MILK DEVELOPMENT COUNCIL PRIORITIES FOR RESEARCH AND TECHNOLOGY TRANSFER FOR THE IMPROVEMENT OF DAIRY CATTLE REPRODUCTION

Transcription

1 MILK DEVELOPMENT COUNCIL PRIORITIES FOR RESEARCH AND TECHNOLOGY TRANSFER FOR THE IMPROVEMENT OF DAIRY CATTLE REPRODUCTION Project No. 97/R1/22

2 MILK DEVELOPMENT COUNCIL PRIORITIES FOR RESEARCH AND TECHNOLOGY TRANSFER FOR THE IMPROVEMENT OF DAIRY CATTLE REPRODUCTION Peter J H Ball, SAC Auchincruive Ayr, KA6 5HW Andrew R Peters Formerly Royal Veterinary College Bolton s Park Potters Bar EN6 1NB

3 CONTENTS Summary... 3 Review of Scientific Literature... 4 Farming press review Survey of MAFF and private Veterinaty Investigation Laboratories Survey of members of the British Cattle Veterinary Association Fertility Databases Ongoing Research New Technologies Conclusions References

4 SUMMARY Information in this report was obtained from a study of recent scientific and technical literature, as well as an interrogation of large fertility databases and the progesterone profile data available from seven years monitoring at SAC. This was supplemented by a survey of Veterinary Investigation Centres and members of the British Cattle Veterinary Association. It is very apparent that fertility problems are a major constraint to efficient dairying in the UK and that they are getting worse, rather than improving. Oestrus detection problems continue to cause severe delays to conception, whilst embryo/foetal loss is on the increase and has major economic consequences. Interactions between fertility problems and disease, nutrition/metabolism and stress have been identified. In some areas, management factors have been identified which indicate potential improvement through farmer awareness. The MDC can contribute to improvements by means of three main strategies: a) Publicity and training, including advisory leaflets emanating from this study with topics including oestrus detection (with a new emphasis on e.g. cow-side tests and false oestrus), service management, embryo/foetal loss (explanation, implications and management strategies) and synchronisation (pros and cons, methods and management tips). b) Strategic research with individual projects, but also a collaborative approach between groups with facilities for e.g. scanning and progesterone monitoring. c) Basic research, especially in areas such as embryo/foetal mortality, with an integration between basic and strategic research and between disciplines (e.g. reproduction; production; nutrition; health and welfare) 3

5 REVIEW OF SCIENTIFIC LITERATURE ON IMPORTANT ASPECTS OF FERTILITY OF DAIRY COWS The review will be dealt with under a number of subheadings as follows: Ovarian function and follicular dynamics New developments in oestrus detection Establishment of pregnancy and embryo mortality Pharmacological treatments The effects of nutrition Metabolic stress in high yielding cows Decision support systems and epidemiological aspects of fertility Diagnostic methods and their improvement Interactions of disease and fertility Genetic aspects of fertility 4

6 1.) Ovarian function and follicular dynamics This account draws heavily on the recent excellent review by Gong and Webb (1996). The formation of the ovarian primordial follicle pool is complete by the time of birth of the heifer and each of the several thousand primordial follicles is potentially capable of producing an ovum for fertilisation and future embryo. However only % will ever develop to this ovulatory stage (Erickson, 1986). Follicles develop from this non-proliferating pool on a continuous basis throughout the life of the cow but the triggering factor for this is unknown. Once this development has started follicles are destined either to ovulate or to undergo atresia (degeneration). It is currently thought that atresia is controlled by a process of programmed cell death or apoptosis (Hsueh et al.,1994). Recent studies using transrectal ultrasound have confirmed that follicle growth and development occurs in a wave like pattern and that there are normally two or three waves per cycle (Sirois and Fortune, 1988; Savio et al., 1988; Knopf et al., 1989). In each wave a cohort of 5-7 follicles grows to a diameter of >5mm (recruitment). One of these begins to grow more rapidly than the others (selection) to a maximum diameter of about 15mm (dominance) and remains at this size for at least 2-3 days before regressing. This is then followed by the emergence of a new wave. If the growth phase of a dominant follicle coincides with luteolysis then this follicle matures further and eventually ovulates releasing the ovum. This follicular wave pattern also occurs during the post-partum period before the first ovulation (Rajamahendran and Taylor, 1990; Boland et al., 1990) and during early pregnancy (Ginther et al., 1989). 5

7 Follicle growth and development are controlled by at least three different interacting mechanisms: the hypothalamo-pituitary -ovarian axis a non-gonadotrophin hormonal axis including growth hormone (somatotrophin), insulin and insulin like growth factors (IGF s) local intraovarian factors. Follicles up to a diameter of about 4 mm seem to be independent of gonadotrophin effects. FSH appears to be involved in recruiting follicles from this pool for further development and LH is involved in the selection of the dominant follicle. Study of the role of the non gonadotrophic hormones came about mainly through interest in the effects of nutrition on reproduction since it became apparent that the metabolic hormones such as growth hormone, insulin and the IGF s can affect ovarian function either directly or through modulating effects of the gonadotrophins on the ovary. Also field studies of the effects of bst showed an increase in the prevalence of twins from about 5% to about 10% (see Gong and Webb, 1996). Detailed studies then showed that treatment of heifers with growth hormone increased the number of small antral follicles between 2 and 5mm in diameter (recruitment), about twofold. However GH treatment had no effect on LH or FSH concentrations or their receptors. Furthermore there is evidence that insulin and IGF1 also increase the gonadotrophin responsive follicle pool and that natural twinning is associated with higher circulating concentrations of IGF1 (Echternkamp et al.,1990). Various local growth factors and their related binding proteins including IGF1, IGF2, transforming growth factors α and β, epidermal growth factor and fibroblast growth factor are probably involved in controlling follicle growth but their roles have not 6

