the first year on pasture, and then blood sampled again at vaccination time prior to second season turnout, and at turnout. Serology was carried out by ELISA for L4 and adult antigen by Intervet UK laboratories according to previous experience. The ELISA is scored for each antigen on a scale of 1 to 9 where 1 is negative, and 9 is maximum. Scores of below 3 are possibly not indicative of lungworm specific antibody, hence 2 is a borderline negative. Outbreaks of disease or recent very heavy exposure are usually accompanied by titres of 7 to 9, although strict correlations are not possible and the titre is used mostly as a guide to antigenic exposure and general immune response. Sampling was as shown in table 1 Table 1 Farm year no Bloods sampled age (m) 1st season wormer wks H -V1 Housing V 1 V 2 Pre turnout A 95/6 10 15 Bolus 11 + + + + B 95/6 10 14 Bolus 11 + + + + C 95/6 10 14 Bolus 10 + + + + D1 95/6 10 15 0 & 8wk 9 + + + + D2 94/5 23 18 Bolus 12 - + + + BCVA 1997 323

CATTLE PRACTICE VOL 5 PART 4 Vaccination was planned with doses 4 weeks apart and finished 2 weeks prior to turnout. The V1 blood sample was therefore a prevaccination titre, V2 the titre after 1st vaccine dose and pre-turnout the titre after second vaccination. In most cases housing was in late December or early January and vaccination was carried out only less than 3 months post housing. Farm D has results for 2 consecutive seasons in 2 completely different groups of calves. Statistics were calculated using a t-test. RESULTS The average titres are shown below in figures 1 and 2. The range per farm against L4 is also shown in table 2, since variation existed within most farm groups. Table 2 Range serological status L4. Farm Housing V1 V2 Pre - turnout A 6 – 9 2 - 9 4 - 9 5 - 9 B 4 – 7 2 - 8 5 - 8 6 - 8 C 2 – 7 2 - 5 7 - 9 7 - 8 D1 2 – 9 2 - 8 5 - 9 4 - 9 D2 Ns 1- 6 3 - 9 4 - 9 BCVA 1997 V1 is pre 1st dose of vaccine and is approximately 6 weeks prior to second season turnout. Fig 1 L4 and Adult Titres L4 and Adult Titres All farm averages All farm averages 0 1 2 3 4 5 6 7 8 Housing Pre V1 Pre V2 Turnout L4 Titre Adul Fig2 Average Serological Status Adult 0 1 2 3 4 5 6 7 8 Housing Pre V1 Pre V2 Turnout Time of Blood Test Titre - Adult Farm A Farm B Farm C Farm D1 Farm D2 Adult titres followed L4 but were usually slightly lower than L4 titres. At housing there is a difference in titres between farms indicative of the degree or recentness of field challenge. These farms had 3-4 months on pasture at the end of their first grazing season without anthelmintic cover according to the manufacturers’ claimed durations of efficacy. The level of antibody to adult lungworm was bordering on negative in some cattle on farm C. There is a consistent reduction in titres in the months of housing prior to vaccination on all farms and consequently on Farm C all but 2 had dropped to borderline negative at time of 1st vaccination. In all, one third of the yearlings tested in this survey were borderline negative (titres 1 or 2) 9-12 weeks after housing. On farm D titres at vaccination were high in one year but borderline negative in most animals the previous year, indicating how immune status can vary on the same farm in different years. Titres consistently rise after first vaccination and slightly further after second. The titre changes over the time periods are indicated in figure 3. Fig 3 Titre Changes Averages - Titre Changes Averages - Status L4 Status L4 -2 -1 0 1 2 3 4 5 6 Post V1 Post V2 Time of Blood Test Titre - L4 Farm A Farm B Farm C Farm D1 Farm D2 Housing to VI 324

CATTLE PRACTICE VOL 5 PART 4 DISCUSSION All these farms showed evidence of exposure to lungworm in the first season. Even in this extremely favourable part of the world for lungworm build up on pasture, one of the 4 farms had evidence of low exposure in late season. Antibody clearly and consistently declines in the short period of housing. At time of turnout for the second season at least one farm’s cattle would have been mostly negative. Whilst this does not mean they would be unprotected, any interference with natural boosting early in the second season might allow immunity to wane to a dangerous level. Such a situation would probably be the case if the animals were treated with anthelmintic at the beginning of the second season. In regions with less favourable climates for end of season exposure and earlier and longer housing periods this effect is likely to be more prevalent. The unpredictability of lungworm challenge is illustrated by Farm D. In 2 consecutive years the titre profile of individual animals at 6 weeks prior to second season turnout (PreV1 sample time) is very different, fig 4. Fig 4 0 2 4 6 8 10 Individual yearling titres Titre 1994/ 5 (10 animals) 1995/ 6 (23 animals) Titre profile Farm D 1994 and 95 6 weeks prior to 2nd season turnout The vaccination of pre- exposed cattle is very effective at boosting immunity. Indeed on these farms it appears a single booster dose was all that was needed. The effect of a single dose in cattle with longer housing periods and less end of season exposure, is uncertain. Essentially immunity to lungworm is only maintained by suitable lungworm (on pasture or in vaccine) challenge each year. It is important to ensure an immunity to lungworm exists at the end of the first season and the best way to ensure this is by vaccination prior to first season turnout. Immunity at the end of the first season should then carry over to the beginning of the next season. Where any possible interference to natural exposure at the beginning of the second season is likely to exist and existing immunity compromised, a single booster dose of vaccine about 4 weeks before turnout will restore optimal immune status. If animals have not been vaccinated prior to first season turnout and therefore possibly not developed an immunity at the end of the first season, a normal 2 dose course should be used. Thereafter, grazing seasons are likely to be within the adult dairy herd which is normally expected to provide sufficient natural exposure to maintain immunity each year. This cannot be guaranteed and the possibility of screening a small sample of adult cows to see if boosting is required is worthy of consideration. ACKNOWLEDGEMENTS Special thanks to Tim Bebbington MRCVS at Castle Vet Group, Launceston, for identifying farms for the survey and carrying out all the blood sampling. Thanks to Jayne Bennett and Nicky Reynolds of Intervet UK Ltd for performing ELISA tests and compiling the results respectively. REFERENCES G David Cattle Practice, Oct 1996 CVL VIDA III,1995 J F Michel and A Mackenzie Res vet Sci, 1965, 6, 344. S M Taylor, T R Mallon, W P Green, Vet Rec, 1990, 126,185 BCVA 1997 325

CATTLE PRACTICE VOL 5 PART 4 BCVA 1997 326

CATTLE PRACTICE VOL 5 PART 4 Experiences with Venereal Campylobacter Infection in Suckler Herds Caldow G.L., Scottish Agricultural College Veterinary Services, Greycrook, St Boswells, TD6 0EU Taylor D.W. , Merlin Veterinary Group, 120 Gala Park, Galashiels, TD1 1EZ ABSTRACT Infertility associated with venereal campylobacter infection has been recognised with increasing frequency in the suckler herds of South East Scotland and North East England. The limitations of the available diagnostic procedures are discussed in relation to the problem of assessing suckler herd infertility. Recent field experience is outlined and two herd investigations are described in detail. It is concluded that many factors act to ensure the clinical manifestation of the disease is so variable that venereal campylobacteriosis should be considered in the differential diagnosis of most cases of infertility in the suckler herd. KEYWORDS: C. fetus, Cattle infertility INTRODUCTION It is more than 50 years since the association between campylobacter organisms and venereal infertility was described (Plastridge and others 1947). In the years immediately after this discovery the disease was recognised as a significant problem for cattle producers in Britain (Hignett 1951). The development and adoption of artificial insemination services provided an effective means of controlling the disease in the dairy herd and after the first flurry of interest only occasional reports have appeared in the UK literature, notably those of MacLaren & Wright 1977; MacLaren and Agumbah 1988; Hewson and others 1985 and Lander 1990a. This encouraged the view that venereal campylobacter infection was an uncommon disease which was of little significance on a national basis. In contrast to this the disease continued to cause interest worldwide particularly in Australia and North America, where research activity was directed at developing vaccines, unravelling the biology of the causal organism and improving diagnostic tests and resulted in the publication of a large number of research communications. It was clear that workers in these countries considered the disease to be a significant problem wherever natural service was used, ie principally beef cow herds, and also that questions remained to be answered concerning the biology of the disease and the significance of diagnostic findings. Disease surveillance data from Britain shows that the number of diagnoses made by the veterinary investigation services have remained at a low level over the past 20 years varying between three and 23 diagnoses per year (Source: VIDA), but prior to this an increase in the number of outbreaks was attributed to increased bull movement between dairy herds (McLaren and Wright 1977). While the national dairy herd has come to rely heavily on artificial insemination the breeding management of suckler herds is unlikely to have altered significantly since the discovery of the disease. Herds with a limited number of breeding bulls have always hired or borrowed bulls when disease or accident has deprived them of a bull. Mature bulls which have already worked in one herd may also be sold and used in a second herd. Therefore the conditions which ensure the maintenance and spread of venereal campylobacter disease continue to operate and it is likely that in the beef herds of Britain the disease remains as much of a problem as it ever was. Recent experience in the south east of Scotland and north east of England offers support to this theory. In addition, difficulties in assessing the significance of diagnostic test results and defining the scale of the clinical problem have made case management a concern. These problems are compounded by the importance that such a diagnosis has for a beef cow herd, where the standard control programmes are costly both financially and in management requirements. This paper seeks to outline the important features of the disease, to describe our recent difficulties in investigating field outbreaks and to suggest a standard approach to herd investigation. AETIOLOGY Campylobacter fetus is a microaerophilic, motile bacterium which has two recognised subspecies: C. fetus subsp venerealis and C. fetus subsp fetus (Berg and others 1971, Véron and Chatelaine 1973). Differentiation has been based on the variation of antigenic and biochemical characteristics of the strains, notably C. fetus subsp venerealis will not grow in 1% glycine whereas C. fetus subsp fetus will. The presence or absence of glycine tolerance is the standard differentiation used by diagnostic laboratories and the differentiation is accepted as being important since C. fetus subsp venerealis is considered to be adapted to the bovine reproductive BCVA 1997 327

CATTLE PRACTICE VOL 5 PART 4 tract and to be the cause of venereal campylobacteriosis, whereas C. fetus subsp fetus is a gut commensal with the ability to cause sporadic fetopathy in cattle. However as long ago as 1971 the inherent capacity for this organism to show antigenic and biochemical variation was demonstrated (Chang and Ogg 1971). Strains identified as C. fetus subsp venerealis could develop resistance to glycine, raising doubts over the value of the classification system. Indeed the subspecies differentiation has been described as arbitrary (Prescott 1990) and causing confusion (Hum and McInnes 1987). In addition experimental infection of clean heifers with strains identified as C. fetus subsp fetus has resulted in clinical disease indistinguishable to that caused by C. fetus subsp venerealis (Schurig and others1973 and MacLaren and Agumbah 1988) and field outbreaks of venereal infections associated with this subspecies have also been identified in Britain (MacLaren & Agumbah 1988). In the course of survey work both subspecies have been isolated from the reproductive tract of cows and bulls (Bawa and others 1991); while a high intestinal carriage rate of C. fetus subsp fetus has been demonstrated in healthy calves and cows, isolations of C. fetus subsp venerealis were also made from faeces (Giacoboni and others 1993). A further source of confusion lies in the description of intermediate biotypes in both subspecies without any clear picture of the clinical significance of these differentiations (Clarke and others 1976 and MacLaren and Agumbah 1988). Therefore on the basis of the above, the isolation of either subspecies from clinical material should neither lead to complacency nor should it constitute a diagnosis of venereal campylobacteriosis on its own. The application of molecular biological techniques to the study of C. fetus is already leading to a greater understanding of genetic variation (Salama and others 1992), of the organisms ability to cause disease (Wang and others 1993) and perhaps in the future to the development of improved diagnostic techniques (Brooks and others 1996). It is to be hoped that these advances will overcome the current problems faced by the diagnostician. CLINICAL DISEASE The clinical aspects of the disease are well reviewed by Clarke (1971) and Dekeyser (1986). Infection in the bull follows natural mating with an infected female, but is unassociated with either clinical signs or pathological changes. Bulls younger than six years of age appear fairly resistant to infection (Clarke 1971) but in older bulls infection becomes established in the lumen of epithelial crypts in the prepuce. It also appears that infected bulls vary in the numbers of viable organisms which can be detected in preputial secretions and this may explain the observed differences in the ability of infected bulls to pass on the disease. In natural service spread of infection is from the penis to the anterior vagina, where the organism becomes established before entering the uterus to cause a mild endometritis and salpingitis. Pathological changes are most pronounced at eight to thirteen weeks after infection (Dekeyser 1986) and have resolved by four to five months. Heifers appear to be more susceptible to the pathological effects of the infection than cows. Where conception has occurred embryonic death and a delayed return to estrus may be observed, but abortions or indeed normal pregnancies can follow. The production of antibody in uterine and vaginal secretions is a key element in the recovery. The most important component is IgG which can be detected at eight weeks post infection, but is largely undetectable by six months. IgA is produced by three to five weeks and is still detectable for up to one year after infection (Corbeil and others 1974). Despite recovery, the development of immunity and the establishment of normal pregnancies, C. fetus has been isolated from vaginal mucus more than a year after infection (MacLaren and Agumbah 1988) creating the potential for cows as well as bulls to maintain the infection in the herd from one breeding season to the next. It has been suggested that after infection a full return to fertility is unlikely (Dekeyser 1986) but this reduction in fertility may be marginal (Clark 1971). The variability of the cows’ response to infection; of bulls’ ability to transmit infection; of age structure and immunity of the herd added to possible variation in pathogenicity between isolates of C. fetus means the severity of any clinical problem is also likely to be very variable. ASSESSING SUCKLER HERD INFERTILITY The introduction of an infected bull to a clean herd could be expected to lead to the well-defined clinical syndrome of irregular, prolonged inter-oestrus intervals. However for the reasons given above this may not happen; furthermore the difficulty in observing or recording services in a suckler herd at pasture means there is often a lack of reproductive records to assess. In many herds it can be up to six months after the end of a prolonged breeding period before the herdsman suspects a problem. The detection of a high barren cow rate at this time offers a major diagnostic challenge. It is essential to have records of calving patterns, length of breeding season, barren rates from previous years and to assess these in relation to bull to cow ratio, age of bull stud, heifer management, cull cow policy and nutritional management before investigating possible infectious causes of infertility. Problem herds without some deficiency in reproductive management are unusual and with the exception of infertility problems related to the use of young bulls, BCVA 1997 328

CATTLE PRACTICE VOL 5 PART 4 uncomplicated bull infertility is unlikely to be excluded on the basis of history alone. Particular attention should be paid to the fertility of first calvers as these will be the most likely to be affected by venereal campylobacteriosis, but it should be recognised that these are also the animals most susceptible to the effects of undernutrition and to other infectious diseases. The common infectious diseases bovine virus diarrhoea virus (BVD), leptospirosis and infectious bovine rhinotracheitis (IBR) can all manifest as an increased barren cow rate. It is certainly possible to exclude any of these infections as a cause of the the infertility if a representative sample of cows is examined serologically and found to be seronegative, however the converse is not true. Therefore in many suckler herds it will not be possible to identify confidently the important factors which have contributed to the infertility and only occasionally will it be possible to exclude the possibility of venereal campylobacteriosis without carrying out appropriate laboratory examinations. DIAGNOSTIC TESTS Demonstration of C. fetus by culture and by the fluorescent antibody test in sheath washings or preputial scrapings are the techniques used in bulls; demonstration of C. fetus in vaginal mucus by culture or demonstration of specific immunoglobulin by the vaginal mucus agglutination test (VMAT) are used in the female. None of the tests is without problems. The sampling technique most frequently used in bulls in Britain is the collection of sheath washings rather than preputial smegma and a method has been described in a BCVA note (BCVA 1995). Provided samples are relatively free from gross contamination and transported to the laboratory within four to six hours we have found phosphate buffered saline (PBS) to be adequate for lavage without the need for transport enrichment medium (TEM) but where immediate delivery cannot be guaranteed the use of TEM has proved to be effective in maintaining the viability of the organism (Lander 1990b and Hum and others 1994). It has been recommended that sheath washings should be screened on at least four occasions before a bull could be considered free of infection (Dufty 1967). As a consequence of the improvement in culture methods (Hum and others 1994) it seems less likely that this recommendation applies now, but nevertheless our experience suggests it is unwise to rely on a single screening. The fluorescent antibody test does not allow the differentiation between the two subspecies of C. fetus (Lander 1990a) and given the improved culture techniques this particular test is of dubious diagnostic value. The collection of vaginal mucus is straightforward using pipettes with the end closed off and perforations in the last few centimetres, a 60 ml syringe and PBS (after the method described by Lander, 1983). As with sheath washings, provided gross faecal contamination of the sample is prevented and samples are delivered to the laboratory within four to six hours of collection, isolation of C. fetus from infected animals should be successful. TEM can be used if time intervals between collection and culture exceed this, but in general our advice is to make every effort to deliver samples to the laboratory immediately after collection. The vaginal mucus agglutination test (VMAT) which has essentially remained unchanged in the past 50 years, uses antigens prepared from C. fetus to detect agglutinins in the vaginal mucus. Immunoglobulin A is the antibody present in these secretions, but where serum or blood contaminate vaginal secretions false positives are likely as serum antibody to C. fetus is present in many cattle (Wilkie and Winter 1971). In addition where cattle are in estrus, large amounts of mucus are produced, IgA is diluted and false negatives will occur. The VMAT can be expected to be positive by seven weeks post infection and may remain positive for at least 10 months after infection (Corbeil and others 1974). Most published reports on experimental infection and field outbreaks indicate the VMAT to be a useful test, however there are exceptions and in one study VMATs did not become positive until 209 days after infection (Hewson and others 1985). At the very least this suggests there can be problems with the test. There is no sensitivity or specificity figure published, but it has been suggested that only 50% of infected females will be positive by VMAT and that 50% of these will be negative by six months post infection (Clarke 1971), leading to the recommendation that at least 10 - 20 cows are sampled in a suspect outbreak. Our limited experiences support this and despite the expense of the test and the time consuming nature of sampling we recommend a minimum of 12 samples are collected. In order to overcome the limitations of the VMAT, work has been carried out to develop an enzyme linked immunoassay in Britain (Hewson and others 1985) and Australia (Hum and others 1991) although at the present time the veterinary investigation services of Britain do not plan to replace the VMAT. CONTROL Artificial insemination offers a simple approach to control. There is no need to remove infected cows and a return to normal herd fertility can be expected. The routine processing of bull semen for artificial insemination does not preclude the transmission of C. fetus, but a suitable disease control programme in a closed bull stud will ensure that semen is maintained free of C. fetus (Philpott 1993). Artificial insemination in the beef herd offers further BCVA 1997 329