8 been delineated in cows. However the expression of these factors and their receptors probably determines the differential fates of follicles within the same gonadotrophin environment (Gong and Webb, 1996). Follicle dominance was initially thought to be due to negative feedback of follicular oestradiol and / or inhibin on FSH secretion. In this way there would be reduced FSH stimulation of the subordinate follicles while the dominant follicle becomes increasingly sensitised to lower levels of FSH or transfers its dependence from FSH to LH. Treatment of cows with steroid depleted follicle fluid suppresses follicle development. However it has now been shown that follicle fluid depleted of both steroids and inhibin can inhibit follicle development without changing FSH, therefore suggesting alternative mechanisms of suppressing subordinate follicle development. Thus follicle dominance results in decreased FSH secretion, direct inhibition of other follicles and an increase in the responsiveness of the dominant follicle. Therefore ovarian follicle growth and development is a highly complex and coordinated process. Better understanding of the mechanisms controlling follicle selection and dominance is pivotal in a better understanding of the factors limiting good reproductive performance from the following points of view: the mechanisms governing the timing of ovulation and oocyte quality with reference to the timing of insemination the control of ovulation by pharmacological treatments the vital relationship between ovulation, luteinisation and the onset of progesterone secretion 7

9 Research to date on patterns of follicle development and the controlling factors has been important in developing more effective pharmacological treatments e.g. the use of antiluteolytic treatments to prevent embryo loss during early pregnancy and the combined control of follicle as well as luteal function in the control of ovulation (see section 4). 2.) New developments in oestrus detection Mottram (1997)has recently produced a detailed report on developments in oestrus detection for the MDC and therefore this account will be brief Failure to detect oestrus is still a major factor causing delays in service, the detection rate being highly correlated with the calving interval (Morant,1983). Average detection rates are around 55% in the UK and have stubbornly remained so for the last 25 years (R. J. Esslemont, personal communication). The methods and aids available e.g. behavioural observations, milk progesterone, mount detectors etc. have been extensively reviewed but none are completely reliable with problems of detection failure and false positives. The problem of oestrus detection is ripe for new major research initiatives. For example, developments in sensing technology offer exciting possibilities for the future e.g. detection of oestrous pheromones by 'electronic noses', and/or on-line milk hormone determinations. 3.) Establishment of pregnancy and embryo mortality This has been the subject of intense research over the last few years and has been reviewed recently by Peters (1996) and what follows draws heavily on that review. 8

10 Approximately 25% of embryos are lost during the first 3 weeks of life. The maintenance of progesterone secretion by a viable corpus luteum is vital to early pregnancy and untimely luteolysis is probably a major cause of embryo loss. In the non-pregnant cow the luteolysin PGF2 α is secreted from endometrial cells following the activation of specific receptors by oxytocin secreted from the corpus luteum. The endometrial oxytocin receptor concentration is increased after about 10 days exposure to progesterone and possibly by oestradiol-17β from waves of ovarian follicle development occurring during the luteal phase of the cycle (see Section 1). During early embryonic life the trophectoderm produces the protein bovine trophoblast interferon (bifn τ ). This protein inhibits PGF2 α secretion and probably constitutes the major signal for maternal recognition of pregnancy. The economic and biological significance of embryo mortality in cattle and other farm species is well recognised. It has been known for many years that in the absence of infectious disease of the reproductive system, it is the most important factor limiting reproductive efficiency in farm livestock. In the United Kingdom alone the cost in losses to farmers is estimated as high as 200 million per year and $1.4 billion in the USA (Gerrits et al., 1979). Normal embryonic development Before considering embryo mortality it is worthwhile reviewing briefly the normal processes involved in embryonic development end establishing some definitions and time relationships (see Figure 1). Ovulation in the cow takes place about 24 hours or so after the onset of behavioural oestrus. Consequently it has been found that artificial insemination is likely to be most successful if performed towards the end or just after oestrus preferably about 6 hours before ovulation (Hunter, 1982). 9

11 10

12 Normal embryonic development and implantation have recently been reviewed by Guillomot (1995). The embryo enters the uterine horn at the morula stage at about 4-5 days after fertilization. The embryo then becomes hollowed to form a blastocyst at days 6-7. The zona pellucida ruptures, resulting in 'hatching' of the embryo after 9-10 days. The blastocyst begins a process of elongation from about day which is accompanied by the secretion of embryonic interferons (see below). Early attachment of the conceptus to the endometrium takes place from about day 19 and actual adhesion is occurring by day The extent and timing of embryo mortality The extent and timing of embryo mortality have been reviewed many times but most recently by Sreenan and Diskin (1986). The main features are summarised in Table 1. Table 1: Approximate pregnancy rates and cumulative embryo/fetal loss in cattle. Data extrapolated from the literature reviewed in the text. Day Pregnancy rate (%) Cumulative loss (%) (calving rate)