CATTLE PRACTICE VOL 5 PART 4 advantages as it provides access to top genetic material and a tool to help in the prevention of dystocia. Indeed many beef herds use artificial insemination and enjoy excellent results, but for those herds where breeding takes place at grass the increased labour requirement can be prohibitive. While synchronisation and artificial insemination programmes for suckler herds in Britain have been described (Penny and others 1997), the use of a follow-up bull is still favoured in spring calving herds. In this situation bulls up to four years of age can be used as they are relatively resistant to infection (Clarke 1971). Vaccination has been used successfully in other countries, but no commercial vaccine is available in Britain. Emergency vaccines can be produced under licence from Veterinary Medicines Directorate (MAFF) and this is the control procedure of choice where artificial insemination is not practicable. A vaccination programme to eradicate infection within a herd is described (Hum and MacInnes 1987), but antibiotic treatment of the bulls has been suggested as an additional safety measure (Hum and others 1993). Although antibiotic treatment of bulls may have a role as an adjunct to the above programmes, on its own it is of no value since re-infection will result as soon as the bull is joined with the herd. Prevention can be achieved by maintaining a strict closed herd policy as cows as well as bulls can introduce infection. In practical terms the purchase of virgin heifers and young bulls should offer little or no risk of introducing infection. The difficult situation is where bulls have to be replaced in a hurry and there is no option but to bring in a proven bull from elsewhere. Screening any such bulls for infection and only accepting clean bulls which are then treated with antibiotics (BCVA 1995) appears the safest approach. At this time screening for BVD and leptospirosis would also be advisable. The bull should also be screened and treated before he is used again on the herd of origin. BCVA 1997 FIELD EXPERIENCES In 1996 SAC Veterinary Services, St Boswells, isolated C. fetus from diagnostic submissions from nine suckler herds. In five of these cases the first isolate was made from abortion material and in all but three herds infertility had not been a complaint prior to the isolation of C. fetus. Follow-up investigation often failed to reveal convincing evidence that venereal campylobacteriosis was a cause of herd infertility, but in three herds a decision was taken to have a vaccine produced, one herd changed to artificial insemination and no action was taken in three others. In two outbreaks (outbreaks A and B) we had the opportunity to explore the problem in more detail. Outbreak A: this occurred in a herd made up of four groups of 100-120 cows each. The management structure was complex and movement of cows and bulls took place between all the groups. Since 1994 C. fetus subsp venerealis had been isolated from abortion material from two of the four units. Poor fertility had been recognised on three of the units for a number of years, manifest as prolonged calving seasons and high numbers of non-pregnant cows. A number of contributing factors had been identified including poor heifer management, undernutrition, inadequate bull power, infertile bulls, cull cow policy, leptospirosis and BVD. The only herd with acceptable reproductive performance was one of those in which campylobacter abortions had occurred. In 1995 when investigating the poor fertility on unit 1 vaginal washings were collected from six empty cows, C. fetus subs venerealis was isolated from one and all gave negative VMAT results. In addition C. fetus subsp venerealis was isolated from the sheath washings of two bulls. By March 1996 further campylobacter abortions had occurred and further sheath washings showed two bulls infected on unit 1 and unit 2. The situation was further complicated at this stage as all of these isolates so far were identified by a reference laboratory as C. fetus subsp fetus. Subsequently these have been identified by a second reference laboratory as C. fetus subsp venerealis as stated above. Reproductive data from two of the four units is summarised in Tables 1, 2 and 3. Table 1: The length of the calving season (weeks), the number of cows calving in first two months of calving season (percent) and barren cows ie the number of cows put to the bull which did not calve (percent) in units 1 and 2 of Outbreak A. Calving spread (wks) Cows calved in 1st 2 months (%) Barren cows (%) Year Unit 1 Unit 2 Unit 1 Unit 2 Unit 1 Unit 2 1994 31 34 58 86 14 4 1995 33 13 60 73 7 7 1996 17 14 88 81 8 7.8 1997 13 13 84 83 8 5 Table 2: The number of cows calving in 3 week intervals throughout the calving season. (percentage) Unit 1 1994 % 1995 % 1996 % 1997 % 1st 3 weeks 13 16 40 59 2nd 3 weeks 18 33 39 19 3rd 3 weeks 46 17 15 11 4th 3 weeks 16 10 6 12 5th 3 weeks 1 4 6th 3 weeks 7th 3 weeks 1 8th 3 weeks 2 2 9th 3 weeks 1 3 10th 3 weeks 1 8 11th 3 weeks 4 330

CATTLE PRACTICE VOL 5 PART 4 Unit 2 1994 1995 1996 1997 1st 3 weeks 38 23 41 46 2nd 3 weeks 37 43 25 34 3rd 3 weeks 13 10 17 7 4th 3 weeks 7 22 11 12 5th 3 weeks 1 6 1 6th 3 weeks 7th 3 weeks 8th 3 weeks 9th 3 weeks 4 BCVA 1997 Table 3: The number of available cows which were mated and conceived in each 3 week period of the breeding season estimated from subsequent calving dates (percentage) 1995 1996 Unit 1 % Unit 2 % Unit 1 % Unit 2 % 1st 3 weeks 37 38 62 38 2nd 3 weeks 57 37 32 45 3rd 3 weeks 52 39 26 18 4th 3 weeks 42 40 36 36 5th 3 weeks 40 10 5 As the barren cows sampled in 1995 had been negative by VMAT it was felt necessary to further explore the possible contribution of C. fetus to the herd infertility. To do so we test mated six maiden heifers from unit 1 with a five year old Blonde d’Aquitaine bull which had sheath washed positive for C. fetus subsp venerealis and six maiden heifers from unit 2 with a six year old Charolais bull also known to be infected. The bulls ran with the heifers for six weeks in two separate groups. Vaginal mucus samples were collected from the heifers at day 0, 56, 112, 167 and 339 relative to the introduction of the bull. They were also examined for pregnancy using an ultrasound scanner at 56 days. A misunderstanding between ourselves and the farm manager led him to dispose of all heifers from unit 2 and those from unit 1 which were not positively identified as being pregnant at the 56 day examination before the third examinations were due. Therefore some of the animals identified as being non-pregnant may have been pregnant. Results are presented in Table 4 All heifers that were retained gave birth to viable calves at full term. At 56 days one heifer was positive by the VMAT and seven were pregnant. The failure to isolate C. fetus from this group was considered more important than a single VMAT positive result. At this time we considered that while the herd was clearly infected, we lacked convincing evidence that venereal campylobacteriosis was a major cause of infertility in these herds. The options for control including artificial insemination programmes, vaccination and running ‘clean’ and ‘dirty’ herds were considered but it was decided these were either too expensive or impractical. However, in 1995 an oestrus synchronisation and artificial insemination programme was implemented in the 120 cow herd of Spring calving cows on unit 1. The stock bulls were run with the cows following AI to sweep up. This was repeated in 1996. From the three week calving figures a figure for cows mated or inseminated which conceived was calculated for AI and for the bulls that ran up on unit 1 and also for unit 2 where natural service continued to be used (Table 3). This statistic is inaccurate as it does not account for cows in anestrus, however it is the best estimate of conception rates that can be used in this situation. In the two years that AI has been used in unit 1 an improvement in the calving pattern has been achieved. The 1995 conception rates to AI were unimpressive but this was in a group of cows with a wide spread in calving dates. However, there appeared to be better conception rates to synchronised AI on unit 1 than to natural service with the follow-up bulls on unit 1 and to the bulls alone on unit 2. The estimated conception rates for natural service are below what is required for a compact calving and the cause of this needs further investigation. Attempts to achieve target condition scores at calving, to improve the health of the bull stud and to increase the bull numbers have led to a reduction in non-pregnant cow numbers and a reduction in the length of the calving periods. Further fertility monitoring is planned this autumn and vaginal mucus samples will be collected from any younger cows which are found to be non-pregnant. Outbreak B this was a 100 cow suckler herd which had no history of reproductive problems. In 1995 there was a calving spread of 14 weeks with 80% calved in the first two months. There were six nonpregnant cows. A bull from this herd was hired to a dairy herd to be used with heifers. One heifer aborted and C. fetus subsp venerealis was isolated from the fetus. The bull was sheath-washed and found to be positive by culture for C. fetus subsp venerealis. The bull was returned to the suckler herd and it was assumed by those investigating the incident at the dairy herd that the bull was the source of the infection. To explore the possibility that the infection might be endemic in the beef herd a group of 20 cows which were due to be culled were test mated with the bull, vaginal washings having been collected prior to introduction of the bull and again at eight weeks. Results are presented in Table 5. A follow-up sample at 14 weeks had been planned, but the farmer made the decision to cull the group early as he feared the bulling activity in the cows would encourage his other bulls to jump fences. The results confirmed 331

CATTLE PRACTICE VOL 5 PART 4 Table 4: Outbreak A: C. fetus subsp venerealis isolations, vaginal mucus agglutination test results and pregnancy diagnoses in 12 heifers test mated with two bulls positive for C. fetus subsp venerealis Unit Cow No Day 0 Day 56 VMAT Culture Pregnancy VMAT Culture Pregnancy One 1 - - - - - 8wks* 2 - - - - - 5wks* 3 - - - - - - 4 - - - - - 7wks* 5 - - - - - - 6 - - - - - 5wks* Two 1 - - - - - - 2 - - - - - - 3 - - - - - 7wks 4 - - - - - 7wks 5 - - - - - - 6 - - - + - 7wks *Monitored throughout pregnancy and found to be consistently negative for C. fetus and vaginal antibody to C. fetus Table 5: Outbreak B: C. fetus subsp venerealis isolations, vaginal mucus agglutination test results and pregnancy diagnoses in 12 cows test mated with one bull positive for C. fetus subsp venerealis Cow No Day 0 Day 62 VMAT Culture Pregnancy VMAT Culture Pregnancy 1 - - - - - - 2 - - - - - 8wks 3 - - - - + - 4 - - - - - 7wks 5 - - - + - - 6 - - - - + -oestrus 7 - - - - + 4wks 8 - - - + + 4wks 9 - - - + + 4wks 10 - - - - - - 11 - - - - - 4wks 12 - - - - + 6wks that the bull was positive for a venereal strain of C. fetus, but indicated that the cows were naive prior to exposure to the bull. DISCUSSION The strains of C. fetus from both outbreaks were indistinguishable on biochemical grounds with findings confirmed by a reference laboratory, yet they appeared to cause very different clinical syndromes. It is possible that despite the presence of endemic infection in the herds involved in outbreak A, the bulls used for test mating were shedding few infective organisms. However as all VMATs carried out in these herds proved to be negative with the single exception of one heifer it appeared that venereal campylobacteriosis was not a problem in these herds. The investigation was hampered by the early disposal of all the heifers from unit 2 and the non-pregnant heifers from unit 1. Fifty-six days after the first possible bull exposure may have been too soon to expect the VMAT to be positive, but the pregnancy diagnosis results did show that the majority of the heifers were mated early in the period. As IgA can be detected as early as three weeks post infection and the VMAT can be expected to be positive by seven weeks (Corbeil and others 1974) this should have been sufficient time. In herd B there were 62 days between first exposure and the collection of vaginal mucus and three of twelve were positive. Similarly, given the success of campylobacter culture in outbreak B, the negative culture results for heifers in outbreak A appear to be a firm indication that venereal spread of infection did not take place. While endemic venereal campylobacteriosis is suspected we remain unsure of the clinical significance of the C. fetus infection BCVA 1997 332

CATTLE PRACTICE VOL 5 PART 4 despite the considerable diagnostic effort employed. In outbreak B the conclusion was that the infection had not been present in the suckler herd, based on the success of infecting mature cows and observing VMAT conversion from negative to positive. This was despite the indication from the dairy herd, to which the bull had been hired, that infection had originated in the suckler herd. It seems certain that had the bull been used in the main herd, venereal infertility would have been a significant problem. These two cases underline the difficulties that can be encountered when investigating possible venereal campylobacteriosis and emphasise the need to carry out a careful investigation before the significance of the isolation of C. fetus can be determined. On the basis of our experiences we believe it is likely that venereal campylobacteriosis is a more widespread problem in beef suckler herds in Britain than has previously been reported. The clinician dealing with suckler herd infertility should therefore be aware of the great variation in the clinical manifestation of herd infection with C. fetus and the difficulties in establishing a diagnosis. We recommend that where C. fetus is identified as the cause of an abortion then the possibility of venereal campylobacteriosis should be considered and where there is evidence of poor reproductive performance that the possibility of venereal campylobacteriosis is investigated. When a herd investigation is carried out at least 12 vaginal mucus samples from barren cows and sheath washings from all bulls should be collected. These samples should be delivered the same day to the diagnostic laboratory in order to have cultures set up within six hours of collection. Where an insufficient number of barren cows is available it would appear prudent to consider collecting samples from pregnant cows which have completed their first mating season in the herd Once a diagnosis has been made careful consideration should be given to the various control options and these should be costed before a programme is decided upon. Finally, it is to be hoped that advances in molecular biology techniques can be applied to the problems of genetic and antigenic variation within the species C. fetus, the relevance of these differences in relation to the clinical disease is further elucidated and improved diagnostic techniques become widely available. Failure to make progress will ensure that the diagnosis of venereal campylobacteriosis remains as much of a problem as it was almost 50 years ago (Hignett 1951) and for much the same reasons. ACKNOWLEDGEMENTS We acknowledge the help and advice from our colleagues and the help and the immense patience of the farmers involved. We thank Alistair Greig and William McKelvey for commenting on the manuscript. Scottish Agricultural Colleges Veterinary Services receives financial support from the Scottish Office Agricultural Environmental and Fisheries Department. REFERENCES Bawa, E.K., Adekeye, J.O., Oyedipe, E.O. and Umoh, J.U. (1991) Prevalence of bovine campylobacteriosis in three states in NigeriaTropical Animal Health and Production 23,157-160 Berg, R.L., Jutila, J.W. and Firehammer, B.D. (1971) A revised classification of Vibrio fetus. American Journal of Veterinary Research, 32, 11-22 British Cattle Veterinary Association (1995) Bull hiring, BCVA, Gloucester Brooks, B.W., Garcia, M.M., Robertson, R.H. and Lior, H. (1996) Electrophoretic and Immunoblot analysis of . fetus lipopolysaccharides Veterinary Microbiology, 51, 105-114 Chang, W. and Ogg, J.E. (1971) Transduction and Mutation to Glycine tolerance in Vibrio fetus. American Journal of Veterinary Research, 32, 649 -653 Clarke, B.L. (1971) Review of Bovine Vibriosis Australian Veterinary Journal, 47,103-107 Clarke, B.L., Dufty, J.H., Monsbourgh, M.J. and Parsonson, I.M. (1976) Immunisation of cattle against vibriosis due to C. fetus subsp fetus biotype intermedius Australian Veterinary Journal 52, 362-365 Corbeil, L.B., Duncan, J.R., Schurig. G.G.D., Hall, C.E. and Winter, A.J. (1974) Bovine venereal vibriosis: variations in immunoglobulin class of antibodies in genital secretions and serum. Infection and Immunity 10, 1084-1090 Dekeyser, P.J. (1986) Bovine Genital Campylobacteriosis in Current Therapy in Theriogenology 2. Editor Morrow, D.A., W.B. Sauders Company, Philadelphia p263-266 Dufty, J.H. (1967) Diagnosis of Vibriosis in the bull Australian Veterinary Journal, 43, 433-437 Giacoboni, G.I., Itoh, K., Hirayama, K., Takahashi, E. and Mitsuoka, T. (1993) Comparison of faecal campylobacter in calves and cattle of different ages and areas in Japan. Journal of Veterinary Medical Science, 55, 555-559 Hewson, P.I., Lander, K.P. and Gill, K.P.W. (1985) Enzymelinked immunoabsorbent assay for antiboies to C. etus in vaginal mucus. Research in Veterinary Science, 38, 41-45 Hignett, P.G., (1951) Vibrio foetus infection in cattle Veterinary Record, 63, 268-269 Hum, S., Brunner, J. and Gardiner, B. (1993) Failure of theraputic vaccination of a bull infected with C. fetus Australian Veterinary Journal 70, 386-387 Hum, S., Brunner, J., McInnes, A., Mendoza, G., Stephens, J. (1994) Evaluation of cultural methods and selective media for the isolation of C. fetus subsp venerealis from cattle. Australian Veterinary Journal, 71, 184-186 Hum, S. and MacInnes, A. (1987) Australian Standard Diagnostic Techniques for Animal Diseases, Edited by Corner, L.A. and Bagust, T.J. CSIRO for the Standing Committee on Agriculture and Resource Management, East Melbourne No 22 Hum, S., Stephens, L.R. and Quinn, C. (1991) Diagnosis by ELISA of bovine abortion due to C. fetus Australian Veterinary Journal 68, 272-275 Lander, K.P. (1990b) The application of a transport and enrichment medium to the diagnosis of C. fetus infection in bulls British Veterinary Journal, 146, 334-340 Lander, K.P. (1983) New Technique for collection of vaginal mucus from cattle Veterinary Record 112, 570 Lander, K.P. (1990a) The development of a transport and enrichment medium for C. fetus . British Veterinary Journal, 146, 327-333 MacLaren, A.P.C. and Agumbah, G.J.O. (1988) Infertility in cattle in South West Scotland caused by an intermediate strain of C. fetus subsp fetus British Veterinary Journal 144, 29-44 MacLaren, A.P.C. and Wright, C.L. (1977) C. fetus infection in dairy herds in South West Scotland, Veterinary Record, 101, 463- 464 BCVA 1997 333

CATTLE PRACTICE VOL 5 PART 4 Penny, C.D., Lowman, B.G., Scott, N.A. and Scott, P.R. (1997) Repeated oestrus synchrony and fixed-time artificial insemination in beef cows Veterinary Record (1997) 140, 496-498 Plastridge, W.N., Williams, L. F. and Petrie, D. (1947) Vibrio abortion in cattle American Journal of Veterinary Research, 8 178- 183 Prescott, J.F. (1990) Diagnostic Procedures in Veterinary Bacteriology Editors Carter, G.R. and Cole, J.R., 5th Edition, Academic Press, London Pages 61 -75 Salama, S.M., Garcia, M.M. and Taylor, D.E. (1992) Differentiation of the subsp of C. fetus by genomic sizing. International Journal of Systematic Baceriology, 42, 446-450 Schurig, G.D., Hall, C.E., Burda, K. Corbeil, L.B., Duncan, J. R. and Winter, A.J. (1973) Persistant Genital Tract Infection with Vibrio fetus fetus associated with serotype alteration of the infecting strain. American Journal of Veterinary Research, 34, 1399-1403 Véron, M,. and Chatelaine, R. (1973) Taxonomic study of the genus Campylobacter Sebald and Véron and designation of the neotype strain for the type species C. fetus (Smith and Taylor) Sebald and Véron International Journal of Systematic Bacteriology, 23, 122-134 Wang, E., Garcia, M.M., Blake, M.S., Pei, Z. and Blaser, M.J. (1993) Shift in S-layer Protein expression Responsible for Antigenic Variation in C. fetus Journal of Bacteriology, 175, 4979-4984 Wilkie, B.N. and Winter, A.J. (1971) Bovine vibriosis: the distribution and specificity of antibodies induced by vaccination and infection and the immunofluorescent localisation of the organism in infected heifers. Canadian Journal of Comparative Medicine 35, 301-312 BCVA 1997 334

CATTLE PRACTICE VOL 5 PART 4 Neospora Caninum Antibody Levels in an Endemically-Infected Dairy Herd Dannatt L. V. I. Centre, Staplake Mount, Starcross, Exeter, Devon, EX6 8PE. ABSTRACT Forty cattle from a 95-cow pedigree Friesian-Holstein dairy herd, which had recently experienced an outbreak of Neospora caninum associated abortion, were blood-sampled every two months over a 12-month period. The Neospora caninum antibody response was analysed by ELISA. Forty per cent of cattle remained seronegative for N. caninum over the sampling period, 35% were seropositive on all 7 time points and 25% of cattle showed variable antibody levels ie at least one positive and one negative result over the 12 months. Antibody levels in both the consistently positive group and the variable group reached a peak 1.5-4 months before calving. These results have implications regarding when cattle should be sampled to optimise detection of Neospora caninum antibodies in a proportion of infected cattle. KEYWORDS : Neospora, cows, antibodies, time. INTRODUCTION Neospora caninum is an apicomplexan protozoan parasite known to cause abortion and stillbirth in cattle both in the United Kingdom and worldwide (Dubey and Lindsay 1996, Trees and others 1994). The full life-cycle of the organism is unknown with transplacental infection the only mode of transmission demonstrated so far (Dubey and others 1992). Diagnosis of abortion is best achieved by histological means but where foetal tissues are unavailable serological examination of the dam’s blood is increasingly being used as an indicator of infection. Interpretation of serological tests can however be problematic. Several studies have indicated that in endemically-infected herds seropositive animals do not necessarily abort but carry their calves to term; such calves may be congenitally infected, probably for life (Paré and others 1996). Antibody levels have been shown to decrease in both naturally and experimentallyinfected animals following abortion and to increase in subsequent pregnancies (Conrad and others 1993, Paré 1997) but nothing is known of the variations in Neospora antibody levels in cattle in the UK under typical management regimes. Fluctuations in antibody levels have implications for diagnosis of Neospora infection on both a herd and an individual basis and this study was set up to investigate the changes in antibody levels over time in a dairy herd following a previous outbreak of abortion due to Neospora caninum. MATERIALS AND METHODS A 95-cow pedigree Friesian-Holstein dairy herd in east Cornwall was selected which had experienced an outbreak of abortion in late summer 1993 with 10 cows aborting over a 6-week period (Dannatt and others 1995). Neospora caninum was demonstrated by immunohistochemistry in the brain of one aborted foetus and 9 cows had significantly raised antibody levels (1/640 or greater) when measured by the immunofluorescent antibody test (IFAT) (Trees and others 1994). Extensive investigations could not incriminate any other abortifacients and blood samples from the remaining 85 milking and dry cows showed the seroprevalence at that time to be 60%. From May 1994 to May 1995 76-89 milking and dry cows were blood sampled 7 times at two-monthly intervals. Forty cows were sampled on all 7 occasions and a total of 125 animals were sampled at least once. Sera were initially examined by the IFAT and later by the Mastazyme Neospora ELISA kit (MAST Diagnostics, Liverpool, UK). Results from the 40 animals sampled on all 7 occasions are considered here. ELISA values were expressed as percentages of high positive control sera to minimise temporal variations. A cut-off value of 20 percent positivity (PP) was used to differentiate positive/negative antibody status and was selected to maximise the sensitivity of the test (Williams & others, in preparation). The seroprevalence calculated at each sampling ranged from 42 - 51%. RESULTS Each animal was allocated to one of three categories: i) animals showing variable (positive/negative) antibody levels (Group A, n=10); ii) animals which showed consistently positive antibody levels on all 7 occasions (Group B, n=14) and iii) animals which showed consistently negative antibody levels (Group C, n=16); Figures 1, 2 and 3 below illustrate the changes in antibody levels for a representative cow in each group. Animals were allocated into Group A if they had at least one positive or negative antibody value on any one of the 7 occasions. A generally rapid rise in antibody levels peaking at 1.5-4 months before calving was the pattern of antibody variation most frequently seen. This was present in 11/24 (46%) of the animals in Groups A BCVA 1997 335

CATTLE PRACTICE VOL 5 PART 4 and B; antibody values in these animals declined thereafter but remained positive. In 5/24 (21%) animals from Groups A and B antibody fluctuations were of much smaller magnitude. In the remaining 8 cows calving was too close to the first or last sampling occasion to allow any definite interpretation to be made. C C Fig. 1 Group A 0 10 20 30 40 50 60 70 80 90 100 5/94 7/94 9/94 11/94 1/95 3/95 5/95 %pp Cow MF Fig. 2 Group B 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 5/94 7/94 9/94 11/94 1/95 3/95 5/95 %pp Cow F4 C Fig. 3 Group C 0 10 20 30 40 50 60 70 80 90 100 5/94 7/94 9/94 11/94 1/95 3/95 5/95 %pp Cow MMM = Calving date Typical antibody patterns of cows DISCUSSION This study shows, firstly, that Neospora caninum antibody levels fluctuate in naturally-infected cattle and that in 25% of animals tested ELISA values fell below the cut-off value for the test on at least one time-point. Secondly, cows with both variable and consistently positive antibody levels showed an increase in these antibody levels in the last half of pregnancy. Fluctuations in Neospora antibody levels over time have also been reported by other workers. Paré and others (1997) monitored antibody levels in the pregnancies of 254 cattle in a dairy herd which had a 60% seroprevalence rate. In this herd 151 cows were found to be seropositive at some time during pregnancy with cow seropositivity during gestation defined as a minimum of two blood samples positive by ELISA. Conrad and others (1993) monitored antibody levels in two cows that were rebred after aborting Neospora-infected foetuses. In both of these animals antibody levels, measured by IFAT, decreased after abortion, peaked at 4-5 months of gestation during their subsequent pregnancies and then remained constant until birth at full-term. Evidence of a similar pattern of antibody variation with peaks in values from 1 - 4 months before calving was also seen in the herd reported by Paré and others (1997). In this herd, statistical analysis of 151 seropositive cows monitored through pregnancy indicated an infected cow was less likely to abort but more likely to produce a congenitally-infected calf if a high antibody value was present in the last third of gestation than if a low antibody value was present; 80% of the seropositive cows in this herd did not abort. In the study by Moen and others (1996) antibody levels rose in the second half of gestation in 58% of 43 animals which had aborted due to Neospora and then successfully rebred; comments were also made in this report on the possible underestimation of Neospora-infected animals which may occur because antibody levels can fall to undetectable values after abortion or calving. In the herd under discussion here a similar pattern of a rise and fall in antibody levels was seen in animals both consistently and variably positive. Lower antibody values generally were exhibited by the latter group and this may be an indication of natural variations in antibody response to antigenic stimulus in individual cows. Consistently seronegative animals can almost certainly be assumed to be uninfected with Neospora (Anderson and others 1997) and calving had no discernible effect on the antibody status of these animals. CONCLUSIONS This study provides information on the variations of Neospora antibody values over time in an endemically-infected herd. The findings have particular implications for the assessment of serological status. Determination of antibody levels from a single blood sample should not be wholly relied on as an indicator of negative status. This phenomenon may be of particular importance in herd investigations and research projects where true evaluation of antibody status is desirable. Conversely, the demonstration of positive antibody levels is more likely to represent an animal’s true Neospora status. Development of tests such as an ELISA for use on bulk milk samples may give a more realistic interpretation of herd antibody status than targeting a specified number of individual animals. ACKNOWLEDGEMENTS The author wishes to thank Dr J. McGarry and Mrs F. Guy of the Veterinary Parasitology Department, University of Liverpool, for carrying out the IFA and BCVA 1997 336

CATTLE PRACTICE VOL 5 PART 4 ELISA tests. Thanks are also due to Dr. Diana Williams for her comments on the manuscript, Professor Sandy Trees and Helen Davison. The technical assistance given by the staff at Starcross VI Centre and the continued co-operation and assistance of everybody on the farm concerned is also gratefully acknowledged. REFERENCES Anderson M.L., Reynolds J.P., Rowe J.D., Sverlow K.W., Packham A.E., Barr B.C. & Conrad P.A. (1997) Journal of the American Veterinary Medical Association 210, 8, 1169. Conrad P.A., Sverlow K., Anderson M., Rowe J., BonDurant R., Tuter G., Breitmeyer R., Palmer C., Thurmond M., Ardans A., Dubey J.P., Duhamel G. & Barr B. (1993) Journal of Veterinary Diagnostic Investigation 5, 572. Dannatt L., Guy. F. & Trees A.J. (1995) Veterinary Record 137, 566. Dubey J.P. & Lindsay D.S. (1996) Veterinary Parasitology 67, 1. Dubey J.P., Lindsay D.S., Anderson M.L., Davis S.W. & Shen S.K. (1992) Journal of the American Veterinary Medical Association 201, 5, 709. Moen A.R., Wouda W. & de Gee A.L.W. (1996) Proceedings of the XIX World Buiatrics Congress, Edinburgh, Scotland. BCVA Publications 198 Paré J., Thurmond M.C. & Hietala S.K. (1996) Canadian Journal of Veterinary Research 60, 2, 133. Paré J., Thurmond M.C. & Hietala S.K. (1997) Journal of Parasitology 83, 1, 82 Trees A.J., Guy F., Low J.C., Roberts J., Buxton D. & Dubey J.P. (1994) Veterinary Record 134, 405. BCVA 1997 337

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CATTLE PRACTICE VOL 5 PART 4 A Comparison of Three Treatments for Endometritis Sheldon I. M. Ark Veterinary Group, 5 Mansel Street, Carmarthen, SA31 1QX ABSTRACT Three commercial preparations for the treatment of bovine endometritis were compared: A, intrauterine infusion of 1500mg oxtytetracycline hydrochloride solution; B, intramuscular injection of 500mcg cloprostenol, a synthetic prostaglandin F2α analogue; and, C, intramuscular injection of 3mg Oestradiol Benzoate per 500kg estimated body weight. A total of 300 cases of endometritis were recorded, and the overall success rate of treatment was 68 per cent. Oxytetracycline, prostaglandin and oestradiol were successful in 73 per cent, 67 per cent and 63 per cent of cases, respectively. Treatment of mild cases was more successful than moderate cases (78 per cent vs. 61 per cent, P<0.01), and more successful than severe cases (78 per cent vs. 44 per cent, P < 0.001). Prostaglandin was more successful if the milk progesterone concentration was > 7ng/ml at the time of treatment (P < 0.05). The presence of a smelly discharge at the time of treatment reduced the success rate by 18 per cent (P < 0.02). The treatment-conception interval for all successful treatments of endometritis by prostaglandin was 18.1 days shorter than for oestradiol (68.3 vs. 86.4 days, P < 0.02); and, for oxytetracycline was 16.2 days shorter than oestradiol (70.2 vs. 86.4 days, P < 0.05). KEYWORDS: Bovine Endometritis Treatment Oxytetracycline Prostaglandin Oestradiol INTRODUCTION Endometritis is defined as an inflammation of the endometrium. It is normally characterised by the presence of a mucopurulent vaginal discharge, 21 days or more after calving and associated with delayed uterine involution. If the incidence of endometritis is 10% and there are 2.68 million dairy cows in the U.K. (Anon., 1992), with 1.4 treatments required per cow (Esslemont and Spincer, 1993) there would be potentially 375,200 treatments for endometritis used every year. The selection of treatments for endometritis has been widely and frequently debated; they are based partly on the published literature, and on the individual preference and experience of the various authors. The three treatments most often used are parenteral prostaglandin F2α or analogues, oestrogen and intrauterine infusion of an oxytetracycline solution. Considering the number of cows treated for endometritis annually, there is little published data on the efficacy of the different products available. Indeed, the present author is not aware of any papers comparing the common available treatments for endometritis, used by veterinary surgeons in the U.K. The use of untreated or placebo-treated control cows with endometritis, in veterinary practice, is difficult to justify ethically, because they have reduced cure rates and reduced fertility compared to treated animals. Steffan and others (1984) reported that only 33% of placebo-treated control cows recovered by 60 days post partum, compared with 49% of cows treated with prostaglandin F2α or antibiotic. Furthermore, a 37% cull rate for the untreated controls, compared with 23% and 32% for cows treated for endometritis with prostaglandin F2α or intrauterine antibiotic, respectively. The number of licensed products available for the treatment of endometritis in cattle is limited. Since The Medicines (Restrictions on the Administration of Veterinary Medicinal Products) Regulations 1994 came into force on the 31 December 1994 only licensed products for the treatment of endometritis may be used. A nil milk withhold period is usually demanded in practice, and so only those products that meet this criterion were considered. The aim of this clinical trial was to evaluate the efficacy of three treatments (prostaglandin PGF2α analogue, intrauterine oxytetracycline, and oestrogen) on clinical cases of endometritis diagnosed in veterinary practice, and their subsequent effect on fertility. MATERIALS AND METHODS Cows with clinical cases of endometritis seen by the author over a 12 month period were used in the trial if they had calved 21 days or more and had a mucopurulent vaginal discharge. Each cow's identity, farm, lactation number, milk yield, calving date, severity of last calving (unassisted, assisted, caesarean / foetotomy), and if foetal membranes were retained for more than 24 hours after the last calving, were recorded on a form. The cow was condition scored using the scale 0-5 (Anon., 1982). Animals with concurrent disease were excluded from the trial, as were cows with any prior treatments. BCVA 1997 339

CATTLE PRACTICE VOL 5 PART 4 A thorough examination of the reproductive system was made of each case and the severity of endometritis was scored (see Table 1) in a similar manner to that used by Murray and others (1990). Table 1 Scoring system for the assessment of the severity of endometritis Clinical sign Point score Vaginal discharge Foul smell 3 No smell 0 Bloody 3 >50ml pus 3 <50ml pus 2 White clumps 1 Normal 0 Diameter of largest uterine horn Primiparous Multiparous cow Large >5.5cm >6.0cm 2 Medium 3.5-5.5cm 4.0-6.0cm 1 Normal <3.5cm <4.0cm 0 Diameter of cervix Primiparous Multiparous cow Large >7.0cm >7.5cm 2 Medium 4.5-7.0cm 5.0-7.5cm 1 Normal <4.5cm< 5.0cm 0 Clinical assessment from total score Severe 8-10 Moderate 4-7 Mild 1-3 Normal 0 BCVA 1997 A vaginal examination was made using a clean gloved hand. Cows with vaginal lacerations were excluded from the trial. The vaginal discharge was examined and scored for consistency and smell. The cervix, uterus and ovaries were palpated per rectum. The presence or absence of a corpus luteum was noted. The diameter of the largest uterine horn and the size of the cervix were scored using similar dimensions to Studer and Morrow (1978). The total clinical score represented an evaluation of the severity of endometritis. Cases with total scores of 1-3, 4-7, and 8-10 were classified as mild, moderate and severe grades, respectively. A milk sample was taken from each cow and stored in a 50 ml container with a preservative (Lactab Mark III, Thompson and Capper) at 4o C for analysis of progesterone concentration. Samples were analysed in duplicate using an enzyme-linked immunosorbent assay by a quantitative method at 405nm (Ovucheck, Cambridge Veterinary Sciences Ltd.; intra-assay variation=10%, inter-assay variation=25%, at 5ng/ml). Cows were given one of the following treatments:- A. Intrauterine infusion of 1500mg oxtytetracycline hydrochloride solution, supplied in 15ml disposable syringes with disposable transcervical catheters (Metrijet 1500, Intervet UK Ltd). B. Intramuscular injection of 500mcg cloprostenol, a synthetic prostaglandin F2α analogue, supplied as a clear acqeous solution containing 263mcg/ml of Cloprostenol Sodium BP (Vet), equivalent to 250mcg/ml cloprostenol (Estrumate, Mallinckrodt). C. Intramuscular injection of 3mg Oestradiol Benzoate per 500kg estimated body weight, supplied as a clear oily sterile solution containing 5mg of Oestradiol Benzoate per ml (Oestradiol Benzoate, Intervet UK Ltd). Treatments were allocated in the order: A, B, C. A was used on the first cow to be treated on each farm, B on the second, C on the third, A on the fourth, and so on. Each cow was re-examined 14 days later, and examined and scored as previously described. The achievement of a score of 0 was regarded as success. If the score was 1 or more the cow was treated again as if it were a new case and a new record made, with a note on the record form indicating that this was the second, or third, treatment as appropriate. The choice of treatment given was determined by the treatment 14 days previously: Previous treatment Current treatment A B B C C A Once a cow had been treated successfully, the following details were recorded when they were available: first service date after treatment, pregnancy rate to first service after treatment, the conception date, and if the cow was culled for failure to conceive. The accuracy of the conception date was confirmed by the subsequent calving date in each case. For each treatment, the success rate was calculated and any differences compared using the Chi-square test; values where P < 0.05 were considered 340

CATTLE PRACTICE VOL 5 PART 4 Table 2 The effect of the clinical severity score and the success rate of first and subsequent treatments for cases of endometritis Treatment Clinical severity score of endometritis Severe Moderate Mild All grades A 7/13 (54%) 23/38 (61%) 50/58 a (86%) 80/109 (73%) B 2/9 (22%) 21/35 (60%) 42/53 (79%) 65/97 (67%) C 5/10 (50%) 21/34 (62%) 33/50 b (66%) 59/94 (63%) All treatments 14/32 c (44%) 65/107 d (61%) 125/161 e (78%) 204/300 (68%) a vs. b P < 0.05 c vs. e P < 0.001 d vs. e P < 0.01 statistically significant. In addition, the effect of the three treatments on the treatment-first service interval, pregnancy rate to first service, culling rate and treatment-conception interval were also compared. Any differences were compared using the Student t-test; values where P < 0.05 were considered statistically significant. Variances were compared using the Fisher F test. RESULTS A total of 300 treated cases of endometritis were recorded with complete case records, of which 225 involved first treatments, 67 second treatments and 8 third, or subsequent, treatments. The overall success rate of the aggregate of treatments for all grades of endometritis was 68% (Table 2). Treatments A, B and C were successful in 73%, 67% and 63% of cases, respectively. There was no statistically significant difference in the success rate of the three treatments, except for mild cases of endometritis where treatment A had a greater success rate than treatment C (86% vs. 66%, P < 0.05). The success rate for all first treatments was 68%; treatments A, B and C were successful in 73%, 68% and 62% of cases, respectively. The success rate for all second treatments was 75%; treatments A, B and C were successful in 77%, 76% and 70% of cases, respectively. The difference in the success of each of the three treatments, as measured by the mean change in the overall clinical score, were not significant. BCVA 1997 There were 53%, 36% and 21% cases of mild, moderate and severe cases of endometritis. The severity score had a statistically significant effect on the success rate of the treatments (Figure 1). Treatment of mild cases of endometritis was more successful than moderate cases ( 78% vs. 61%, P < 0.01), and more successful than severe cases (78% vs. 44%, P < 0.001). Where only the first treatments were considered, mild cases were more successfully treated than severe cases (79% vs. 44%, P < 0.001). The presence of a smelly discharge at the time of treatment reduced the success rate of treatment by 18% (54% vs. 72%, P < 0.02, Table 3). Figure 1 The percent success rate of treatement for endometritis and the clinical severity score 0 10 20 30 40 50 60 70 80 90 Mild Moderate Severe Table 3 The effect of the presence or absence of a smelly discharge at the time of treatment on the success rate of treatment Smell No Smell 32/59 (54%) a 173/241 (72%) b a vs. b P < 0.02 Treatment B had a 79% success rate if the milk progesterone concentration was > 7ng/ml at the time of treatment (Table 4), compared with 58% success if the concentration was < 7ng/ml (P < 0.05). It was assumed that a milk progesterone concentration of > 7ng/ml indicated the presence of an active CL. The interval from calving to treatment had no effect on the success rate of treatment, except for treatment B, where the success rate was 23% greater for cows treated more than 42 days post partum (80% vs. 57%, P < 0.05). The success rate of treatment of endometritis when the cow had a history of an assisted calving, or, a history of retained foetal membranes, was 71% and 72%, respectively, compared with 64% for cows with a normal puerperium; the differences were not significant. Condition score at the time of treatment had no effect. Success rates of 71%, 66%, and 54% 341