13 Due to the small size and inaccessibility of embryos there are severe practical difficulties in detecting and monitoring the stages of early development. Most studies have involved the method of slaughter of groups of cows/heifers at a series of time points after service. Sreenan and Diskin (1986) reviewed the published data and concluded that fertilisation rates for both cows and heifers were very similar at 90% and 88% respectively. However the studies reviewed were all carried out under experimental conditions and therefore fertilisation rates may well be lower under practical farm conditions. Since the proportion of animals which calve to a given insemination (the calving rate) is about 55%, a further loss of 30-35% occurs between the time of fertilisation and parturition. Losses after day 42 (fetal losses) have been estimated at about 5% (Boyd et al.,1969). Therefore approximately 20-25% inseminations fail during the embryonic period i.e. between days 1 and 42. Within this period most loss has occurred before day 25 with the most substantial losses occurring between days 8 and 13 (8-9%) and between days 14 and 19 (13-15%) (Sreenan and Diskin, 1986), although there are also losses later in pregnancy (Ball, 1997). Causes of embryo loss and reproductive expectancy Despite many years of study the causes of embryo loss in farm animals are poorly understood. Definitions and diagnosis of problems are notoriously imprecise, particularly in attempting to differentiate between what is normal and what is abnormal i.e. pathological. For example in abattoir surveys most cows culled as infertile do not have any macroscopically visible pathological changes (Dawson, 1975). Nevertheless only 55% of cows conceive successfully to first insemination. Similarly studies have shown that most but not all cows have an equal chance of conception at each consecutive service. Of course a small proportion will repeat due to pathological abnormalities. 12

14 Repeat breeders are cows which fail to conceive after several (usually 3 or more) services (Ayalon, 1978; 1984) but which appear normal on superficial clinical examination (see review by Lafi and Kaneene, 1988).. These animals are often regarded as abnormal but firm evidence is limited. To this extent the term repeat breeder is unsatisfactory since it does not distinguish between possible causes related to the cow, bull, environment or management. However there are indications that some older cows may become less fertile associated with some endocrine changes, e.g. Shelton et al. (1990) showed evidence of luteal inadequacy in cows culled for failure to conceive successfully to repeated inseminations. It was suggested by Bishop (1964) that as in humans, the majority of embryo loss in cattle occurs because of genetic abnormalities. These embryos are eliminated early in their life at a 'low biological cost'. However cytogenetic studies have shown that gross chromosomal defects can account for only 3-5% of losses (Gayerie de Abreu et al., 1984). Table 2 shows the expected reproductive performance of a herd of 100 cows with a 60% per cent calving rate to first insemination. Table 2: Reproductive expectations for 100 cows each with a 60 per cent probability of calving to each service Order number of inseminations Number of inseminations Number of calves born Number of returns to service Total

15 On average 60% of first services, second services and so on will result in a successful full term pregnancy. Therefore 6.4% of cows will require 4 or more services even though there may be no detectable abnormality. Thus about 1.65 services are needed for each calf born. Luteal function during early pregnancy Plasma and milk progesterone concentrations rise during the first few days of pregnancy in an identical manner to that in the early luteal phase of the non-pregnant animal (Figure 2). In the pregnant cow high concentrations are maintained for the duration of the pregnancy and are regarded as being essential for the maintenance of pregnancy. The synthesis and release of PGF2α normally associated with luteolysis are much reduced if not abolished in the pregnant state. Many workers have studied progesterone concentrations in pregnant and nonpregnant cows to determine whether these affect fertility. There are conflicting reports as to when progesterone concentrations in pregnant cows begin to diverge from the non-pregnant. Some authors have claimed that levels are different as early as day 10 (e.g. Hansel, 1981) whereas others have reported no significant differences until day 18 after insemination (Bulman and Lamming, 1978). It is well established now that the embryo secretes antiluteolytic substances from around day 13 which are probably responsible for differences in progesterone patterns between pregnant and non-pregnant after this time (see below). 14

16 15

17 Lamming et al. (1989) have published the largest database on milk progesterone concentrations in pregnant and non-pregnant cows. This included 1600 cows kept under UK farm conditions. Whilst milk progesterone data may not give a truly quantitative estimate of circulating concentrations at a specific time, they do provide a valuable insight into differences in patterns between cows maintaining pregnancy and those which do not. It should be borne in mind that these data are now 20 years old in which time there have been major changes in the genotype as well as in the nutritional and general management of the national dairy herd. These authors found that milk progesterone concentrations in pregnant and non-pregnant cows rose in parallel and were not significantly different until day 9 after which time they diverged, the concentrations in pregnant cows remaining higher (Figure 2). Lamming et al. (1989) also compared milk progesterone concentrations in pregnant, inseminated-notpregnant and non-inseminated animals. They found lower concentrations in inseminated-not-pregnant than non-inseminated cows but could not explain this difference. They also reported a significant 'dip' in progesterone concentrations of the pregnant group on day 11, followed by a rise which was thought to reflect a 'rescue' effect of the corpus luteum by the embryo at this stage. Whilst it is clear that maintenance of the corpus luteum and continued progesterone secretion are essential for the maintenance of pregnancy, the importance of the concentrations or patterns per se are more equivocal. Veterinarians have used supplementary progesterone in an effort to support pregnancies in a variety of species over the years. However this practice has never been validated satisfactorily and there are known disadvantages. Interest has recently been focused on the rate of rise of progesterone concentrations from the post-ovulatory period up to day 10 of pregnancy. Shelton et al. (1990) reported that the rate of rise in progesterone concentrations was lower in the post-ovulatory period in cows selected for subfertility than in pregnant or non-pregnant heifers. They also showed evidence of a reduced progesterone response to exogenous hormones of luteal cells from older cows in vitro. 16