CATTLE PRACTICE VOL 5 PART 4 were achieved for cows in lactation 1, 2-4, and >4, respectively; the differences were not significant. The treatment-first service intervals for A, B, and C, were 47.0 (SD ± 24.6), 52.3 (SD ± 33.4), and 59.7 (SD ± 35.9) days, respectively; however, the differences were not significant. The treatmentconception interval for all successful treatments of endometritis (Table 5) by product B was 18.1 days shorter than for product C (68.3 vs. 86.4 days, P < 0.02); and, for product A was 16.2 days shorter than C (70.2 vs. 86.4 days, P < 0.05). There was no effect of treatment on the first service pregnancy rate, or culling rate, following successful treatment (Table 6). Table 4 The effect of milk progesterone concentration at the time of treatment on the success rate of treatment Treatment Milk Progesterone Concentration < 7 ng/ml > 7 ng/ml A 45/62 (73%) 29/42 (69%) B 33/57 (58%) a 34/43 (79%) b C 34/56 (61%) 26/40 (65%) All treatments 112/175 (66%) 89/125 (70%) BCVA 1997 a vs. b P < 0.05 Table 5 The effect of three products on the treatment-conception interval for successful treatments Product Mean (days) ±SD SE A 70.2 a 33.3 4.2 B 68.3 b 37.1 5.3 C 86.4 c 38.1 5.5 a vs. c P < 0.05 b vs. c P<0.0 Table 6 The effect of successful treatment on the first service pregnancy rate and the culling rate for failure to conceive Treatment Pregnancy rate (%) Culling rate (%) A 48.7 18.4 B 52.5 15.3 C 39.0 16.9 All treatments 46.9 17.0 DISCUSSION The concept of a clinical score for endometritis was introduced by Murray and others (1990), and the scoring procedure was modified in the current study. Specific dimensions for the diameter of the cervix and largest uterine horn, and a specific volume of pus in the vagina, were used to make the assessment more objective, in agreement with previous investigators (Studer and Morrow, 1978). One assessor was used to score all the cases, at the time of treatment and 2 weeks later, in this trial because Murray and others (1990) found considerable variation between veterinary surgeons. The overall success rate of treatment of endometritis was 68%, which is the same as that found by Murray and others (1990). Oxytetracycline, prostaglandin and oestradiol had clinical success rates of 73%, 68% and 62%, respectively, after first treatment , and 73%, 67%, and 63%, respectively, for all treatments. The differences in success were not statistically significant. The success rate of second treatment was 75% for all cases with little difference between the treatments. Spontaneous cure rates of 33% (Steffan and others, 1984) are considerably less than the treatment cure rates reported here. In previous reports the success rate of prostaglandin F2α, or analogues, ranged from 42% to 92%. There are few trials reporting on the effectiveness of oestrogens or intrauterine oxytetracycline. However, in one trial Olson and others (1986) used oestradiol 17 βcypionate with a success rate of 50-60%, and a number of trials have assessed other intrauterine antibiotics with a success of 49% (Steffan and others, 1984) and 69% (Murray and others, 1990). Clinical success can be influenced by factors other than the treatment. Clinical severity, the smell of the vaginal discharge, and the presence of a CL can be important. Mild cases had a 78% clinical cure rate, compared with 61% for moderate cases (P < 0.01) and 44% for severe cases (P < 0.001). For first treatments only, there was a similar trend. The present study supports previous reports that increased clinical severity of endometritis reduced the success rate of treatment (Murray and others, 1990; Chaffaux and others, 1991). The higher success rate for repeat treatments reported here and elsewhere is probably a reflection of the higher proportion of cases classified as "mild" at the time of the second treatment. The use of a scoring system by veterinary surgeons when treating clinical cases of endometritis in the field would be useful for indicating the probable requirement for a repeat examination and treatment 14 days later. The presence of a smelly vaginal discharge reduced the success rate by 18% (54% vs. 72%, P < 0.02). Smell is probably related to the presence of A. pyogenes and anaerobic bacteria; these organisms have been shown to be synergistic and cause more severe cases of endometritis (Ruder and others, 1981). The current author used a milk progesterone concentrations of > 7ng/ml as indicating the presence of an active CL, and this was associated with greater success rates for prostaglandin (P < 0.05). This value is similar to that used by a number of authors, including Jackson (1977), Coulson (1978) and Murray and others (1990) who also found the presence of an active CL was beneficial to the 342

CATTLE PRACTICE VOL 5 PART 4 outcome of prostaglandin treatment for endometritis, due to the effect of luteolysis and subsequent oestrus. However, not all investigations have confirmed this; Pepper and Dobson (1987) found no effect of high or low milk progesterone at the time of treatment. Where a CL was not found to be important for the outcome of treatment, it was postulated that the prostaglandin had a direct myometrial effect (Young and others, 1984). Following a history of retained foetal membranes or assistance at calving there was a reduced success rate, although the effect was not significant. This trend agrees with Murray and others (1990) who reported that previous retained foetal membranes significantly reduced the success rate; though, in their study, this may have been associated with the high proportion of cows treated less than 21 days post partum. The treatment-conception interval for prostaglandin was 18.1 days shorter than for oestradiol (68.3 vs. 86.4 days, P < 0.02); and, for oxytetracycline was 16.2 days shorter than oestradiol (70.2 vs. 86.4 days, P < 0.05). This agrees with Vujosevic and others (1979) who found a reduced calving-conception interval for cloprostenol treated cows compared with those treated with an intrauterine antiseptic. Similarly, Steffan and others (1984) reported a 24 and 16 day reduction in calving-conception intervals for cows treated with prostaglandin and intrauterine antibiotic, respectively, compared with untreated cows; which was similarly confirmed by Chaffaux and other (1991). There was no significant effect of treatment on the subsequent calving intervals, first service pregnancy rate, or culling rate, following successful treatment. In summary, the current trial found that oxytetracycline, prostaglandin and oestradiol treatments had no statistical difference in the cure rates for severe or moderate endometritis, but oxytetracycline was better than oestradiol for mild cases. Prostaglandin is more successful in the presence of an active CL, or if the calving-treatment interval was 42 days or more. Furthermore, the success rate of treatment is reduced as the severity grade of endometritis increases, or in the presence of a smelly vaginal discharge. Finally, oxytetracycline and prostaglandin are superior to oestradiol, in terms of the treatment-conception interval. On the basis of the cost of an increased calving-conception interval, culling for failure to conceive (Esslemont & Spincer, 1993), and the cost of treatment, oxytetracycline and oestradiol would cost £19.59 and £48.37 more than prostaglandin, respectively, for each case. ACKNOWLEDGEMENTS This paper is based on work carried out in partial fulfilment of the requirements for the examination for the RCVS Diploma in Cattle Health and Production. I would like to thank Professor David Noakes for his advice, guidance, comment and encouragement. The staff of the Royal Veterinary College kindly provided the milk progesterone assay. REFERENCES Anonymous (1982) Ministry of Agriculture, Fisheries and Food Leaflet P612: Condition Scoring of Dairy Cows. ADAS Publications, London. Chaffaux, St., Lakhdissi, H. & Thibier, M. (1991) Etude epidemiologique et clinique des endometrites post-puerperales chez les vaches laitieres. Recueil de Medecine Veterinaire Special Reproduction des Ruminants, 167, 349-358. Coulson, A. (1978) Treatment of metritis in cattle with prostaglandin F2a. Veterinary Record, 103, 359. Esslemont, R.J. & Spincer, I. (1993) Dairy report number 2. The incidence and cost of disease in dairy herds. University of Reading. Jackson, P.S. (1977) Treatment of chronic post partum endometritis in cattle with cloprostenol. Veterinary Record, 101, 441-443. Murray, R.D., Allison, J.D. & Gard, R.P. (1990) Bovine endometritis: Comparative efficacy of alfaprostol and intrauterine therapies, and other factors influencing clinical success. Veterinary Record, 127, 86-90. Olson, J.D., Bretzlaff, K.N., Mortimer, R.G. & Ball, L. (1986) The Metritis-Pyometra Complex. In "Current Therapy in Theriogenology 2" pp 227-236. Ed. D.A. Morrow. W. B. Saunders, Philadelphia. Pepper, R.T. & Dobson, H. (1987) Preliminary results of treatment and endocrinology of chronic endometritis in the dairy cow. Veterinary Record, 120, 53-56. Ruder, C.A., Sasser, R.G., Williams, R.J., Ely, J.K., Bull, R.C. & Butler, J.E. (1981) Uterine infections in the postpartum cow: II Possible synergistic effect of Fusobacter necrophorum and Corynebacterium pyogenes. Theriogenology, 15, 573-580. Sheldon, I.M. (1996) A comparison of three commercial treatments for bovine endometritis that require nil withdrawl of milk for human consumption. Diploma in Cattle Health and Production thesis. Royal College of Veterinary Surgeons, London. Steffan, J., Adriamanga, S. & Thibier, M. (1984) Treatment of metritis with antibiotics or prostaglandin F2α and influence of ovarian cyclicity in dairy cows. American Journal of Veterinary Research, 45, 1090-1094. Studer, E. & Morrow, D.A. (1978) Postpartum Evaluation of Bovine Reproductive Potential: Comparison of Findings from Genital Tract Examination per Rectum, Uterine Culture, and Endometrial Biopsy. Journal of the American Veterinary Medical Association, 172, 489-494. Tennant, B. & Peddicord, R.G. (1968) The influence of delayed uterine involution and endometritis on bovine fertility. Cornell Veterinarian, 58, 185-192. Vujosevic, J. Zeremski, M. & Kuzmanov, D. (1979) Use of a prostaglandin analogue (cloprostenol) and intrauterine infusion therapy in delayed involution in cows. Veterinarski Glasnik, 34, 41-46. Young, I.M., Anderson, D.B. & Plenderleith, R.W.J. (1984) Increased Conception Rate in Dairy Cows after Early Post Partum Administration of Prostaglandin F2 -alpha THAM. Veterinary Record, 115, 429-431. BCVA 1997 343

CATTLE PRACTICE VOL 5 PART 4 Septic Physitis, Arthritis and Osteomyelitis Probably Caused by Salmonella typhimurium DT104 in Beef Suckler Calves Blake N., P.R. Scott and G.A. Munroe., Dept. of Veterinary Clinical Studies, Royal (Dick) School of Veterinary Studies, Easter Bush, Roslin, Midlothian, EH25 9RG, ABSTRACT Salmonella typhimurium DT 104 was isolated as the cause of severe diarrhoea and collapse in two 10 to 14 dayold calves in a herd of 120 autumn-calving suckler cows. One calf died despite intensive therapy. The other calf recovered but presented with 8/10 lameness three weeks later. Radiographic examination revealed septic metaphyseal osteomyelitis and physitis of the distal tibia and tarsal bones including the calcaneus, and septic arthritis of the tibio-tarsal and intertarsal joints. Another calf, aged three weeks, became quadriplegic and was euthanased after four days on the basis of a presumptive diagnosis of cervical vertebral body abscess. Septic physitis was confirmed radiologically in two more calves aged two and three months-old, in the younger calf infection had extended from the distal femoral growth plate into the stifle joint. KEYWORDS: Salmonella typhimurium, Septic Physitis, Septic Arthritis, Cattle, Lameness. INTRODUCTION Septic conditions including polyarthritis, meningitis and omphalophlebitis, caused by high environmental bacterial challenge and facilitated by failure of passive antibody transfer, remain depressingly common in agricultural practice. Advances in veterinary knowledge and introduction of a wide range of antimicrobial drugs has had little effect on the incidence of perinatal diseases caused by bacteria; indeed the perinatal calf mortality rate has not changed since the 1950’s. Of great concern is the current salmonellosis epidemic in the United Kingdom caused by S. typhimurium DT104. This report describes the clinical symptoms and radiological findings in three of four calves with septic physitis, two of which also developed associated septic arthritis which was probably caused by S. typhimurium DT104. MATERIALS AND METHODS Farm: The farm comprised a 200 cow beef suckler herd with 140 cows calving from September to December; the remainder calved from March until June. The cows were predominantly Normandy cross Blue-Grey cows bred to Charolais bulls by artificial insemination after observed oestrus. Cows had been vaccinated against rotavirus and enterotoxigenic E. coli (Rotavec K99; Mallinkrodt) approximately two months prior to calving. The farm often encountered a scour problem caused by cryptosporidiosis in late season calves and baquiloprim-sulphonamide boluses (Zaquilan; Mallinkrodt) were administered within the first few days of life with apparent good success. RESULTS In early December two 10 to 14 day-old calves were presented with lethargy and weakness and a history of profuse diarrhoea of two days’duration which had been treated with baquiloprim-sulphonamide boluses and oral fluids (Effydral; Fort Dodge). The faeces were very fluid with some small milk clots but no blood. Faecal samples were collected into 60 mls sample pots and sent for laboratory examination. A diagnosis of mixed rotavirus and cryptosporidiosis leading to a severe metabolic acidosis was made. The calves had jugular catheters inserted and 3 litres of normal saline containing 600 mEq of added bicarbonate (48 g of sodium bicarbonate) infused over four to six hours. Both calves received amoxycillin-clavulanic acid (Synulox; Pfizer) by intramuscular injection, and flunixin meglumine by intravenous injection (Finadyne; Schering Plough). One calf deteriorated and was re-presented 12 hours later in lateral recumbency with injected scleral vessels, dorsiflexion of the neck, and dorsomedial strabismus. The calf was hyperaesthetic and handling induced opisthotonus. A diagnosis of meningitis was made and the treatment changed to florfenicol (Nuflor; Schering Plough) but the calf died within hours. Salmonella typhimurium DT104 was recovered from the faecal samples of both calves. The remaining calf made an uneventful recovery but was noted to be 6/10 to 8/10 lame three weeks later and was treated with long-acting oxytetracycline (Engemycin 10%; Mycofarm) administered intramuscularly. No improvement in the lameness was noted and the calf was examined by a veterinary surgeon when a diagnosis of septic arthritis of the hock joint was reached. Over the next three months the calf received three separate seven day courses of amoxycillin-clavulanic acid and a 10 day course of enrofloxacin (Baytril 10%; Bayer) but no consistent improvement was noted and the calf remained ill-thriven. A diagnosis of septic arthritis associated with the prior samonellosis was made and a hopeless prognosis was afforded. During late December a four week-old calf presented with sudden onset severe lameness (9/10 lame) and BCVA 1997 345

CATTLE PRACTICE VOL 5 PART 4 was treated by the farmer with procaine penicillin (Depocillin; Mycofarm). No obvious lesion was detected on veterinary examination but pain was present on palpation of the left stifle and the course of penicillin treatment was continued. No improvement was observed after 10 days and further examination revealed marked thickening of the joint capsule with associated heat and pain. The antibiotic treatment was changed to amoxycillin-clavulanic acid with little improvement. During December a three month-old calf was presented with sudden onset lameness of two days’ duration and slight soft tissue swelling over the distal metatarsal/fetlock joint region. The lameness had markedly improved since it had been first noted and the calf was only 2/10 lame. A traumatic injury was suspected but the possibility of an infective condition could not be discounted and a course of amoxycillinclavulanic acid was commenced. The calf’s condition deteriorated and was re-presented three weeks later when 5/10 lame. At this stage there was heat and swelling of the distal metatarsus and a diagnosis of septic physitis was reached. A three week-old calf was presented with quadriparesis of two days’ duration. Examination revealed normal mentation, no cranial nerve signs but upper motor neuron signs of the thoracic and pelvic limbs indicating a cervical spinal lesion. A diagnosis of a cervical vertebral body abscess was made and the calf was destroyed for animal welfare reasons two days later. Radiological findings Radiographic examination was undertaken of the three calves to evaluate potential treatment options including surgery. Calf A; Latero-medial and dorsoplantar radiographs were taken of the right hock region. These revealed marked soft tissue swelling, with extensive mottled areas within the distal tibia, talus, calcaneus and central tarsal bone consistent with advanced osteomyelitis of these bones. In addition there was evidence of periarticular changes in the tibiotarsal and proximal intertarsal joints probably as a result of septic arthritis. Calf B; Latero-medial and cranio-caudal radiographs were taken of the left stifle region. There was marked soft tissue swelling and joint distension of the joints of the stifle. Calf C; Latero-medial and dorsoplantar radiographs were taken of the distal left metatarsus and fetlock joint. There was marked widening of the distal metatarsal physis with areas of lysis surrounded by bone sclerosis, especially in the distal metaphyseal region. These changes were typical of a septic physitis. Calf B; Joint lavage After sacrococcygeal extradural lignocaine injection, lavage of the femorotibial joint was undertaken through two arthrotomy incisions with 2 litres of Hartmann’s solution with 4 mls of Pevidine antiseptic solution added. Previous arthrocentesis had yielded thin brown turbid fluid. DISCUSSION The extent of S. typhimurium DT 104 infection in this beef herd was not established as faecal cultures were not collected from all calves or cows. Diarrhoea/dysentery was not prevalent in the calves, however S. typhimurium DT 104 was recovered from two diarrhoeic calves which became recumbent, one of which subsequently died of septicaemia; the other calf developed septic physitis/arthritis. Septic physitis was confirmed radiologically in two calves aged two and three months-old, in the younger calf infection had extended from the distal femoral growth plate into the stifle joint. Septic osteomyelitis of the distal tibial growth plate and tarsal bones including the calcaneus, and septic arthritis of the tibio-tarsal and intertarsal joint was confirmed in one calf from which S. typhimurium DT 104 had been isolated three months previously. No attempt was made to culture joint fluid samples because of prior antibiotic therapy, and the general poor success rate of such cultures. Cervical vertebral body abscessation is a common manifestation of S. dublin infection and may be the only manifestation of this disease in a herd. The choice of antibiotic treatment for suspected septic physitis was based upon the authors’ previous good results achieved with high dose procaine penicillin therapy (44,000 iu/kg for up to three weeks). Such cases had occurred singly on farms and had tended to affect calves more than four months-old. The poor treatment response in this study could be related to either the delay in presentation of affected calves, inappropriate antibiotic therapy, or the resistance pattern of the S. typhimurium DT104 isolate although this was not determined. In order to commence antibiotic therapy quickly Madigan and House (1996) have recommended that all cases of lameness in neonates less than 45 days should be regarded as septic until proven otherwise. While this recommendation may result in some unnecessary antibiotic treatments, in the current circumstances this would appear to be very sound advice. CONCLUSION The occurrence of cases of septic physitis, vertebral body abscessation, and septicaemia/meningitis in young beef calves was putatively attributed to S. typhimurium DT104. Two calves were destroyed for welfare reasons and the response to prolonged antibiotic therapy was poor in the remaining two calves. REFERENCES Madigan, J.E. & House, J.K. (1996) Infectious lameness. In: Large Animal Internal Medicine. Ed. Smith, B.P. Mosby, Philadephia. 2nd Edition, p 418. BCVA 1997 346