18 In a recent study by Mann (1996) where embryos were collected from pregnant cows 16 days after insemination, large embryos were found in cows with normal post ovulatory rises in progesterone concentrationswhile small underdeveloped embryos were found in cows where the rise in progesterone was late and the peak concentrations were low. Furthermore the luteolytic signal was inhibited in the cows with large embryos whereas in the cows with small embryos the luteolytic signal was developing. This suggests that the post ovulatory rise in progesterone is crucial in maximising the growth of the embryo so that it is capable of secreting IFN s in time to prevent luteolysis. Therefore a substantial proportion of embryo loss may be explained by a deficiency in the rate of rise of progesterone in the first few days of pregnancy due either to slower luteinisation or a delay in ovulation. Review of milk progesterone data on over 1300 cows has confirmed a wide variation in the length of the follicular phase (Lamming and Darwash, 1995). These authors have demonstrated a negative correlation between this 'interluteal' phase and the pregnancy rate, i.e. as the interluteal phase increases the subsequent pregnancy rate decreases. Although it is not clear whether the extended phase represents a delay in ovulation or a subsequent delay in progesterone rise, it could indicate a delay in embryo development and associated delay in its antiluteolytic effects as discussed above. There are therefore some inconsistencies as to whether the rate of rise in progesterone concentrations is different between those cows that conceive successfully and those that do not with some data suggesting a difference and others not (see for example Figure 2). 17

19 Maternal recognition of pregnancy Short (1969) originated the term 'maternal recognition of pregnancy' as an expression of the fact that luteal function is maintained in early pregnancy and that the normal luteolytic mechanism is inhibited. It was subsequently discovered that in ruminants specific proteins secreted by the embryonic trophoblast are mediators of this maternal recognition (Bazer et al., 1986), which occurs in the cow at around day 15. Ovine and bovine trophoblast proteins (otp-1 and btp-1) are proteins of molecular weight 22,000-24,000, consisting of 172 amino acids now recognised as interferons (IFN) belonging to the IFN-α family (Roberts et al., 1991). They possess the antiviral, immunomodulatory and antiproliferative activity of IFN-α and bind to the same receptor. However they have since been reclassified in the separate family of IFN-tau (Roberts et al., 1992) as they have certain distinctive properties. In particular the ruminant trophoblast interferons have a high amino acid sequence homology to each other that is distinct from the other IFN's α, β and w and they are poorly induced by viruses. The ovine and bovine IFN-tau molecules have about 80% amino acid sequence homology, whereas there is about 50% homology between IFN-α and IFNtau (Roberts et al., 1992). There are 4 or 5 IFN-tau genes in sheep and cattle whose promoter regions are highly conserved and distinct from other type 1 IFN's. These genes are not induced by viruses and are expressed only in the trophectoderm layer from the time of blastocyst hatching until implantation (see review by Mathialagan and Roberts, 1994). Both otp-1 and btp-1 (IFN-tau) are secreted by the conceptus coincident with the blocking of luteolysis (Roberts, 1989). Hernandez-Ledezma et al. (1992) studied the expression of btp-1 (IFN-tau) in embryos derived by fertilization in vitro. IFN production began at the expanded blastocyst stage on days 8-9 just prior to rupture of the zona pellucida and hatching. Transcription of the DNA was confined to the trophectoderm layer. Initial IFN expression does not require the uterine environment but continued viability and 18

20 expression requires exposure to the uterus. Expression ceases after day 19. Assal- Meliani et al., (1993) showed that ovine IFN-tau inhibits lymphocyte proliferation and suggested that it acts by preventing immunological rejection of the early embryo. It has been suggested that some embryo mortality i.e. that between days 14 and 19 and caused by luteal failure, occurs because certain embryos develop more slowly than normal and do not produce enough IFN to prevent luteolysis and maintain the pregnancy (see above). Hernandez-Ledezma et al. (1993) studied the expression of bifn's in vitro and correlated production with embryo quality and stage of development. Assessment of embryo quality in embryo transfer technology is somewhat subjective, based on shape, size, cell number, cellular integrity, appearance of the nucleus and cytoplasm and other less tangible criteria (Lindner and Wright, 1983). Hernandez-Ledezma et al., (1993) therefore suggested that IFN-tau production may be a useful criterion for selection of embryos for transfer i.e. as an index of embryo viability. It has been hypothesised that supplementation of IFN-tau to slower developing embryos might enhance their chances of survival (Thatcher et al., 1995). However until recently insufficient amounts of IFN-tau have been available for such experiments so several have been carried out using IFN-a. Experiments published to date are summarised in Table 3 and are described below. Helmer et al., (1989) infused an enriched preparation of btp-1 into the uterus of cyclic cattle and extended luteal function and consequently the interoestrous interval. They were also able to demonstrate reduced concentrations of PGF2α in the vena cava between days 12 and