CATTLE PRACTICE VOL 5 PART 4 Sporadic Milk Drop in Dairy Cows R.F. Gunning Veterinary Laboratories Agency, Veterinary Investigation Centre, Langford, Bristol. INTRODUCTION The potential causes of milk drop are legion; indeed there can be few disorders of the lactating cow in which some loss of milk is not a clinical feature. However in many cases careful clinical examination, with or without laboratory back-up, will elucidate the underlying cause. Sudden outbreaks of epidemic agalactia are not uncommon and often follow the introduction to an immunologically-naive herd of an infectious agent. Infectious bovine rhinotracheitis virus (IBRV)and Leptospira hardjo are notable examples, and bovine respiratory syncytial virus (BRSV) has recently been added to the list of infectious agents causing epidemic milk drop (Pritchard & Fishwick, 1997). Andrews (1997) has recently drawn attention to the potential influence of total mixed rations on the alimentary tract of cattle, and it is quite possible that epidemic milk drop might ensue from errors in feed formulation or the incorporation of an inappropriate ingredient into the total ration. With increasing frequency Veterinary Investigation Centres receive reports from cattle practitioners of a sporadic milk drop syndrome in dairy herds. Typically only one or two cows are affected at any one time but, over a season, the accumulated number of cases may be quite considerable and this is the cause for concern. In several herds no satisfactory explanation for this phenomenon has been found and all attempts at identifying an infectious or metabolic cause have failed. THE CLINICAL PICTURE By definition, sporadic milk drop occurs sporadically but in the course of a year, or even during one winter, as many as 20% of a herd may be affected. There is no precise clinical definition but in individual cases the classic sign is sudden onset agalactia. Some cows have total milk loss but partial loss is more usual. It occurs at any stage of lactation but is perhaps more common during the first three months. Accompanying signs are variable but most cases are dull and inappetent, and some weight loss may be observed. In some affected herds cows are nonpyrexic, while in others high fevers are usual; but typically a mild fever (39.0-39.5°C) is the norm. Increased respiratory rate and mucopurulent nasal discharge are sometimes observed but these are inconsistent signs. Neutrophilia and a raised serum haptoglobin value have been detected in some of the blood samples examined. Recovery within seven to 10 days, with or without treatment, is usual although relapses are a significant feature in some herds. There is no age predilection. Sporadic milk drop syndrome is typically a problem of the winter months, but may occur in the summer. It may occur in successive years on the same farm. There are some marked differences between herds in the clinical histories - for example, pyrexia versus no pyrexia, respiratory signs versus no respiratory signs, recurrent cases versus no recurrence - and it therefore seems highly unlikely that the sporadic milk drop syndrome is a single entity. However, within any one affected herd the majority of cases do seem to have a fairly consistent clinical presentation and may have a common aetiology. DIFFERENTIAL DIAGNOSIS Individual cases may show seroconversion to one or more infectious agents, such as IBRV, BRSV, parainfluenzavirus-3, bovine virus diarrhoea virus and L. hardjo, but in the herds under consideration affected cows do not exhibit a consistent serological response, and therefore no single agent can be incriminated. Colleagues within the Veterinary Laboratories Agency have identified Chlamydia psittaci as a cause of bovine abortion in the UK (Holliman and others, 1994) but the role of chlamydia in other cattle disease syndromes, including milk drop, remains unresolved. Davies and Hartley (1997) reported recently an apparent outbreak of traumatic reticulitis in a dairy herd in which the presenting signs were milk drop and vague malaise. The source of the wire was disintegrating car tyres on the top of a silage clamp and this is now recognised as a significant threat to cattle health on many farms. Unfortunately an unequivocal diagnosis of traumatic reticulitis is not possible unless the animal dies or surgery is performed. In some cases inappetence, and therefore milk drop, may be due to indigestion, and there is a suggestion that there is a nutritional component to the problem. In most affected herds cattle are fed maize silage, usually as part of a total mixed ration, and the possibility of transient acidosis has been raised. The use of computerised milk recording on some farms may have revealed cases that might otherwise have gone undetected. THE FUTURE Together with colleagues in practice, the author will take a closer look at some herds in which sporadic milk drop is an on-going problem. The first objective is to define more precisely the clinical picture and patterns of disease in these herds. BCVA 1997 347

CATTLE PRACTICE VOL 5 PART 4 Further attempts will be made to establish the cause or causes of the syndrome. REFERENCES Andrews, A. H. (1997) Cattle Practice 5, (3), 175 Davies, I. H. & Hartley, C. R. (1997) Veterinary Record 141, 54 Holliman, A., Daniel, R.G., Parr, J.G., Griffiths, P.C., Bevan, B.J., Martin, T.C., Hewinson, R.G., Dawson, M. & Munro, R. (1994) Veterinary Record 134, 500 Pritchard, G.C. & Fishwick, J. (1997) Veterinary Record 141, 13 BCVA 1997 348

CATTLE PRACTICE VOL 5 PART 4 Early Pregnancy in the Cow Mann G E., Cattle Fertility Research Group, University of Nottingham,Sutton Bonington, Loughborough, LE12 5RD INTRODUCTION Failure of early pregnancy represents one of the major constraints to profitability in the UK dairy industry. In successfully mated cows as many as 95% of eggs may be fertilised but approximately 30% of the embryos formed will be lost by day 25 following mating. In economic terms this early embryo loss costs over £200 million pa. Much of this loss results from a failure in the mechanisms responsible for the establishment of pregnancy. In the cow this establishment of pregnancy depends on the effective functioning of an endocrine communication system between the mother and the embryo. This system underpins the "decision", by the cow, to either maintain the corpus luteum and thus the pregnancy or to undergo luteolysis and reovulate, generating a new opportunity to become pregnant. Understanding the mechanisms underlying this process is of paramount importance in generating remedial therapies with which to combat the major economic problem of early pregnancy failure. THE OESTRUS CYCLE AND EARLY PREGNANCY In the cow, ovulation is followed by the formation of a corpus luteum which secretes progesterone during the ensuing 18 day luteal phase. At the end of the luteal phase, a mechanism known as luteolysis causes the corpus luteum to regress and progesterone production to decline. This fall in progesterone allows the development of a new ovulatory follicle during the 3 day follicular phase. This follicle produces large quantities of the hormone oestradiol which first induces behavioural oestrus and then the LH surge responsible for ovulation. In cyclic cows luteolysis is caused by the release of large luteolytic episodes of prostaglandin F2∝ by the uterus. These luteolytic releases of prostaglandin F2∝ are produced in response to the binding of ovarian oxytocin to newly developed oxytocin receptors on the uterine endometrium (for review see Wathes & Lamming, 1995). In the pregnant cow the continued development of the embryo requires the continued secretion of progesterone and so luteolysis must be prevented and corpus luteum progesterone secretion maintained. The embryo achieves this though the secretion of a protein, trophoblast interferon, which acts on the uterine endometrium to inhibit the development of oxytocin receptors (for review see Flint et al., 1992). With no receptors in the target tissue, oxytocin is unable to induce luteolytic episodes of prostaglandin F2∝ release and so luteolysis is prevented. Thus, the success of early pregnancy in the cow depends on a fine balance between the development of the mechanism to destroy the corpus luteum in the cow, and the production of the embryonic signal to prevent this. The two potential outcomes of early pregnancy are illustrated in Figure 1 Figure 1 Mean milk progesterone concentrations in inseminated cows in which pregnancy was successfully established ( ; n=28) or in which early pregnancy failed ( ; n=24). Note the different levels of progesterone between days 12 - 18. (data from Mann et al., 1994) 0 5 10 15 20 25 0 3 6 9 12 15 18 21 24 27 30 Days from insemination Milk Progesterone (ng/ml) where the luteolytic fall in progesterone is clearly prevented in those cows in which insemination was successful. THE CONTROL OF EARLY PREGNANCY A strong or early signal from the mother for the corpus luteum to regress or a weak or delayed signal from the embryo to prevent the loss of the corpus luteum will result in the failure of the pregnancy. Both the mechanism of luteolysis in the mother and the production of the antiluteolytic signal by the embryo are controlled by the maternal hormone environment, and in particular ovarian progesterone and oestradiol production. The Role of Progesterone In mated animals that subsequently undergo luteolysis it is now well established that both plasma and milk progesterone concentrations are lower during the second half of the luteal phase than in those cows which become pregnant (Lukesewska & Hansel, 1980, Lamming et al., 1989; Mann et al., 1995). These studies demonstrate that progesterone BCVA 1997 349

CATTLE PRACTICE VOL 5 PART 4 is a vital factor associated with the success or failure of pregnancy, but do not determined whether differences in progesterone are the actual cause of early pregnancy failure or merely an indirect effect of other factors leading to the failure of pregnancy. We have previously found that a low level of progesterone during the luteal phase results in the development of a strong luteolytic signal (Mann & Lamming, 1995a). A strong luteolytic signal reduces the chance of an embryo being able to inhibit luteolysis, and thus we have demonstrated a direct adverse effect of a low progesterone level on the potential outcome of early pregnancy. More recently we have demonstrated that the pattern and level of progesterone during early pregnancy has a marked effect on both the development of the embryo and the inhibition of the luteolytic mechanism (Mann et al., 1996). We found that in cows with poorly developed embryos, progesterone showed both a late post ovulatory rise and a low final luteal phase level compared to cows with well developed embryos (Figure 2). In this study, well developed embryos were found to be producing significant quantities of the antiluteolytic trophoblast interferon signal while poorly developed embryos were not. In the cows with well developed embryos, producing a strong interferon signal, luteolytic prostaglandin F2∝ release was fully inhibited while in cows with poorly developed embryos large releases of luteolytic prostaglandin F2∝ were present. Figure 2 Post ovulatory progesterone patterns leading to good embryo development and successful inhibition of luteolysis ( ; n=5) or poor embryo development and failed inhibition of luteolysis ( ; n=5). 0 1 2 3 4 5 6 7 8 9 10 0 2 4 6 8 10 12 14 16 Day of cycle Plasma Prog. (ng/ml) In summary, it appears that both a late post ovulatory progesterone rise, and low luteal phase progesterone can cause early pregnancy failure through mechanisms including both increased strength of the luteolytic signal and reduced embryo development and inadequate production of the antiluteolytic interferon signal. The Role of Oestradiol As well as the level of progesterone, the level of oestradiol is also involved in the control of the luteolytic mechanism in the cow, lower levels of oestradiol resulting in a weaker luteolytic signal (Mann & Lamming, 1995b). Furthermore, if oestradiol levels are sufficiently inhibited during the luteal phase, luteolysis can be delayed (Villa-Godoy et al, 1885). It would seem likely, therefore, that as well as lower levels of progesterone, higher levels of oestradiol might also be associated with early embryo loss. However, during later stages of the luteal phase, when the luteolytic mechanism is developing, plasma oestradiol concentration do not appear to differ between those cows in which early pregnancy is successful and those in which pregnancy fails. In a recent study on early embryo development we did observe major differences in oestradiol levels, early in the luteal phase, between inseminated cows with and without an embryo (Figure 3). Figure 3 Post ovulatory plasma oestradiol concentrations in inseminated cows with ( ; n=15) or without ( ; n=5) an embryo present on day 16. 0 1 2 3 4 5 6 0 2 4 6 8 10 12 14 16 Day of cycle Plasma Oestradiol (pg/ml) In this study we found that in mated cows subsequently found to possess no embryo on day 16, oestradiol was elevated on days 1 and 2 of the cycle, indicating that ovulation had been delayed in these animals. Despite an apparent lack of any major differences in oestradiol levels during the luteal phase between cows in which pregnancy is successfully established and cows in which pregnancy fails, manipulation of BCVA 1997 350

CATTLE PRACTICE VOL 5 PART 4 oestradiol concentrations has proved successful in improving pregnancy rates. Treatment of cows on day 12 post insemination with Receptal, a preparation of the gonadotrophin releasing hormone analogue buserelin, has been shown to give a 12% improvement in calving rate (Peters et al., 1992). We have demonstrated that this effect of Receptal is mediated through a fall in plasma oestradiol concentration (Figure 4) (Mann et al 1995), the fall in oestradiol leading to a reduction in luteolytic prostaglandin F2∝ release (Mann & Picton, 1995). Figure 4 Plasma oestradiol concentrations in cows (n=5) injected during consecutive oestrous cycles (arrow) with either 2.5ml Receptal ( ) or control saline ( ). Note the lower oestradiol levels in the Receptal treated cycle on days 13 - 15. (From Mann & Lamming 1995c) 0 0.5 1 1.5 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Day of cycle Plasma Oestradiol (pg/ml) Receptal APPROACHES TO REDUCING EARLY EMBRYO LOSS In view of the critical role of the interaction between the development of the luteolytic mechanism and the production of an antiluteolytic signal by the embryo in the establishment of pregnancy, 3 possible approaches could be taken to reduce early embryo loss: (i) reduce the strength of the mechanism causing the corpus luteum to regress (ii) strengthen the embryonic signal (interferon) protecting the corpus luteum (iii) improve the synchrony between the embryo and the uterus As the ovarian hormones play a key role in the control of these processes, manipulation of ovarian hormone levels, either indirectly through the use of products like Receptal, or directly through, for example, progesterone supplementation may represent the most appropriate approach to improving fertility that is currently available. However, it is important that short term reliance on such therapies does not deflect from the need for a longer term approach to improving the underlying level of national dairy cow fertility. ACKNOWLEDGEMENTS The financial support of MAFF, MMB, MDC and Hoechst in the execution of many of the studies referred to in this paper is gratefully acknowledged. REFERENCES Flint A.P.F., Stewart H.J., Lamming G.E. & Payne J.H. (1992). Role of the oxytocin receptor in the choice between cyclicity and gestation in ruminants. Journal of Reproduction & Fertility Supplement 45, 53-58. Lamming G.E., Darwash A.O. & Back H.L. (1989). Corpus luteum function in dairy cows and embryo mortality. Journal of Reproduction & Fertility Supplement 37, 245-252. Lukesewska J. & Hansel W. (1980). Corpus luteum maintenance during early pregnancy in the cow. Journal of Reproduction & Fertility 59, 485-493. Mann G E. & Lamming G.E. (1995a). Progesterone inhibition of the development of the luteolytic signal in the cow. Journal of Reproduction & Fertility 104, 1-5. Mann G.E. & Lamming,G.E. (1995b). Effect of the level of oestradiol on oxytocin-induced prostaglandin Ffifi release in the cow. Journal of Endocrinology 145, 175-180. Mann G.E. & Lamming G.E. (1995c). Effects of treatment with buserelin on plasma concentrations of oestradiol and progesterone and cycle length in the cow. British Veterinary Journal, 151, 27- 432. Mann G.E. & Picton H.M. (1995). The effects on the ovary and uterus of treating cows with Buserelin on day 12 of the oestrous cycle. Journal of Reproduction & Fertility Abstract Series No. 15; abstract 61. Mann G.E., Lamming G.E. & Fray M.D. (1995). Plasma oestradiol and progesterone during early pregnancy in the cow and the effects of treatment with buserelin. Animal Reproduction Science 37, 121-131. Mann G.E., Mann S.J. & Lamming G.E. (1996). The interrelationship between the maternal hormone environment and the embryo during the early stages of pregnancy. Journal of Reproduction & Fertility Abstract Series No. 17; abstract 55. Peters A.R., Drew S.B., Mann G.E., Lamming G.E. & Beck N.F.G. (1992). Experimental and practical approaches to the establishment and maintenance of pregnancy. Journal of Physiology & Pharmacology 43 (Supplement 1), 143-152. Villa-Godoy A., Ireland J.J., Wortman J.A., Ames N.K., Hughes T.L. & Fogwell R.L. (1985). Effect of ovarian follicles on luteal regression in heifers. Journal of Animal Science 60, 519-52. Wathes D.C. and Lamming G.E. (1995) The oxytocin receptor, luteolysis and the maintenance of pregnancy Journal of Reproduction & Fertility Supplement 49 53-67. BCVA 1997 351

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CATTLE PRACTICE VOL 5 PART 4 Strategic Milk Progesterone Monitoring Darwash A O., Lamming G E, Cattle Fertility Research Group, University of Nottingham INTRODUCTION The two main ovarian hormones oestradiol (E2) and progesterone (P4), are present in measurable quantities in milk. Progesterone, the dominant hormone during the oestrous cycle and early pregnancy can be easily quantified using radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). This has made it possible to monitor, using non-invasive techniques, patterns of ovarian activity, to determine the nature of hormone differences which lead to sub-fertility, to assess the success or failure to a particular service and to appraise the efficacy of hormonal treatment. Strategic milk progesterone monitoring may best be defined as a milk sampling protocol for P4 determination aimed at characterizing spontaneous or induced events during a specific stage of the reproductive cycle in postpartum cows. Supported by AFRC and MMB, we initiated the use of the progesterone assay in monitoring postpartum reproductive activity in cows during 1975-1982. Based on the analysis of results obtained from these studies and due to the constant replacement of British Friesian with Holstein-Friesian cows the, MDC and MAFF are currently financing a 3-year research project to investigate short- and long-term measures to identify and tackle the serious problem of subfertility in dairy herds. The objective of this paper is to present preliminary results of field trials testing the efficacy of strategic milk progesterone monitoring as an aid to fertility management. Due to costs and time involved in determining P4 concentrations, a minimum number of samples needs to be defined for a meaningful analysis of the cow’s reproductive state. These are presented below in ascending order. 1. To determine the appropriate time for AI Number of samples needed : 2 The precision of oestrus detection is essential to ensure the success of AI. In order to confirm whether AI has actually occurred at the appropriate time in relation to oestrus and ovulation two samples are needed: one on Day 0 and the other on Day 5 post AI. Fig.1 shows the variation between animals in P4 levels on Days 0 and 5 after AI in five herds currently been monitored. From Fig.1(A), we can deduce that approximately 11% of animals had atypically high progesterone levels (> 2 ng/ml); indicating late insemination in relation to oestrus and ovulation. Furthermore, Fig. 1(B) shows that 19.5% and 12.8% of animals had atypically low (< 3ng/ml) and atypically high (>10 ng/ml) P4 values respectively, on Day 5 post service, which indicates either the occurrence of AI at an inappropriate time, delayed ovulation or the formation of an incompetent corpus luteum. A computer programme is being developed to devise verifiable rules for the classification of the correct time of insemination and determine the reliability of Day 24 sample in confirming pregnancy or whether particular cows which are not likely to be pregnant should be observed for oestrus 18-20 days after AI. 2. To determine the suitability of a host to embryo transfer (ET). Number of samples needed: 2-3 ( on days 3-7 after oestrus ) Transferring embryos on day 7 in synchronized or naturally cycling animals may be a costly business if the host has not ovulated and the uterus is not subjected to sufficient P4 level essential for embryo survival. At least two samples showing P4 > 3 ng/ml between days 3-7 are needed to confirm the occurrence of ovulation and the competence of the corpus luteum (CL). As it was indicated in the previous section, the critical magnitude of P4 essential for successful pregnancy needs further investigation and verification by actual calving. Fig BCVA 1997 353

CATTLE PRACTICE VOL 5 PART 4 2. shows a large variation between animals in P4 levels after oestrus. BCVA 1997 3. To assess the efficacy of hormone treatment: Number of samples: 3 during the first week following treatment If veterinary treatment is the option selected following the diagnosis of subfertility, then a threesample protocol during the first week after treatment may reveal whether it was successful in terminating atypical ovarian patterns and in inducing oestrus or ovulation, or whether an additional intervention is required (See Fig. 3) 4. To characterize ovarian activity prior to AI : Number of samples needed: 6-9 on days 30-55 postpartum This protocol was devised to cover at least one oestrous cycle using three times a week sampling regime. Regardless whether ovulation has previously occurred or not, P4 levels lower than 3 ng/ml for longer than 12 days, detected in 15.4% of animals in the current survey, were a result of delayed ovulation. The occurrence of irregular oestrus behaviour during a prolonged absence of luteal activity indicates the presence of a follicular cyst. The estimated time of ovulation and heat in the rest (82.4%) of animals identified as having typical cycles were regularly made known to the respective farm managers (Fig. 4). Another 2.2% were shown to have prolonged luteal activity (P4 > 3ng/ml, for > 19 days, this being appropriate for prostaglandin treatment to induce luteolysis). Based on P4 identification of atypical hormonal patterns the field veterinary surgeon is informed so that he has access to the hormone profiles in making a decision about treatment. 5. To quantify the incidence of silent ovulation Number of samples needed: 18-27 Milk progesterone monitoring offers an objective and accurate means of assessing the extent of silent ovulation in postpartum cows. This is essential if the effects of yield, stress, environmental temperature, negative energy balance or sire influence (genetics) on the ability of cows to exhibit visible symptoms of oestrus is to be evaluated. Aside from the assumed variation between animals in the sensitivity of the brain to endogenous oestradiol levels, we do not know of any work on how to intensify symptoms of oestrus. A number of workers have implicated P4 levels during the previous cycle as a cause of silent ovulation, but this is not confirmed in animals providing profiles as shown in Fig.5. 354

CATTLE PRACTICE VOL 5 PART 4 This cow was showing a regular cyclic pattern with typical P4 levels known in other cows to create visible signs of oestrus. Such a profile indicates the need for careful scrutiny for oestrus following the decline in P4 levels and thereafter, to investigate appropriate veterinary intervention. From the work presented in last year’s symposium, it seems that there is a genetic component to the variation found between animals in their capacity to show oestrus. Furthermore, the next paper on the genetic and environmental factors associated with subfertility may be viewed carefully. SUMMARY We have tested the efficacy of progesterone monitoring in characterizing reproductive parameters essential for a meaningful approach to high reproductive performance. There is a clear need to develop the necessary technology to enable farm management to utilize the information generated by the these monitoring protocols. We feel that on-line milk progesterone monitoring with relevant computer software programmes will greatly assist the fertility management of dairy herds. Furthermore, the timely availability of progesterone profiles to the herd veterinary surgeon will assist in identifying cows requiring remedial treatments as well as information to judge its efficacy. Information is available to enable scientists to specify the hormone profiles required pre- and post-insemination to ensure high fertility. Measurement of the relevant hormone patterns provides the only objective and non-invasive method currently available to assess the reproductive status of the dairy cow. ACKNOWLEDGEMENT The authors wish to thank all co-operating farms for their help in sample collection and the supply of information on their herds, to MDC and MAFF for the financial support, to Malinkrodt, Inter-Ag and AKZO-NOBEL for the donation of ESTRUMATE, CIDRs, and Fertagyl, respectively. BCVA 1997 355

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CATTLE PRACTICE VOL 5 PART 4 Subfertility in Dairy Cattle: Potential Genetic and Environmental Influences. Lamming G E1 , Darwash A O1 and Woolliams J A2 1 Cattle Fertility Research Group, University of Nottingham 2 Roslin Research Institute, Midlothian, UK INTRODUCTION For a dairy cow to conceive and maintain pregnancy it is imperative that synchrony is achieved between a number of physiological and managerial processes. These currently include visible manifestation of oestrus, a timely natural mating or alternatively artificial insemination by skilled technician using semen of fertile bulls, shedding of an ovum capable of being fertilised and the secretion of adequate progesterone levels essential for optimum tubal and uterine environments to maintain the developing embryo. There is convincing evidence that in UK herds these ideals are not been achieved causing a serious decline in the fertility status of dairy cows. In the future we must use both short-and long-term measures to improve their fertility, the former by employing new technology, the later by promoting the application of new genetic techniques. The ideal fertility model is for the animal to conceive as a heifer at 15 months of age and to achieve a target calving pattern for at least 5 lactations. Alas, this is not attainable for the majority of cows causing a considerable loss to dairy farmer and the industry. Furthermore, there is a large variation between cows in their fertility level, which constitutes an enigma for farmers, veterinary practitioners, AI technicians and scientists. In our field studies covering 1800 cows during 2500 lactation’s, we found wide variation between cows, the most fertile animal had a 90% conception to first service for 10 lactation’s while the least was a cow that required 10 services in order to conceive. From our analysis about one third of all cows in UK herds show various types of subfertility; with a large proportion of these problems directly attributable to defective endocrine patterns. On a national level, as it is shown in Fig.1 for US herds in New York State (Butler, 1997), the significant increase in the average annual milk yield for Holstein-Friesian cows during the last 35 years has been accompanied by a consistent downward trend in average conception rate (CR) to a particular service, estimated at approximately 1% decrease every 2 years. Although no comparable extensive UK data are available, the trend is expected to be similar (we estimate about 1% decline per 3 years). This is of serious concern. Furthermore, during the last 25 years there has been a significant replacement of Friesian cows with Holstein-Friesian blood and this may have increased the rate of decline in herd fertility. The aim of our studies is to analyse the causes of subfertility in cattle and to suggest a long-term approach to achieve improved reproductive efficiency based on the results of our field studies. THE RATE OF GENETIC PROGRESS FOR SELECTED TRAITS During the last five decades, animal breeders and geneticists have successfully used genetic techniques to improve the milk production efficiency of the dairy cow. In order to accomplish this and particularly now, a fast genetic gain is pursued by single trait selection for yield. This was assisted mainly by the respectable level of heritability for milk yield which is estimated at 25% (Bourdon, 1997) to 35% (Swalve, 1995). Since the conventional reproductive traits examined (interval to first service, calving intervals and total conception) characteristically have low heritability of 0 to 10% (Hays et al., 1992; Campos et al. 1994; Marti and Funk 1994), there has been no selection for fertility. Cows with low milk production (even though fertile) are usually culled, while subfertile high yielding animals are given every opportunity to become pregnant to stay in the herd. Over the years, this practice must have caused an increase in the frequency of undesirable genes controlling endocrine mechanisms associated with subfertility. We believe this philosophy of failure to select for fertility traits needs to be examined and measures employed to reverse this serious trend to lower fertility. In wild bovidae, natural selection operates freely and there is a natural discrimination against subfertility while in domesticated cattle the current husbandry practice certainly does not select for fertility and may indeed BCVA 1997 357

CATTLE PRACTICE VOL 5 PART 4 select for decreased reproductive performance. Our estimate of the impact from insemination data indicates that currently about 40% of inseminations are not followed by calving and that only 58% of the UK cattle become pregnant after two services, out of which about 8% are usually lost due to late embryo mortality leaving,50% actually producing live calves. EFFECTS OF ENVIRONMENTAL FACTORS ON FERTILITY The literature is rich with references detailing the effects of the management, season, yield and nutrition on fertility in the dairy cows. Subfertility can be easily induced if the animal is wrongly diagnosed in heat and inseminated at an inappropriate time (Table 1) or by unskilled AI technicians using semen of subfertile bulls. Table 1: Recorded success or failure to 3657 inseminations in 2400 postpartum cows Parameter No. % Fertile (lst. service) 1334 36.5 Fertile (subsequent services) 810 22.1 Service during luteal phase 66 1.8 Service during pregnancy 50 1.4 Late embryo mortality 244 6.7 Wasted inseminations: assumed fertilisation failure (5%) 1153 31.5 Total 3657 100 BCVA 1997 High ambient temperatures are known to reduce fertility (Thatcher et al., 1978). However, the reports on the effects of yield and energy balance on fertility have been equivocal due to vagueness in the definition of fertility and in the inherent capacities of some animals to remain in positive energy balance. A number of studies have shown a negative correlation between production and reproduction (Hansen et al., 1983, Faust et al., 1988 and Oltenacu et al., 1991). Contrary to these findings, no effects of high milk production on the reproductive efficiency (Nieuwhof et al., 1989) or ovarian function (Harrison et al., 1990) were observed in postpartum cows, while, Hermas et al., 1987, Seykora and McDaniel 1983, and Philipson 1981, went further to state that a substantial additive genetic variance does exist and that profitable breeding programmes should include selection for reproductive traits. Since we assume that dairy cows are fed sufficient maintenance and production rations as per NCR standards, the variation between animals in their fertility levels may be attributed to the large variation between them in their capacity to ingest feedstuff, and or their inherent endogenous hormonal levels and the corresponding receptors concentrations in target organs. We anticipate Prof. Webb’s paper may clarify this relationship. Heritable endocrine mechanisms controlling reproductive events A number of reproductive processes have been shown to be heritable. These include the oestrogen receptor concentrations in the porcine uterus (Rothchild 1996), ovulation rate in the Merinos (Piper et al., 1988) and the response to GnRH in beef cattle (McKinnon et al., 1993). Other reproductive disorders e.g. cystic ovarian disease and the incidence of silent heat (ovulation without oestrus) were also shown to be heritable (Johansson, 1961). The reason for low heritability estimates of fertility parameters reported in the past could be due to the measurement of reproductive traits (interval to first postpartum service and conception rate to first service) that are highly influenced by the management. Incorporating reproductive traits in the selection index of animals Three main requirements are essential for a trait to be incorporated into a selection index: a) It must be heritable b) have an economic value and c) its relationship with other index parameters is known. At the University of Nottingham, a thorough analysis of milk progesterone database has identified the return to postpartum cyclicity as a heritable trait (h2 =0.21, Darwash et al., 1997a ). This trait was also found to be favourably associated with a number of fertility parameters (see fig. 2) and not unfavourably associated with yield. (Darwash et al., 1997b ). Our ability to measure accurately the inherent hormonal patterns had helped to produce objective estimates of heritability of a number of reproductive processes which are based on the endocrine patterns of the cow and not based on the herdsman’s subjective observations. Currently an additional number of reproductive parameters influenced by endocrine mechanisms, 358

CATTLE PRACTICE VOL 5 PART 4 e.g. silent heat (as Fig 5 Darwash this conference), corpus luteum competence, delayed ovulation and persistent corpora lutea are being monitored in a structured database from an identified reference population. BCVA 1997 Milk samples are being collected thrice weekly for progesterone determination from approximately 1000 animals of 60 sires families. Hormone monitoring increases the precision in defining and quantifying traits for the heritability estimates. Furthermore, in co-operation with Roslin Research Institute, we are implementing a research protocol for estimating the heritability of specific endogenous patterns in heifers potentially to be incorporated into testing procedures for dairy bulls. The association between a favourable pattern and future reproductive performance may assist in selecting fertile animals at an early stage. From selected animals under study, blood, semen and milk samples are being collected and stored for micro satellite analysis of QTL associated with favourable traits. SUMMARY An extensive programme of monitoring milk progesterone concentrations in dairy cows has permitted definition of the postpartum hormone profiles associated with normal fertility as well as patterns causing subfertility. In the short-term, the use of this data will permit an accurate identification of animals requiring remedial veterinary treatments and assess the response to such intervention in terms of inducing normal cycling patterns and improved conception rate. However, a long-term solution to correct the apparent declining level of fertility in UK dairy cows will require the identification of heritable traits associated with high fertility and the application of this knowledge to the development of a new multi trait selection procedure to ensure that heritable endocrine factors associated with improved reproductive performance of cows can be identified and used in a national cattle breeding programme. ACKNOWLEDGEMENTS This work is supported by a MAFF/MDC Linked Grant, ‘Subfertility in Dairy Cattle: Causes and Strategies for Remedial Treatment’. REFERENCES Bourdon R.M. 1977. Understanding animal breeding. Prentice Hall, N.J. pp 523. Butler W.R. and Smith R.D. 1989. Interrelationships between energy balance and postpartum reproductive function in dairy cattle. J. Dairy Sci. 72:767-783. Butler R.W. Cherney D.J.R. and Elrod C.C. 1995. Milk urea nitrogen (MUN) analysis: field trial results on conception rates and dietary inputs. Proceedings of the Cornell Nutrition Conference, Rochester, NY p89-95. Butler R W. 1997 Personal communication Campos M.S. Wilcox C. J. Becerril C.M. and Diz A. 1994. Genetic parameters for yield and reproductive traits of Holstein and Jersey cattle in Florida. J. Dairy Sci. 77:867. Darwash A.O. Lamming G.E. and Woolliams A.J. 1997a . Estimation of genetic variation in the interval from calving to postpartum ovulation of dairy cows. J. Dairy Sci. 80:1227-1234. Darwash A.O. Lamming G.E. and Woolliams A.J. 1997b . The phenotypic association between the interval to postpartum ovulation and traditional measures of fertility. Animal Science 65:9 - 16. Faust M.A. McDaniel B.T. Robinson O.W. and Britt J.W.. 1988. Environmental and yield effects on reproduction in primiparous Holsteins. J. Dairy Sci. 71: 3092. Foote R.H. 1996. A review: Dairy cattle reproductive physiology and management-past progress and future prospects. J. Dairy Sci. 79:980-990. Hayes J.F.R. Cue R.I. and Monardes H.G. 1992. Estimates of repeatability of reproductive measures in Canadian Holsteins. J. Dairy Sci. 75:1701. Hansen L.B. Freeman A.E. and Berger P.J. 1983. Yield and fertility relationships in dairy cattle. J. Dairy Sci. 66:293. Harrison R.O. Ford S.P.,Young J.W. Conely A.J. and Freeman A.E. 1990. Increased production versus reproduction and energy balance in high producing dairy cows. J. Dairy Sci. 62:1922. Johansson I. 1961. Genetic aspects of dairy cattle breeding. University of Illinois Press, pp 259. Marti C.F. and D.A. Funk. 1994. Relationship between production and days open at different levels of herd production. J. Dairy Sci. 77:1682. Mckinnon M.J., Norbert N.J., Meyer K., Burrow H.M., Bryan R.R. and Hetzel D.J.S. 1991. Genetic parameters for testosterone response to GnRH stimulation and scrotal circumference in tropical beef bulls. Livest. Prod. Sci. 29:297. Nieuwhof G.J., Powel R.L and Norman H.D.. 1989. Ages at calving and calving interval in cattle in the United States. J. Dairy Sci. 72:685 Oltenacu P.A., Frick A. and Lindhe B.. 1991. Relationship of fertility to milk yield in Swedish cattle. J. Dairy Sci. 74:264. Philipson J. 1981. Genetic aspects of female fertility in dairy cattle. Livestock Prod. Sci. 8:307. Piper L.R., Bindon B.M., Davis G.H. and Elsen G.M.. 1988. Control of litter size: major genes and industry utilisation of the Boorola F. Gene. Proc. 3rd. World congr. Sheep and Beef Cattle Breeding. Vol. 2, PP 589-609. (Paris, France). Rothschild M.F., Jacobson C., Vaske D.A., Tuggle C., Wang L., Short T., Ekhardt G., Saski S., Vincent A., McClaren D.G., Sowthwood O., Van der Steen H. Mileham A. and Plastow G. 1966. The estrogen receptor locus is associated with a major gene influencing litter size in pigs. Proc. Natl. Acad. Sci., USA 93:201- 205 Seykora A.J. and B.T. McDaniel B.T. 1983. Heritability and correlations of lactation yield and fertility for Holsteins. J. Dairy Sci. 66:1486. 359

CATTLE PRACTICE VOL 5 PART 4 Swalve H.H. 1995. The effects of test-day model on the estimation of genetic parameters and breeding values for dairy traits. J. Dairy Sci. 78:929-938. Thatcher W.W., Roman-Ponce H. and Buffington D.E. 1978. Large dairy herd management. Wilcox C.J. and H.H. Van Horn. Ed. University Presses, Florida, Gainesville, Florida, U.S.A. BCVA 1997 360

CATTLE PRACTICE VOL 5 PART 4 Nutritional Influence on Subfertility in Cattle Webb R1 , Garnsworthy P C1 , Gong J G2 , Gutierrez C G2 , Logue D3 , Crawshaw W M3 and Robinson J J4 . 1 University of Nottingham, Sutton Bonington, Loughborough, LE12 5RD; 2 Roslin Institute, Edinburgh, EH25 9PS; 3 SAC, Auchincruive, Ayr, KA6 5HW; 4 SAC, Craibstone, Bucksburn Estate, Aberdeen, AB21 9AY. ABSTRACT One of the most effective methods for enhancing milk production efficiency in dairy cows is to increase the yield per animal. Significant progress has been achieved towards this end by the dairy industry through intense selection, assisted by the application of new reproductive techniques such as artificial insemination (AI) and multiple ovulation and embryo transfer (MOET). Many of today’s dairy cows are capable of producing over 9000 litres of milk during a 305-day lactation. Unfortunately, the increase in milk yield has been accompanied by some negative consequences, such as an increase in the incidence of metabolic diseases, lameness and mastitis and a reduction in reproductive performance. In addition, this has given rise to public concern that the high yielding dairy cow may be under a state of metabolic stress during peak lactation and therefore the welfare and performance of other body functions are compromised. It has become increasingly evident that fertility across the whole dairy industry, over the last few decades, has been declining continuously, with increasing milk yield. The reason for this decline in fertility is not well understood, although a nutritional influence on the initiation of oestrous cycles, follicular growth, oocyte quality and early embryonic development has been implicated. This paper will review some of the possible sites in the reproductive system where nutrition may impair function and the possible mechanisms involved. INTRODUCTION The effects of nutrition on the reproduction of livestock are well known. In The Origin of Species, Darwin relates that domestic animals reproduce more efficiently than their wild ancestors, speculating that a readily available food supply was, at least in part, responsible for this effect. Correlative data in wild animal species showed that they reproduce at times of the year when more nutrients are available. The same trend is seen in bovine species, a species that generally reproduces throughout the year (Aleksiev et al. 1988). However, in tropical areas where seasonal variations in food quantity and quality are acute, reproductive performance of cattle is profoundly affected. Analysis of reproductive records of rangeland grazing cattle over 15 years, showed that calvings tended to cluster during the spring, indicating higher fertility during the summer months, when grass growth was at its maximal (Enriquez et al. 1993). A range of reproductive parameters are known to be affected by deficient nutrition. Puberty can be delayed by impairing the growth of animals (Robinson, 1990). In cycling heifers, reduction of energy intake led to anoestrus when body weight fell by approximately 20% (Imakawa et al. 1986; Richards et al. 1989; Rhodes et al. 1995). First ovulation postpartum (and thus calving interval) can be prolonged by poor nutrition in both dairy (Whitaker et al. 1993; Beam & Butler, 1997) and beef cattle (Short & Adams, 1988; Jolly et al. 1995). Body energy reserves and therefore body condition score at the time of parturition was the single most important determinant of the length of postpartum anoestrus in beef cattle (Wright et al. 1992; Bishop et al. 1994) and cows calving in poor body condition remained in anoestrus for a longer period. In contrast, body condition score per se does not appear to have a major influence as reproductive efficiency in dairy cattle (Garnsworthy and Haresign 1989), but the extent of the negative energy balance, caused by the high demands of early lactation (Lucy et al. 1991; Veerkamp et al. 1994), determined the interval from parturition to first ovulation postpartum (Butler & Smith, 1989; Swanson et al. 1989; Canfield & Butler, 1990). and was linked to reduced conception rates (Garnsworthy and Haresign, 1989). Follicular development in dairy cattle is re-established early during the postpartum period (Savio et al. 1990; Beam & Butler, 1997). However, the first ovulation postpartum was delayed until the energy balance of the animal had passed its nadir and although still negative, had begun to recover (Zurek et al. 1995; Senatore et al. 1996; Beam & Butler, 1997). In conclusion, compared with thermoregulation, locomotion, growth, cellular maintenance and lactation, nutritional requirements for reproductive functions occupy a low physiological priority when energy is restricted. Hence, reproduction is arrested well before other more vital functions are compromised. Because of genetic selection, assisted by the application of new reproductive techniques such as artificial insemination (AI) and multiple ovulation and embryo transfer (MOET), many of today’s dairy cows are capable of producing over BCVA 1997 361