21 Table 3: Summary of published IFN supplementation experiments in sheep and cattle. Author Species IFN used Route Improvement in pregnancy rate Effects in non-pregnant animals Nephew et al (1990) Sheep rbifn- Ι1 1 im +16% Martinod et al (1991) Sheep rbifn- Ι1 1 im +5.6% Schalue-Francis et al (1991) Sheep rbifn- Ι1 1 im +21% Helmer et al (1989) Cows enriched btp-1 im NA Barros et al (1992a) Cows rbifn- Ι1 1 im -11% Barros et al (1992b) Cows rbifn- Ι1 1 im NA Salfen et al (1995) Cows rbifn- Ι1 1 iu NA Meyer et al (1995) Cows rifntau * iu NA Decreased PGFM; extended luteal phase Decreased LH; decreased progesterone Decreased PGFM; extended luteal phase Decreased PGFM; extended luteal phase * both ovine and bovine IFNtau were used to equivalent effect. Note: treatments were given at various stages after oestrus but usually twice daily betweem days 11 and 19 im = intra-muscular; iu = intra-uterine 20

22 Injection of sheep with recombinant bovine IFN-α resulted in increased pregnancy rates, lower PGF concentrations and extended cycle lengths in non-pregnant animals (Nephew et al., 1990; Martinod et al., 1991; Schalue-Francis et al., 1991; Parkinson et al., 1992). Although injection of bifn-α extended luteal lifespan in non-pregnant heifers it decreased rather than increased pregnancy rates, despite the use of various treatment regimes between days 11 and 19 after service and a range of doses (Barros et al., 1992a). It was concluded that other non-specific effects of IFN-α were responsible e.g. hyperthermia which occurs following treatment with this protein (Biggers et al., 1987; Newton et al., 1990). However bifn-α is also known to decrease circulating progesterone concentrations possibly due to acute effects on LH secretion (Barros et al., 1992b), as well as suppression of hypothalamic neuronal activity and leukopaenia. The reason for the difference in response of the pregnant ewe and cow to rbifn-α is not clear, but IFN-α may not be pyrogenic in sheep. Oxytocin, oxytocin receptors and luteal function Wathes and Lamming (1995) have recently reviewed the role of luteal oxytocin and its endometrial receptor in controlling the corpus luteum. During luteal regression, pulses of oxytocin are thought to stimulate synthesis and pulsatile release of PGF2α, following an increase in endometrial oxytocin receptors (OTR). In early pregnancy OTR synthesis and consequently PGF2α release are inhibited at least in part, by conceptus interferon production. OTR's are present in anoestrus, oestrus, in the late luteal phase and for most of pregnancy. Overall, plasma oxytocin concentrations parallel changes in plasma progesterone (Figure 2). They are basal at oestrus and rise from about day 2 of the cycle peaking around day 9 (Wathes and Denning-Kendall, 1992) and falling from about days before the onset of luteolysis (see Wathes et al., 1993). The pattern is similar in the pregnant animal with plasma concentrations falling days after insemination. 21

23 Plasma concentrations of PGF2α are low for most of the cycle but pulsatile release may start as early as day 13 with pulse frequency increasing until luteolysis is wellestablished by day 17 or so. In non-pregnant ewes most oxytocin pulses occur in association with PGF2α whereas in pregnancy most are not associated with PGF2α (Hooper et al., 1986). The pattern is assumed to be similar in cattle. Endometrial OTR concentrations are low during the luteal phase of the cycle from about day 5 until about day 17 when they begin to increase peaking at oestrus (see Wathes and Lamming, 1995). The early increase in OTR's is limited to the luminal epithelium (Stevenson et al., 1994) and only occurs in non-pregnant animals. This increase occurs before PGF2α is released and progesterone concentrations begin to decline. The receptors then spread to the rest of the endometrial tissue; the maximum oxytocin concentrations therefore not being reached until luteolysis is complete. Early in the luteal phase progesterone down-regulates the OTR, preventing an increase in concentration for at least 10 days. Treatment of ovariectomised ewes with progesterone decreases endometrial OTR's, but concentrations begin to rise again after days, still in the face of high progesterone concentrations (Vallet et al., 1990). Evidence in vitro suggests that oestradiol-17β also increases the concentration of OTR's but the role of follicular oestradiol in vivo is not clear (Wathes and Lamming, 1995). Oxytocin concentrations may also regulate the OTR since oxytocin infusion during mid-cycle can prevent luteolysis (Flint and Sheldrick, 1985). The size of the PGF2α response to oxytocin challenge is correlated to the endometrial OTR concentration in cows (Lamming and Mann,1995). However the physiological release of PGF2α may not be correlated to the OTR concentration. The PGF2α response to oxytocin may require only a minor increase in OTR concentrations. Also 22

24 ovariectomised ewes have high OTR concentrations and do not respond to oxytocin challenge (Wathes and Lamming, 1995). Control of PGF2α synthesis may also operate downstream to the oxytocin/otr receptor interaction and may involve the phospholipase C pathway controlling the synthesis of the PG precursor arachidonic acid. Progesterone clearly blocks the increase in OTR for about the first days of the luteal phase but the mechanism of this prolonged effect followed by up-regulation is unclear. McKracken et al. (1981) proposed that the increasing progesterone concentrations in the luteal phase down-regulates its own receptor for about 10 days. However there is no evidence either that progesterone receptors increase prior to the OTR increase or that progesterone treatment reduces its own receptor (Wathes and Lamming, 1995). These authors have proposed that progesterone stimulates an endometrial inhibitor of the OTR which has a half life of several days or that a sequence of intracellular events is induced which results in an increase in OTR concentrations after several days. Wathes and Lamming (1995) have suggested a model for the control of the OTR by steroid hormones in the sheep and this is summarised in Figure 3. The area of early embryonic development and mortality is the subject of an MDC / MAFF link grant under the sustainable livestock systems programme with the University of Nottingham, Royal Veterinary College and Roslin Institute. 23

25 24

26 Later or detectable embryo or fetal loss comprises that which occurs between about day 20 and 60 and occurs in a smaller proportion of cows 5-8%, (Ball, 1997). However it is potentially more serious because it causes a longer delay in the calving to successful conception interval. Losses appear to be concentrated between days 30 and 40. Some may be due to genetic factors but most are considered to be due to uterine factors. Also of significant importance are insemination during pregnancy, inadvertent PG injection, pregnancy diagnosis per rectum, and age. Interferons, oxytocin receptors and luteal function During early pregnancy the rise in OTR concentrations is inhibited probably by the action of embryo-derived IFN. Certainly IFN-tau infusion prevents the increase in OTR's in the non-pregnant animal (Mirando et al., 1993). The mechanism of action of IFN may involve suppression of both oestradiol and oxytocin receptors probably at the transcriptional level (Bazer et al., 1994). IFN-tau binds to specific endometrial receptors and the signal transduction system may involve activation of tyrosine kinase and protein phosphorylation as for IFN's in other tissues (Thatcher et al., 1995). These authors have further proposed that IFN-tau inhibits the enzyme PG synthetase by mobilising linoleic acid, a specific inhibitor of this enzyme and which is present in higher concentrations in the endometrium during early pernancy. This results in a redirection of arachadonic acid metabolism from PG's and leukotrienes to epoxy derivatives. Thus linoleic acid is thought to act as a competitive inhibitor of arachadonic acid for PG synthetase. Thatcher et al. (1995) fed heifers either yellow grease (20% linoleic acid) or tallow (2% linoleic acid) (see section 5). The yellow grease group had lower PGF responses to oxytocin challenge and the luteolytic response was slower. They also suggested that other fatty acids eicosapentenoic and docosahexanoic, present in fish oil, may inhibit PGF2α synthesis. 25

27 IFN-tau also induces the 2' 5' oligoadenylate synthetase system which is involved in the antiviral and antiproliferative actions of IFN's (Short et al., 1991). However the mechanism of action of IFN-tau may be even more complex since bifn-a has been shown to stimulate progesterone production by luteal cells in vitro, whilst not affecting oxytocin output (Luck et al., 1992). Also Thibodeaux et al. (1994) showed that extracts from trophoblasts could stimulate progesterone production in vitro. Therefore it is possible that embryonic IFN's exert luteotrophic and antiluteolytic effects at multiple sites. Furthermore other maternal products may interact with embryo-derived IFN's. For example Battye et al. (1993) have suggested that platelet activating factor (PAF) may act in concert with IFN to prevent luteolysis. PAF stimulates PGE secretion in endometrial stromal cells (Tiemann et al., 1995). Although PGE has been shown to be luteotrophic in some circumstances this was not confirmed either in vitro or in vivo in cattle (Shelton et al., 1990; Parkinson et al., 1991) so its exact role is unclear. Imakawa et al., (1993) showed that maternal granulocyte macrophage colony stimulating factor (GM-CSF) may be involved in stimulating embryo IFN production. A summary of the control of PGF2α production and its inhibition during pregnancy is shown in Figure 4. 26

28 27

29 Prospects for prevention of embryo mortality Recent research both in terms of physiological mechanisms and pharmacological treatments (see section 4), has mostly focused on the period of maternal recognition of pregnancy or the antiluteolytic effect. It has been estimated that 13-15% of pregnancies are lost around this time i.e.days or thereabouts, probably related to a failure of the antiluteolytic IFN-tau secretory mechanism. Experiments where gonadotrophic agents hcg or GnRH have been administered with positive effect have achieved results consistent with the degree of embryo loss thought to occur at that time (see section 4). Experiments with IFN-tau supplementation in cattle have not yet been published but those with IFN-α in sheep suggest improvements of a similar magnitude. It should be remembered however that substantial embryo losses (at least 8-9% of pregnancies) occur before this period and are therefore unrelated to lack of IFN production, but some other mechanism(s) possibly including the pattern of progesterone secretion in the first few days after ovulation. Equally the 10% or so failures of fertilization merit research attention. Early experiments where IFN-tau has been supplemented as an antiluteolytic agent are encouraging and the availability of the recombinant molecule may make possible the development of commercial products to improve pregnancy rates on-farm. However if some embryos die because they fail to produce sufficient IFN, we should first fully understand the reasons why. The genetic control of embryo IFN production should be fully elucidated. Otherwise by assisting the survival of IFN-deficient embryos we may risk selection of future breeding stock with even lower fertility than at present. 28