CATTLE PRACTICE VOL 5 PART 4 9000 litres of milk during a 305-day lactation. This has given rise to public concern that the high yielding dairy cow may be under a state of metabolic stress during peak lactation and therefore welfare and performance of other body functions are compromised, although this has yet to be clarified. This paper will discuss some of the possible physiological sites where nutrition, including lactation induced negative energy balance, may impinge on reproductive function in cattle and the mechanisms involved. Sites of Influence in the Reproductive System In cattle, as in most other mammalian species, it has been well established that ovarian follicle development is primarily controlled by an integrated feedback system encompassing gonadotrophinreleasing hormone (GnRH) from the hypothalamus, follicle-stimulating hormone (FSH) and luteinising hormone (LH) from the pituitary gland and steroids (oestrogens, androgens and progestins) and proteins (inhibin-related proteins) secreted from the ovaries (see Webb et al. 1992, 1994; Gong & Webb, 1996). Follicular development is primarily controlled by the co-ordinated action of gonadotrophins, (see Campbell et al. 1995). The gonadotrophin requirements for follicular growth in cattle, were recently determined by Gong et al. (1996a) who used long-term treatment with a GnRH-agonist to suppress either LH release or both LH and FSH release from the pituitary gland. They showed that without pulsatile release of LH, follicle development could proceed, but was halted when the dominant follicle reached 7-9 mm in diameter. When FSH concentrations were suppressed after a long period of GnRH-agonist treatment, follicle growth stopped at 4 mm in diameter. Therefore, since follicle development depends on sustained support by gonadotrophins, changes in gonadotrophin secretion caused by nutrition would affect follicular development. The recent development in ovarian ultrasonography has established that ovarian folliculogenesis in cattle is characterized by a continuous turnover in follicular waves (see Webb et al. 1992; see Gong & Webb, 1996). Each wave of follicle development involves the simultaneous growth of a cohort of 5-7 antral follicles from the growing pool to >5 mm in diameter and then the selection of one of these follicles to grow rapidly while other cohort follicles regress. The selected follicle continues to grow to become a dominant follicle with a diameter of about 15 mm and remains at maximum size for 2-3 days before regression, followed by a new wave of follicle development. Approximately 80% of cattle have 3 waves of follicular growth and development during each oestrous cycle. If the growth phase or early plateau stage of a dominant follicle coincides with luteolysis, it undergoes rapid preovulatory maturation and eventually ovulates. As well as during the oestrous cycle, continuous turnover in follicular waves occurs at almost every physiological stage in cattle, including the post-partum anoestrus period (Boland et al. 1991), before puberty (Adams et al. 1994) and during most of pregnancy (Ginther et al. 1996). Following ovulation, fertilisation will occur if cattle are inseminated at the appropriate time in relation to ovulation. Following fertilisation optimum progesterone, from the newly formed CL is essential in order to provide optimal oviductal and uterine environments for embryonic growth and development. However, conception rates of HolsteinFriesian cows to a particular service have been steadily declining in the US by approximately 1% every 3 years, whilst during the same period the breed average for milk yield has almost doubled (see Darwash & Lamming, 1997). Therefore, the possible sites where inadequate nutrition may have detrimental effects on reproductive function include: 1) The hypothalamus/pituitary gland to impair gonadotrophin release with the subsequent effect of delaying ovulation or causing abnormal follicular growth. 2) Direct effect on the ovaries, where follicular growth patterns may be altered. Also corpus luteum function may be influenced. 3) Since the follicle is the vehicle for the oocyte, the quality of the oocyte may be reduced, with subsequent negative effects on embryo survival. 4) Embryo survival and development may also be impaired due to an inadequate uterine environment. Effect of Nutrition on Hypothalamic/ Pituitary Function Circulating FSH concentrations in intact animals do not appear to be affected by nutrition. Rhodes et al. (1995) found no changes in FSH concentrations in undernourished heifers even after they had lost 17% of their body weight. Although some studies were unable to find differences in the pattern of LH secretion in heifers (Enright et al. 1994; Rhodes et al. 1995), there seems to be a consensus that significant changes in body weight or nutrient supply can alter the pattern of LH secretion. This was shown first by Gonzales-Padilla et al. (1975) and corroborated by Yelich et al. (1996) in feedrestricted heifers. In beef cows body condition at parturition was found to be directly related to LH pulse frequency after weaning (Bishop et al. 1994). Low level of feeding appears to have very little effect on fat cows, but quite a large effect (27 day delay in ovulation) in thin cows and is associated with reduced LH pulse frequency (Wright et al. 1992). In dairy cows, Canfield & Butler (1990) observed that baseline and mean concentration of LH and LH pulse frequency BCVA 1997 362

CATTLE PRACTICE VOL 5 PART 4 were higher in cows after the energy balance nadir compared to before the energy balance nadir. Consequently, nutrition appears to affect pituitary gonadotrophin secretion and LH pulse frequency in particular, preventing the final maturation of a potentially ovulatory follicle. However, in most cases the pituitary gland is capable of releasing substantial amounts of LH and FSH after GnRH stimulation and the amount of LH released in respsonse to GnRH did not change when heifers with initial high body condition score (BCS) were losing weight. However, the LH response to GnRH was reduced in heifers that lost weight from an already low BCS (Roberson et al. 1992). Taken together, these results indicate that the response to GnRH is reduced once a compromising BCS is reached. This is illustrated by the finding that the start of the first follicular wave on the ovaries does not differ for post-partum suckler beef cows on a low feeding level (80 MJ) from those on a high diet (120 MJ), yet first ovulation was delayed by 25 days by low feeding (Stagg et al. 1995). Secondly, synthesis and storage of gonadotrophins, or sensitivity/response of the pituitary gland to GnRH, are reduced when BCS is low. In conclusion, FSH concentrations in intact animals are not acutely influenced by nutritional status. However, LH concentrations and pulse frequency appeared to decrease when animals were restricted to sub-maintenance nutrition. In high-yielding dairy cows the size of the energy deficit in the first 2 to 3 weeks after calving is closely correlated with the interval to first oestrus (Butler & Smith, 1989). For example, in a recent series of studies at the Roslin Institute, where the dairy herd have been selected for genetic merit for milk yield, the High line animals produce 20% more milk than the Low line cows and have a significant delay in return to oestrous cycles. Ovarian function In cattle ovaries, regardless of their reproductive status, follicles in all stages of development are present. As discussed, antral follicular develoment proceeds in a typical wave-like pattern initiated by the growth of a number of small (2-5 mm) follicles, from which only a handful escape atresia and grow beyond 4-5 mm in diameter. Finally only one large follicle (>8 mm) normally emerges as the dominant follicle. The dominant follicle present at the regression of the corpus luteum will induce the preovulatory peak of gonadotrophins, ovulate and luteinise. This normal pattern of follicular development has been shown to be altered by nutritional deficiencies. Influence of Nutrition and/or Lactation In beef cattle, the diameter of the preovulatory follicle decreases when animals are losing weight (Murphy et al. 1991; Grimand et al. 1995; Rhodes et al. 1995). Furthermore, the reduction in follicle diameter was positively correlated with weight loss until the animals ceased ovulating (Rhodes et al. 1995). In the studies of Murphy et al. (1991) and Spicer et al. (1991), showing decreased diameter of the dominant follicle, the number of follicles >5 mm was not altered by body weight loss. However, reduction in the number of small (<6 mm) follicles was observed under more extreme undernutrition (Gutierrez, 1992). In contrast, an increase in diameter of the largest follicle was seen when heifers were gaining weight (Rhodes et al. 1995; Murphy et al. 1991; Spicer et al. 1991). These follicles were also dominant as the number of subordinate follicles was reduced as the first ovulation postpartum approached (Gutierrez et al. 1994). The negative energy balance in dairy cows, immediately after calving, is caused by the large energy demands of lactation, together with low appetite in the immediate postpartum period (Butler & Smith, 1989; Swanson, 1989). Waves of follicular development in postpartum cows are normally initiated early after calving, following an increase of plasma FSH within the first 5 days postpartum (Beam & Butler, 1997). During this period, the extent of the energy balance deficit affects follicular development. Compared with non-lactating cows (de la Sota et al. 1993), lactating cows had a lower energy balance, reflected in lower plasma concentrations of glucose, insulin and insulin-like growth factor-1 (IGF-1), but higher concentrations of non-esterified fatty acids (NEFA). The lower energy balance of the lactating cows was accompanied by fewer follicles of <15 mm in diameter (de la Sota et al. 1993) compared to non-lactating cows, indicating that low energy balance decreased the number of small follicles progressing into the large diameter categories. However, in other studies, lower energy balance in lactating cows was associated with higher numbers of small (3-5 mm) and medium-sized (6-9 mm) follicles (Lucy et al. 1991a,b). Furthermore, when cows were fed low or high energy diets, the growth rate of preovulatory follicles in ‘low energy diet’ cows was slower than that in ‘high energy diet’ cows (Murphy et al. 1991; Lucy et al. 1992). Effects of BST Paradoxically, treatment of lactating cows with bovine somatotrophin (BST), which increases milk production and causes a decline in energy balance, also stimulates an increase in the number of follicles reaching larger size categories with a consequent decline in small (<5 mm) follicle numbers (de la Sota et al. 1993; Lucy et al. 1993b). The explanation for the increase in the number of follicles progressing to larger follicle size categories is not clear, but could be due to changes in circulating concentrations of metabolic hormones and metabolites (ie glucose, insulin, IGF-I, IGF-binding proteins) induced by BST in lactating cows. Indeed, recent work has shown that factors classically thought to be involved BCVA 1997 363

CATTLE PRACTICE VOL 5 PART 4 in the regulation of metabolic processes, such as growth hormone, insulin and IGF’s, have a pronounced influence on ovarian follicle development in cattle. This may represent part of the mechanism underlying well recognized, but poorly understood, effects of nutritional and other environmental factors on reproductive function in cattle. Pharmacological administration of BST to heifers increased the number of small follicles (<5 mm), but did not alter the number of follicles (>5 mm) or the dynamics of follicle turnover (Gong et al. 1991; 1993a). Furthermore, GH increased follicle number in the absence of changes in circulating concentrations of FSH and LH and gonadotrophin binding to theca and granulosa cells (Gong et al. 1991). Similarly, in dairy cows, BST treatment increased the number of follicles without altering gonadotrophin levels (de la Sota et al. 1993b). However, increased follicle numbers were seen in the larger size categories (>5 mm) (de la Sota et al. 1993; Lucy et al. 1993b) and in one study (Kirby et al. 1997) follicular turnover was enhanced by earlier onset of atresia of the first dominant follicle. In addition, Gong et al. (1993b) showed that the small (<5 mm) follicles induced by BST stimulation were responsive to exogenous gonadotrophin stimulation, thus increasing the response to superovulatory treatments compared to heifers not treated with BST. Moreover, low GH levels may also affect follicular development. Cohick et al. (1996) immunised prepubertal heifers against GH releasing hormone, thereby reducing the circulating concentrations of GH, and found that only 22% of heifers developed follicles above 7 mm in diameter, compared with 77% of control heifers. Recently, increased dietary intake has been associated with increased small follicle recruitment during the first follicular wave of the oestrous cycle in Holstein-Friesan heifers (Gutierrez et al. 1997a). This increase in small follicle numbers was rapidly lost as soon as dietary treatments were terminated. The number of medium-size follicles increased in all groups, at the time when numbers of small follicles were decreasing. Taken together, these results indicate that nutrition appears to affect, mainly, the recruitment of small follicles, but not follicle selection and dominance. As discussed already, similar observations were made by Gong et al. (1993) following BST treatment. Interestingly, BST is unlikely to have a direct effect on the ovaries to cause the increment in small follicle number seen in these studies. Although BST has been associated with increases in follicle number (Gong et al. 1993a; de la Sota et al. 1993), there is evidence that ovarian follicles do not possess GH receptors (Lucy et al. 1993a). In addition, if animals are fed above their nutritional requirements, GH decreases, whilst IGF-I and insulin increase (Breier et al. 1986; Clarke et al. 1993; Thissen et al. 1994). Insulin and/or IGF-I therefore, seem to be important mediators in GH action. When insulin concentration is low in underfed animals, there is a decline in GH receptor capacity, with GH resistance. Insulin administration can reverse this effect (Pell & Bates, 1990). In contrast, when insulin concentration is high during overfeeding, IGF-I production increases. This is despite a decline in GH secretion and is probably due to enhanced binding of GH to its receptor, caused by insulin. Alternatively, there is evidence that high dietary protein intake can increase the production of IGF-I by the liver (Elsasser et al. 1989). Although paradoxically, in periods of undernutrition, GH increases while IGF-I decreases (Thissen et al. 1994; Armstrong et al. 1993; Spicer et al. 1992). Therefore, the effects of BST administration on follicular development are likely to be mediated by changes in either insulin or IGF-1 or both. It is likely that insulin and/or IGF-1 act by altering the response of follicular cells to gonadotrophins, as shown by recent in vitro studies (Gutierrez et al. 1997b). This also indicates that a change in circulating gonadotrophin concentrations is not mandatory. Seasonal Influences Nutrition, as affected by season, may also influence the normal development of follicles. At SAC Auchincruive, two balanced herds of dairy cows of high genetic merit are maintained under two distinct management systems calving in either spring or autumn. One herd is managed under an intensive or high input system to yield an average of 9000 litres of milk, while the second herd is maintained under an extensive or low input system to produce 5000 litres per lactation respectively. More cows from the autumn calving cohort developed follicular cysts than cows in the spring calving cohort. Despite the relatively high (≈10%) occurrence of follicular cysts in the UK dairy herd, accounting for significant losses, the underlying mechanisms leading to the development of ovarian cysts have not been fully elucidated (see Garverick, 1997). However, it is not inconceivable that the stress of a change in diet at different times of the year, coupled with high milk production, could lead to a greater predisposition for abnormal follicular development. Oocyte quality and embryo survival Recent studies have indicated that zygotic development can be influenced by the ovarian environment within which the maternal gamete develops prior to ovulation and that nutrition has the capacity to modify this environment. Indeed, it is becoming more apparent that nutritionally-mediated effects on oocyte maturation and the early development of the embryo are central to embryo survival and the programming of the feotal growth trajectory. In a recent study (McEvoy et al. 1997) it was shown that maternal nutrition, prior to oocyte BCVA 1997 364

CATTLE PRACTICE VOL 5 PART 4 collection, influences the subsequent developmental capacity of oocytes harvested from follicles of different sizes. The results demonstrate that energy intake by oocyte donors, but not dietary protein concentrations, influenced blastocyst production in vitro. Also, that the dietary effects on blastocyst yields occurred in 2-4 mm follicles only. This is in agreement with the observations discussed above that nutrition affects the growth of small follicles. Other factors are also known to affect fertilization rates and embryo quality. For example, selenium deficiency is known to increase embryo mortality around the time of implantation in sheep (Piper et al. 1980). Selenium is also involved before this time in uterine contractions at oestrus, sperm transport and fertilization, particularly noticeable in superovulated animals (Hurley & Doane, 1989). There are reports of normal fertility in spontaneously ovulating animals, yet very poor fertilization rate and embryo quality following superovulation and these are correctable by selenium administration (cited by Hurley & Doane, 1989). In the case of dietary protein, diets high in rumen degradable protein lead to an excess of rumen ammonia, which before it is converted to urea by the liver and excreted in the urine, causes alterations in uterine pH and in endometrial Na and K fluxes and these lead to reductions in embryo survival. Under practical conditions the reduction appears to be around 15 to 20%. The detrimental effect of this increase in ammonia on protein synthesis in day 4 embryos has been clearly shown (Robinson & McEvoy, 1996). However, these are data from experiments with sheep and need to be extended to cattle. Detoxification of the ammonia imposes a demand for additional glucose and this may be the reason why dietary supplements of fish meal, that contain undegraded dietary protein, improve fertility (Armstrong et al. 1990). They may alleviate the adverse effects of high rumen degradable protein on embryo survival by enhancing gluconeogenesis for the hepatic conversion of ammonia to urea or supplying preformed amino acids. However, fish meal also supplies essential fatty acids and their antiluteolytic properties may also enhance embryo survival (Coelho et al. 1997). The effects of plane of nutrition and rumen degraded versus undegraded dietary proteins on the quality of the oocyte and the subsequent ‘down-stream’ effects on the health and viability of the embryo is now attracting our research interest. Initial findings imply that through its effects on the intrafollicular environment and oocyte maturation, inappropriate nutrition can impart a legacy of developmental retardation leading to decreased embryo survival. CONCLUSIONS The initiation of normal oestrous cycles postpartum is delayed in dairy cows with a higher genetic merit for milk production, confirming that intense selection towards high milk yield can compromise reproductive performance. This result supports the previous observations that the intervals from calving to the first ovulation postpartum and to conception are often extended in lactating cows with a high milk output. Therefore, it appears that when such animals are intensively selected on a single trait, negative consequences can occur. A selection programme based on multiple indices may be a more viable approach in the future. In addition, although a nutritional influence on oocyte quality, with ensuing negative effects on early embryonic development and subsequent foetal loss has been suggested, systemic studies are required to examine if this reduction in fertility is due to an affect of metabolic changes associated with high milk output on the quality of oocytes prior to ovulation, since the follicle is the vehicle for the oocyte. In conclusion, identification of the mechanisms and key sites of action responsible for compromised reproductive function, including abnormal follicular development, will enable the identification of possible indices for future multiple-trait selection programmes. ACKNOWLEDGEMENTS The work carried out by the co-authors was supported by MAFF DS0206, SOAEFD, BBSRC, British Council and Conacyt (Mexico). REFERENCES Adams GP, Evans ACO and Rawlings NC. (1994). Follicle waves and circulating gonadotgrophins in 8-month-old prepubertal heifers. J. Reprod. Fertil. 100, 27-33. Aleksiev AI, Peeva T, Vasilev M and Folikhronov O. (1988). Effect of season of calving on reproductive performance and productivity of buffalo cows. Zhivotnov'd Nauki 25, 3-10. Armstrong JD, Cohick WS, Harvey RW, Heimer EP and Campbell RM. (1993). Effect of feed restriction on serum somatotropin, insulin-like growth factor-1 (IGF-1) and binding proteins in cyclic heifers actively immunized against growth hormone releasing factor. Dom. Anim. Endocrinol. 10, 315-324. Armstrong JD, Goodall EA, Gordon FJ, Rice DA and McGaughey WJ (1990). The effect of levels of concentrate offered and inclusion of maize gluten or fish meal in the concentrate on reproductive performance and blood parameters of dairy cows. Anim.Prod. 50, 1-10. Beam SW and Butler WR. (1997). Energy balance and ovarian follicle development prior to the first ovulation postpartum in dairy cows receiving three levels of dietary fat. Biol. Reprod. 56, 133-142. Bishop DK, Wettemann RP and Spicer LJ. (1994). Body energy reserves influence the onset of luteal activity after early weaning of beef cows. J. Anim. Sci. 72, 2703-2708. Boland MP, Goulding D and Roche JF (1991) The use of ultrasound to monitor ovarian function in farm animals. Ag. Biotech. News Inform. 2, 841-844. Breier BH, Bass JJ, Butler JH and Gluckman PD. (1986). The somatotrophic axis in young steers: influence of nutritional status on pulsatile release of growth hormone and circulating concentrations of insulin-like growth factor 1. J. Endocrinol. 111, 209-215. Butler WR and Smith RD. (1989). Interrelationship between energy balance and postpartum reproductive function in dairy cattle. J. Dairy Sci. 72, 767-783. Campbell BK, Scaramuzzi RJ and Webb R (1995). Control of antral follicle development and selection in sheep and cattle. J. Reprod. Fertil. Suppl 49, 335-350. BCVA 1997 365

CATTLE PRACTICE VOL 5 PART 4 Canfield RW and Butler WR. (1990). Energy balance and pulsatile LH secretion in early postpartum dairy cattle. Dom. Anim. Endocrinol. 7, 323-330. Coelho S, Ambrose JD, Binelli M, Burke J, Staples CR, Thatcher MJ and Thatcher WW (1997). Menhaden fish meal attenuates estradiol- and oxytocin-induced uterine secretion of PGF2∝ in lactating dairy cattle. Theriogenology 47, 143. Clarke IJ, Fletcher TP, Pomares CC, Holmes JHG, Dunshea F, Thomas GB, Tilbrook AJ, Walton PE and Galloway DB. (1993). Effect of high-protein feed supplements on concentrations of growth hormone (GH), insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 in plasma and on the amounts of GH and messanger RNA for GH in the pituitary glands of adults rams. J. Endocrinol. 138, 421-427. Cohick WS, Armstrong JD, Withacre MD, Lucy MC, Harvey RW and Campbell RM. (1996). Ovarian expression of insulin-like growth factor-I (IGF-I), IGF binding proteins, and growth hormone (GH) receptor in heifers actively immunized against GHreleasing factor. Endocrinology 137, 1670-1677. Darwash AO and Lamming GE (1997) Abnormal Ovarian Patterns as a cause of subfertility in dairy cows: protocols for early detection and treatment. Cattle Pract. 5, 3-7. de la Sota RL, Lucy MC, Staples CR and Thatcher WW. (1993). Effects of recombinant bovine somatotropin (Sometribove) on ovarian function in lactating and nonlactating dairy cows. J. Dairy Sci. 76, 1002-1013. Elsasser TH, Rumsey TS and Hammond AC. (1989). Influence of diet on basal and growth hormone-stimulated plasma concentrations of IGF-1 in beef cattle. J. Anim. Sci. 67, 128-141. Enright WJ, Spicer LJ, Prendiville DJ, Murphy MG and Campbell RM. (1994). Interaction between dietary intake and ovariectomy on concentrations of insulin-like growth factor-I, GH and LH in plasma of heifers. Theriogenology 41, 1231-1240. Enriquez BA, Galina CS, Navarro RR and Gutierrez C. (1993). Estimacion de la epoca mas propicia para un empadre estacional en ganado cebu bajo condiciones de tropico humedo. Avan. Invest. Agropec. 2, 101-114. Garnsworthy, P.C. and Haresign, W. (1989) Fertility and nutrition. In Dairy Cow Nutrition – The Veterinary Angles (Ed.A.T.Chamberlain) pp23-34. University of Reading, Department of Agriculture. Garverick HA. (1997). Ovarian follicular cysts in dairy cows. J.Dairy Sci. 80, 995-1004. Ginther OJ, Kot K, Kulick LJ, Martin S and Wiltbank MC. (1996). Relationship between FSH and ovarian follicular waves during the last six months of pregnancy. J.Reprod.Fert. 108, 271-279. Gong JG, Bramley TA and Webb R. (1991). The effect of recombinant bovine somatotropin on ovarian function in heifers: Follicular populations and peripheral hormones. Biol. Reprod. 45, 941-949. Gong JG, Bramley TA and Webb R. (1993a). The effect of recombinant bovine somatotrophin on ovarian follicular growth and development in heifers. J. Reprod. Fertil. 97, 247-254. Gong JG, Bramley T, Wilmut I and Webb R. (1993b). Effect of recombinant bovine somatotropin on superovulatory response to pregnant mare serum gonadotropin in heifers. Biol. Reprod. 48, 1141-1149. Gong JG and Webb R (1996) Control of ovarian follicle development in domestic ruminants: its manipulation to increase ovulation rate and improve reproductive performance. Anim. Breeding Abst. 64, 195-204. Gong JG, Campbell BK, Bramley TA, Gutierrez CG, Peters AR and Webb R. (1996). Suppression in the secretion of folliclestimulating hormone and luteinizing hormone, and ovarian follicle development in heifers continuously infused with a gonadotropinreleasing hormone agonist. Biol. Reprod. 55, 68-74. Gonzalez-Padilla E, Wiltban JN and Niswender GD. (1975). Puberty in beef heifers. 1. The interrelationship between pitiutary, hypotalamic and ovarian hormones. J. Anim. Sci. 40, 1091-1104. Grimard B, Humblot P, Ponter AA, Mialot JP, Sauvant D and Thibier M. (1995). Influence of postpartum energy restriction on energy status, plasma LH and oestradiol secretion and follicular development in suckled beef cows. J. Reprod. Fertil. 104, 173- 179. Gutierrez CG. (1992). Comparacion de la foliculogenesis y ciclos estrales de novillonas cebu y cebu-holstein durante los meses de marzo a junio en el tropico humedo de mexico. MSc thesis, Universidad Nacional Autonoma de Mexico. Gutierrez CG, Galina CS, Zarco L and Rubio I. (1994). Pattern of follicular growth during prepuberal anoestrous and transition from anoestrous to oestrous cycles in Bos indicus heifers. Adv. Agri. Res. 3, 1-11. Gutierrez CG, Oldham J, Bramley TA, Gong JG, Campbell BK and Webb R (1997a) The recruitment of ovarian follicles is enhanced by increased dietary intake in heifers. J. Anim. Sci. 7,1876-1884 Gutierrez CG, Campbell BK and Webb R (1997b). Development of a long-term bovine granulosa cell culture system: induction and maintenance of estradiol production, response to folliclestimulating hormone and morphological characteristics. Biol.Reprod. 56, 608-616. Hurley WL and Doane RM (1989). Recent developments in the roles of vitamins and minerals in reproduction. J.Dairy Sc. 72, 784-804. Imakawa K, Day ML, Garcia-Winder M, Zalesky DD, Kittok RJ, Schanbacher BD and KInder JE. (1986). Endocrine changes during restoration of estrous cycles following induction of anestrus by restricted nutrient intake in beef heifers. J. Anim. Sci. 63, 565-571. Jolly PD, McDougall S, Fitzpatrick LA, Macmillan KL and Entwistle KW. (1995). Physiological effects of undernutrition on postpartum anoestrus in cows. J. Reprod. Fertil. 49, 477-492. Lucy MC, Staples CR, Michel FM and Thatcher WW. (1991a). Energy Balance and size and number of ovarian follicles detected by ultrasonography in early postpartum dairy cows. J. Dairy Sci. 74, 473-482. Lucy MC, Staples CR, Michel FM and Thatcher WW. (1991b). Effect of feeding calcium soaps to early postpartum dairy cows on plasma prostaglandin F2fi, luteinizing hormone, and follicular growth. J. Dairy Sci. 74, 483-489. Lucy MC, Beck J, Staples CR, Head HH, DelaSota RL and Thatcher WW. (1992). Follicular dynamics, plasma metabolites, hormones and insulin-like growth factor (IGF-1) in lactating cows with positive or negative energy balance during the preovulatory period. Reprod. Nutr. Dev. 32, 331-341. Lucy MC, Collier RJ, Kitchell ML, Dibner JJ, Hauser SD and Krivi GG. (1993a). Immnunohistochemical and nucleic acid analysis of somatotropin receptor populations in the bovine ovary. Biol. Reprod. 48, 1219-1227. Lucy MC, DelaSota RL, Staples CR and Thatcher WW. (1993b). Ovarian follicular populations in lactating dairy cows treated with recombinant bovine somatotropin (Sometribove) or saline and fed diets differing in fat content and energy. J. Dairy Sci. 76, 1014- 1027. McEvoy TG, Sinclair KD, Staines ME, Robinson JJ, Armstrong DG and Webb R (1997) In vitro blastocyst production in relation to energy and protein intake prior to oocyte collection. J. Reprod. Fert. Abstr. Ser. Murphy MG, Enright WJ, Crowe MA, McConnell K, Spicer LJ, Boland MP and Roche JF. (1991). Effect of dietary intake on pattern of growth of dominant follicles during the oestrous cycle in beef heifers. J. Reprod. Fertil. 92, 333-338. Pell JM and Bates PC. (1990). The nutritional regulation of growth hormone action. Nutrit. Res. Rev. 3, 163-192. Piper LR, Bindon BM, Wilkins JF, Cow RJ, Curtis YM and Cheers MA (1980). The effect of selenium treatment on the fertility of Merino sheep. Proc.Aust.Soc.Anim.Prod. 13,241-244. Rhodes FM, Fitzpatrick LA, Entwistle KW and De'ath G. (1995). Sequential changes in ovarian follicular dynamics in Bos indicus heifers before and after nutritional anoestrus. J. Reprod. Fertil. 104, 41-49. Richards MW, Wettemann RP and Schoenemann HM. (1989). Nutritional anestrus in beef cows: body weight change, body condition, luteinizing hormone in serum and ovarian activity. J. Anim. Sci. 67, 1520-1526. Roberson MS, Stumpf TT, Wolfe MW, Cupp AS, Kojima N, Werth LA, Kittok RJ and Kinder JE. (1992). Circulating gonadotrophins during a period of restricted energy intake in BCVA 1997 366

CATTLE PRACTICE VOL 5 PART 4 relation to body condition in heifers. J. Reprod. Fertil. 96, 461- 469. Robinson JJ. (1990). Nutrition in the reproduction of farm animals. Nutrit. Res. Rev. 3, 253-276. Robinson JJ and McEvoy TG (1996). Feeding level and rumen degradable nitrogen effects on embryo survival. In “Techniques for Gamete Manipulation and Storage”. ICAR Satellite Symposium, Hamilton, New Zealand. Savio JD, Boland MP, Hynes N and Roche JF. (1990). Resumption of follicular activity in the early post-partum period of dairy cows. J. Reprod. Fertil. 88, 569-579. Senatore EM, Butler WR and Oltenacu PA. (1996). Relationships between energy balance and post-partum ovarian activity and fertility in first lactation dairy cows. Anim. Sci. 62, 17-23. Short RE and Adams DC. (1988). Nutritional and hormonal interrelationships in beef cattle reproduction. Can. J. Anim. Sci. 68, 29-39. Spicer LJ, Enright WJ, Murphy MG and Roche JF. (1991). Effect of dietary intake on concentrations of insulin-like growth factor-1 in plasma and follicular fluid, and ovarian function in heifers. Dom. Anim. Endocrinol. 8, 431-437. Spicer L, Crowe MA, Prendiville DJ, Goulding D and Enright WJ. (1992). Systemic but not intraovarian concentrations of insulinlike growth factor-I are affected by short-term fasting. Biol. Reprod. 46, 920-925. Stagg K, Diskin MG, Sreenan JM and Roche JF (1995). Follicular development in long-term anoestrous suckler beef cows fed two levels of energy post-partum. Anim.Reprod.Sci. 38,49-61. Swanson LV. (1989). Discussion - Interactions of nutrition and reproduction. J. Dairy Sci. 72, 805-814. Thissen JP, Ketelslegers JM and Underwood LE. (1994). Nutritional Regulation of the insulin-like growth factors. Endocr. Rev. 15, 80-101. Veerkamp RF, Simm G and Oldham JD. (1994). Effects of interaction between genotype and feeding system on milk production, feed intake, efficiency and body tissue mobilization in dairy cows. Livest. Prod. Sci. 39, 229-241. Webb R, Gong JG, Law AS and Rusbridge SM. (1992). Control of ovarian function in cattle. J. Reprod. Fertil. Suppl. 45, 141-156. Webb R, Gong JG and Bramley TA. (1994). Role of growth hormone and intrafollicuar peptides in follicle development in cattle. Theriogenology 41, 25-30. Whitaker DA, Smith EJ, Rosa GOd and Kelly JM. (1993). Some effects of nutrition and management on the fertility of dairy cattle. Vet. Rec. 133, 61-64. Wright IA, Rhind SM, Smith AJ and Whyte TK. (1992). Effects of body condition and estradiol on luteinizing hormone secretion in post-partum beef cows. Dom. Anim. Endocrinol. 9, 305-312. Yelich JV, Wettemann RP, Marston TT and Spicer LJ. (1996). Luteinizing hormone, growth hormone, insulin-like growth factorI, insulin and metabolites before puberty in heifers fed to gain at two rates. Dom. Anim. Endocrinol. 13, 325-338. Zurek E, Foxcroft GR and Kennelly JJ. (1995). Metabolic status and interval to first ovulation in postpartum dairy cows. J. Dairy Sci. 78, 1909-1920. BCVA 1997 367

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CATTLE PRACTICE VOL 5 PART 4 The Advantages of Milk Progesterone Monitoring Lamming J., The Grange, Market Rasen, Lincs - Farmer We first became involved with the University of Nottingham milk progesterone investigations in 1974 when the use of the technique for infertility studies was under scrutiny. We were involved in a trial to test the benefits of prostaglandin with or without a progesterone releasing device (PRID) to allow fixed time insemination. By 1979 when the trials stopped we were convinced of the benefits progesterone monitoring gave and were therefore pleased to co-operate again when the trials started in 1993. The Farm and the Dairy Herd The 95 acre farm was purchased in 1959 and the herd started with 24 purchased animals. Since then all the herd is home bred, with cow numbers increased to about 100 in 1981. When quotas came in we were producing about 100,000 litres above quota, but this has been purchased with cow numbers now running about 95, producing 7,200 litres per cow. Generally about 24 animals are removed each year, increasingly for non reproductive problems. We use an all-year calving pattern commencing with heifers calving at two years of age. The Current Experiments Currently we are engaged in the ‘genetic’ trial and in the ‘early induction of ovulation and oestrous cyclicity’. The former has resulted in delayed veterinary treatment for subfertility in a small number of cows, while the latter has induced early oestrous cycles and provided an ability to reduce calving interval to below one year. The major feature of profiling has been to identify clearly the approximately 30% of cows which have reproductive problems usually involving more than one cause, and clearly running in families. We have experienced regularly each year between 8 and 15% embryo loss, some due to leptospirosis requiring regular vaccination. Until recently subfertility has been the major reason for culling, but recently due to an improved first service pregnancy rate, we are now able to cull more for other reasons. The Major Advantages of Recent Developments We currently use a computer programme and make extensive use of the progesterone profiles provided. Since starting the recent trials two years ago no cows have received remedial veterinary treatment other than four animals for ovarian cysts. We regularly use Kamar heat detectors placed four days post BCVA 1997 calving and renewed when triggered. Knowledge that the cows are cycling leads to a clearer definition between normal and problem cows. The Advantages of Progesterone Profiling 1. We believe we are in control of fertility; 2. During the last two years we appear only to have missed five oestrous periods; 3. Progesterone profiles show which animals are not cycling, 60% have no problems at all, the remaining 40% require careful observation; 4. Fertility is satisfactory from July to December, then falls gradually; 5. Profiling to determine the fertility status of an animal allows us to use the best semen on fertile cows and to dispose of the progeny of subfertile animals; 6. The fertility improvements are illustrated in the following table; HERD MONITOR - JOHN LAMMING YEAR 1979 1995 1997 Herd size 75 90 93 Days to 1ST service 68.7 65.0 63.0 Calving to conception 96.2 94.0 74.0 Services/pregnancy 1.8 1.7 1.5 % Pregnancy to 1 service 44.6 59.0* 70.5* 2 20.3 23.0* 24.2* 3 13.5 12.0* 1.2* 3+ 5.4 6.0* 4.2* Calving index (days) 379 380 368 Number culled 24 24 22 Culled for subfertility ? 19 11 *Comparable pregnancy rates based on positive Day 24 milk progesterone concentration later confirmed by Day 60 pregnancy rate. Subjected to later embryo loss, see text. 369

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CATTLE PRACTICE VOL 5 PART 4 The Advantages of Milk Progesterone Monitoring Dally A E H., Farms Manager, Rangemore Estate, Burton Upon Trent, East Staffordshire INTRODUCTION Skilled breeding management is vital for efficient livestock production, be it for milk or meat. This paper focuses on how milk progesterone monitoring has affected these block calving herds. Rangemore Estate covers 2500 acres near Burton Upon Trent in East Staffordshire. It consists of two dairy units milking 214 and 217 cows and producing 7538 and 7891 litres, respectively. Quota presently stands at 3.167 million litres. Each unit calves within a two block calving period February/March and mid July/mid September with one herd running one month behind the other. Why take part in the monitoring programmes? The main reproductive concern for this farm was replacement rates, re-absorption and accuracy of heat detection. What were the advantages? i) Concentrates the mind The milk sampling and subsequent feedback concentrates the mind on the work needed, to look out for signs of oestrus and getting cows back in calf. This has been confirmed by the results from last Autumns serving. In the first 3 weeks of service 93% of animals were served. This reduces the number of animals that need to be watched. ii) Silent heat There are some cows that even with the best observation, tail paint and KAMAR heat detectors do not show signs of heat. These animals via progesterone monitoring can be predicted as to when is the best time to inseminate them. iii) Missed Heat Man is fallible, observation may be poor, heat detectors may have come off or the cow may be believed to be in calf so not much time is spent on her. A drop in progesterone can trigger questions to be asked, thus saving another 3 weeks until she next comes into heat. iv) Identification of potential problem cows Progesterone monitoring can highlight problem cows that due to cystic ovaries, irregular ovarian activity or a number of other problems may not be cycling correctly. By highlighting these irregularities early, these animals can be identified, treated and hopefully the problems rectified. v) Reduce semen wastage With the progesterone monitoring available, analysis can show when the chances of conception are likely to be low. In these cases cheap semen can be used allowing more expensive semen available to use on those animals more likely to conceive. This could increase genetic merit faster. vi) Supports the vet By having feed back on progesterone profiles, the vets job, analysis and subsequent treatment is helped greatly. This way only the real problem cows are put forward to the vet saving his time and the farms money. vii) Helps the herdsman Progesterone monitoring helps to reduce the pressure on the herdsman during the block-serving period. For example, if he/she knows from the monitoring that 75% of animals are expected to show heat in the last 7 days of the first 3 week period worrying is reduced. viii) Uses MDC/MAFF funding This research is using MDC/MAFF funding that will be of benefit to the dairy industry Drawbacks Being part of a research project, the farm has had to stick to the parameters laid down. This led to some cows that would have been treated earlier for acyclity not being treated until they had finished the trial, consequently one or two cows have not conceived within the desired block. There is a lot of work involved within the trials, this needing first rate staff to undertake the extra workload. The speed that results are returned has a delay on it due to the laboratory techniques, travelling time and weekends. There is also the frustration of knowing what is now possible but not yet being able to use that knowledge CONCLUSION An online progesterone monitoring system will be of definite benefit to the dairy farmer, his cows and his vet, hopefully confirming to the herdsman when to serve the cow and whether she has a problem or is in calf. However, this should by no means minimise the importance of good visual observation of all cattle. This online progesterone monitoring system however will also need software available to interpret profiles in the parlour, saving valuable time. BCVA 1997 371

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