30 4.) Pharmacological treatments i) Embryo mortality Numerous attempts have been made to prevent embryo mortality by exogenous hormones including progesterone, interferons, GnRH and hcg, all of which have produced variable results. These are reviewed below. Ashworth et al. (1989) reported a positive association between the periovulatory progesterone concentration and embryo survival in ewes. Injection of progesterone from days 1-4 post-oestrus increased the rate of embryo development as shown by their increased length when recovered at day 14 (Garrett et al., 1988). Similarly increases in fetal weight have been reported in sheep following progesterone treatment between days 1 and 3 (Kleemann et al. 1994). Such treatment appears to advance uterine development by stimulating production of a series of proteins (Garrett et al., 1989; Lawson et al., 1983; see review by Thatcher et al., 1994). Trials to assess the effects of supplemental progesterone on pregnancy rates are extremely difficult to interpret with significant improvements (e.g. Robinson et al., 1989) reductions (e.g. Van Cleef et al., 1989) or no effect (e.g. Diskin and Sreenan, 1986) being reported. Reviewing the effects of supplemental progesterone, Diskin and Sreenan (1986) suggested that the available data were totally inconclusive and that a better understanding of the physiological control of early pregnancy was necessary before practical application of such treatments could advance. The evidence suggests that progesterone secretion could be a limiting factor to embryonic development during the first few days of pregnancy and supplementation may be beneficial where background fertility levels are low (e.g. Robinson et al., 1989). However progesterone supplementation can advance the timing of the 29

31 luteolytic signal (Burke et al., 1994) and by increasing peripheral progesterone concentrations could reduce pituitary luteotrophic support due to increased negative feedback. Taken together the data suggest that progesterone supplementation may be advantageous in some circumstances but further attention to the rate and timing of delivery is clearly necessary in order to control this exquisitely sensitive period (Thatcher et al., 1994). The role of luteotrophic treatments i) Human chorionic gonadotrophin (hcg) hcg has been used under practical farm conditions at oestrus as a 'holding' injection, to improve the chances of pregnancy, particularly in repeat breeder cows. Such treatment has been reported to increase pregnancy rates (Brown et al., 1973) but in other studies there was no effect (Hansel et al., 1976; Echternkamp and Maurer, 1983). hcg stimulates ovulation and development of accessory corpora lutea when administered in the luteal phase (Price and Webb, 1989; Rajamehendran and Sianangama, 1991). The response was greatest between days 4 and 7 and between days 14 and 16, relative to other times. This was subsequently believed to coincide with the presence of a dominant follicle, at least in cows exhibiting three follicular waves during a cycle (see Webb et al., 1992). In these studies hcg did not extend the length of the cycle indicating that the new corpora lutea are either non-functional or responsive to PGF2α. hcg caused an increase in progesterone concentrations when injected on day 4 of the cycle (Breuel et al., 1989) and increased pregnancy rates although numbers of animals were very small. In the current author's view the most promising approach with hcg has been injection on day 11.5 after service in sheep (Nephew et al., 1994). This 30

32 treatment resulted in higher protein concentrations and interferon tau concentrations in uterine flushings and increased pregnancy rates. Conversely treatment of cows and heifers with hcg on day 5 after insemination did not improve pregnancy rates (Schmitt et al., 1995). As with progesterone treatment it is clear that timing may be all important. Treatment between days 11 and 13 may be particularly significant because this is the normal time of maternal recognition of pregnancy and is clearly a critical period from the point of view of embryo survival. Peters et al. (1998a) have recently carried out studies of the effects of hcg on day 12 of the oestrous cycle, showing that it increases the diameter of the corpus luteum and the circulating concentrations of progesterone for several days and, it delays the emergence of the subsequent follicular wave. thereby reducing oestradiol concentrations. ii) Gonadotrohin releasing hormone (GnRH) Again GnRH has been used on the day of service as a 'holding' injection for first service and problem cows but its efficacy has been equivocal. The rationale has presumably been to induce ovulation at the appropriate time relative to insemination and to stimulate luteinization thereby improving the chances of successful fertilization and embryo survival. Lucy and Stevenson (1986) reported lower progesterone concentrations after GnRH treatment at oestrus whereas Mee et al. (1993) from the same research group reported higher progesterone concentrations as a result of formation of a higher proportion of large luteal cells. There has been recent interest in improving methods of the control of ovulation by the use of GnRH to reprogramme follicle development prior to luteolytic (PGF2α) 31

33 treatment (e.g. Pursley et al., 1995; see section 4ii) This treatment is designed to improve the synchrony of follicular maturation and hence ovulation after PGF2α. Ovulation can be further controlled by a second injection of a GnRH analogue during the preovulatory period. Preliminary data in this area (Peters et al. 1998b;c) showed that GnRH treatment hours after PGF2α can advance and synchronise the preovulatory oestradiol peak, ovulation and post-ovulatory luteal development and associated rise in progesterone concentrations. The implications of this for improved embryo survival following insemination remain to be determined. In recent reviews of the efficacy of GnRH at oestrus in increasing pregnancy rates (Mee et al., 1990; Stevenson et al., 1990), an overall six or seven per cent improvement in pregnancy rates was found (see Table 4). Table 4: Effect of GnRH at the time of insemination on the pregnancy rate of cows. Data summarised from reviews by Mee et al. (1990) and Stevenson et al. (1990) Number of herds Number of cows Pregnancy Rate % Control cows Treated cows % improved First service >60 11, Repeat service 80 3, Such marginal improvements emerging from these meta-analyses of data from almost 15,000 cows illustrate why the efficacy has been equivocal in much smaller individual studies. In a study where buserelin was injected in dairy cows on the day of first insemination (Drew and Peters,1994) the overall response was a nonsignificant 6.1% improvement in pregnancy rate, a figure very close to those cited in the above reviews. Also there was a significant negative correlation between the 32

34 apparent percentage improvement in pregnancy rate in each herd and the corresponding fertility of the control cows in that herd (see Figure 5). In other words the lower the background fertility in a given herd, the higher the percentage improvement. A different approach was developed by MacMillan, Taufa and Day (1986) in New Zealand. Cows were injected with the GnRH agonist buserelin either between days 1 and 3, 4 and 6, 7 and 10, or 11 and 13 after insemination. Only treatment between days 11 and 13 resulted in any improvement in pregnancy rates. This or similar regimes have now been repeated in several countries and the 33

35 results are summarised in Table 5. In the United Kingdom two trials reported improvements of 9.4 and 12.0% respectively (Sheldon and Dobson, 1993; Drew and Peters, 1994), whereas larger studies notably in Australia and Ireland (Jubb et al., 1990; Ryan et al., 1994) failed to find any differences. Table 5: Summary of published data on the effect of GnRH analogues given on days 11 to 13 after insemination. Author Number of Percent pregnant Percent cows Control Treated improved MacMillan et al. (1986) Drew and Peters (1994) Sheldon and Dobson (1993) Jubb et al. (1990) Drost and Thatcher (1992)? Ryan et al.(1994) Clearly further work is necessary to understand the discrepancies between these differing results. The stage at which GnRH was injected in these studies (days 11 to 13) is the approximate time of maternal recognition (see above) and is therefore critical in terms of embryo survival. In view of these conflicting trial results worldwide, such a treatment could only show potential if a real physiological effect could be established. Thatcher et al. (1989) injected buserelin at three-day intervals into cyclic heifers during the luteal phase, starting at day 12. Progesterone concentrations were maintained at luteal levels for as long as the injections continued i.e. until day 48 after the preceding oestrus. This 34

36 indicates that buserelin exerts a continued luteotrophic or antiluteolytic effect under these circumstances. As discussed above, injection of hcg during the luteal phase of the cycle can induce ovulation of the dominant follicle (Price and Webb, 1989). It is now well known that cows exhibit either two or three distinct waves of ovarian follicular development during each cycle (see section 1) and three-wave cycles appear to be the most common. Each wave is associated with an increase in oestradiol-17β secretion. Price and Webb (1989) obtained the highest proportion of ovulations when cows were injected between days 4 and 7, or 14 and 16, periods which coincide broadly with the time of maximum follicle diameters in ovarian cycles of three follicular waves. Webb et al. (1992) injected non-pregnant cows with GnRH on day 6 of the cycle and 75% ovulated with the subsequent formation of accessory corpora lutea. This raises the possibility that treatment at 12 days after insemination induces ovulation and accessory corpora lutea or at least luteinisation of follicles. Apart from inducing additional progesterone secretion this could also result in a 'down regulation' of oestradiol production. The latter has been demonstrated by Mann et al. (1995) who injected cows with buserelin 12 days after insemination. Plasma oestradiol concentrations were reduced compared to control animals. The implication of this response is that oestradiol may cause 'up regulation' of endometrial OTR's in the late luteal phase which in turn control the synthesis and secretion of the luteolysin PGF2α (see section 3). Therefore a reduction in oestradiol concentrations at this time might inhibit the luteolytic mechanism thus allowing some pregnancies to continue. In fact it has been shown that buserelin treatment suppresses pulses of the PGF2α metabolite 13, 14 dihydro 15 keto PGF2α (PGFM) from about day 13 onwards (G.E. Mann, personal communication), confirming an antiluteolytic effect of the GnRH analogue. Most studies found no evidence of extension of oestrous cycles in non-pregnant cows treated with GnRH analogues. Thus Mann et al. (1995) concluded that GnRH treatment weakens rather than delays the luteolytic signal. These authors argue that faced with a reduced maternal luteolytic drive, an embryo may then be more likely to 35

37 be able to produce a sufficient antiluteolytic signal to prevent luteal regression and allow the pregnancy to continue. Thus the treatment may be regarded as buying the embryo time to grow big enough to produce its own antiluteolytic signal so that pregnancy is maintained. The timing of buserelin/gnrh treatment appears critical since treatment at other times after insemination did not have any effect (MacMillan et al., 1986; Drew and Peters, 1994). If the above explanation concerning the prevention of the rise in oestradiol is true then only treatment at around days 11 to 13 would be effective as this is the time of the second follicular peak, maximum oestradiol and beginning of infrequent pulses of PGF2α secretion, also coinciding with normal embryonic IFN production. Recently, sufficient supplies of recombinant IFN-tau have become available to allow experiments in large animals (Ott et al., 1991). Meyer et al., (1995) gave intrauterine infusions of recombinant ovine or bovine IFN-tau to non-pregnant cows. This resulted in extension of the oestrous cycle and abolished the oxytocin-induced PGF2α secretion on day 17. IFN-tau should be more effective than IFN-a in preventing embryo mortality as there should be no side-effects as discussed in section 3 above. ii) Improvement of synchronisation techniques The use of PG's was originally envisaged to allow the synchronisation of the cycles of groups of animals so that they could all be inseminated at fixed times with an expectation of normal fertility. It has since become apparent that responses are more variable than anticipated. The fixed times recommended in the product data sheets are usually 72 and 96 hours or 72 and 90 hours after PG injection. Although it has been suggested that fertility following a single fixed time insemination is equivalent to that after two (Young and Henderson, 1981), this would not be the general view. Indeed 36