The frequency of early and late embryonic mortality was determined following 171 inseminations performed between January and December 1998 in 6 herds located in Reunion Island. Milk was sampled 23 to 24 days after Alfor progesterone determination in cows not observed to return to oestrus after AI. Cows were also blood sampled at between 30 and 45 days post-AI to measure Pregnancy Specific Protein B. Overall pregnancy rate was 25.1% (range between herds 12.5% to 42.3%). Early embryonic mortality or fertilisation failures and late embryonic mortality occurred in 57.3% (range between herds 52.9% to 61.6%) and 17.5% (range between herds 3.8% to 33.4%) of cows inseminated, respectively. Nonpregnant cows, not detected in oestrus by Day 24 after AI were recorded to returned to oestrus, on average 66 ±9.6 days or more than 3 cycles after AI. The pregnancy rate was depressed when cows were inseminated during the wet season compared with cows inseminated during the dry season. We conclude that fertilisation failures or early embryonic mortality was the main cause of reproductive failure in inseminated cows in tropical climates. The incidence was twice that obtained in studies conducted under more temperate climatic conditions.

Fertility in dairy cows has decreased during the past 50 years and current management procedures have done little to rectify this situation. Following intense selection for increased milk production, there are significantly more problems in establishing pregnancy at the appropriate time. In recent years, major strides have been made in the development of new embryo-based biotechnologies, including in vitro embryo production, ovum pick-up, cryopreservation of gametes and embryos, cloning and sexing of gametes. The objectives of this paper are to review current progress relating to embryo-based technologies in cattle.

The consequences of peri-parturient Monensin administration were studied in two dairy herds in ≥ 2 parity HF cows (1) calving in moderate body condition (score, BCS: 3.25-3.75) and producing ≥8500 kg FCM in the previous lactation (Herd K), as well as (2) in those being overfed before calving (BCS: ≥3.75), but producing less milk (5-8000 kg FCM) (Herd S). In both herds on days 249-256 of gestation half of the cows received an intraruminal constant release capsule of Monensin (Rumensin® capsule, ELANCO) (Monensin-K and Monensin-S cows), the others remained untreated controls (Control-K and Control-S cows). Blood samples were taken on day 1-3 after calving and again 4 times with 7 days intervals to assay certain hormones and metabolites. On days 1-3 and again on days 28-35 also the ACTH induced Cortisol response and TRH induced T4/T3 responses were determined. The resumption of ovarian cyclicity was followed by milk progesterone profiles. In the first 10 weeks after calving the milk production of Monensin-K cows (n=13) did not differ from that of Control-K cows (n=14). The cows lost their body weight continuously in the first 10 weeks of lactation in Herd S, whereas only in the first 5 weeks in Herd K. Monensin only slightly influenced the degree of weight loss (on both farms), and the circulating leptin level (determined only in Herd S). Significantly lower insulin, IGF-1, T4, T3, glucose and cholesterol levels as well as less elevated βOH-butyrate and non-esterified fatty acid concentrations were seen in Control-S (n=13) than in Monensin-S (n=ll) cows, first of all in the first two weeks after calving. These ionophor-related differences were less obvious or disappeared in Herd-K. Monensin shortened the postpartum acyclic and anestrous periods as well as the calving to re-conception interval but this effect was significant only in the Herd S.

Coming at a time of increasing concern about the declining fertility of high-yielding cows the organisers of this conference are to be congratulated on achieving a well-balanced programme representative of the breadth and depth of the science that is now being directed to the problem of dairy cow infertility. From a starting point of uncertainty about how to deal with the problem, delegates came away very well informed scientifically and, if perhaps with varying views as to the best way forward, more aware of the multidisciplinary research and development approach now being used to investigate it. The purpose of this perspective is to capture, along with the broad issues, the relevant technical detail of the papers and discussions in a way that hopefully provides a balanced overview of the conference.

The involvement of adrenocortical and thyroid hormones in the pathogenesis of ketosis as well as the ovarian consequences of this metabolic disorder were studied in ≥2 parity cows (n=199) in 3 large scale dairy herds. To compare the plasma / serum concentrations of certain hormones [Cortisol, thyroxin (T4), triiodo-thyronine (T3), insulin, isulin-like growth factor-1 (IGF-l)J and metabolites [glucose (G), acetoacetic acid (ACAC), βOH-butyrate (BHB), non-esterified fatty acid (NEFA), trigliceride (TG), total cholesterol (TCh)J, and the activity of aspartate aminotransferase (AST), blood samples were taken 1 to 3 days after calving and again at 7-day intervals on four other occasions. The ACTH-challenged Cortisol responsiveness and the TRH-induced T4/T3 increase were determined between days 1 to 3 and again between days 28 to 35. The resumption of ovarian cyclicity was followed up by individual progesterone (P4) profiles based on milk samples taken 3 times a week for about 80 to 85 days postpartum. A concentration of 1 mmol/l of BHB level was estimated as a border between hyper- (>1 mmol/l) and normal ketonaemic (<1 mmol/l) conditions. 5 different ketone patterns were distinguished: (1) non-ketotic (n=98; normal ketonaemia in all samples), (2) early type ketosis (n=45; hyperketonemia was detected only in the first week after calving), (3) late type (lacta-tional) ketosis (n=11; after a normal ketonaemic period increasing hyperketonaemia was detected in the 5th, or in the 4th and 5th weeks), (4) temporary ketosis (n=ll; hyperketonaemia was detected for 1-2 weeks in the 2nd and 3rd or in the 3rd and 4th weeks); (5) long-lasting ketosis (n=34; hyperketonaemia had been detected since calving for 4 to 5 weeks or until death or emergency slaughtering). Simultaneously with the hyperketonaemic stage increased NEFA, ACAC, depressed TCh, glucose and decreased insulin, IGF-1, T4 and T3 concentrations were detected in almost all the cases. Obvious metabolic and endocrine alterations were found, however, only in long-lasting ketosis. The TRH-stimulated T4 and T3 responses remained almost unaffected proving intact thyroid function in early and late type as well as in temporary ketosis. Depressed thyroid response and delayed onset of cyclic ovarian function were detected only in cases of long-lasting ketosis. The cows characterized by lower than normal (< mean-SD of non-ketotic cows) ACTH-stimulated cortisol response on days 1-3 after calving showed poorer chancefor spontaneous recovery. There was a significant negative correlation between the IGF-1 level in the 1st week after calving and the duration of the postpartum acyclic period. In late type (lactational) ketosis the cessation of ovarian cyclicity was the most characteristic genital malfunction.

The postpartum changes in plasma leptin levels of dairy cows ovulated within 35 days after calving were compared to of those with delayed onset of cyclicity in two experiments. The cows were sampled for leptin, insulin, IGF-1, thyroid hormones (T4, T3, rT3), Cortisol and also for some metabolites (glucose, NEFA, BHB and TCH) on day 1-3 after calving and again four times 7 days apart (Exp. 1), or in weeks 1, 2, 3, 5 and 10 postpartum (Exp. 2). In Exp. 1 also the standard low dose ACTH-induced Cortisol response and TRH-stimulated T4/T3 responses were determined on days 1-3 and 28-35. In cows of Exp. 1 the leptin level varied within a wide range with a mean of about 4 ng/ml HE in the first samples. Up to week 5 it remained unchanged in cows ovulated within day 35 but reduced in those with delayed onset of cyclicity, resulting in significant difference between the two group means.

In cows of Exp. 2 the week 1 leptin levels were lower and less variable than that of Exp. 1. In weeks 2 and 3 a slightly increasing tendency of leptin pattern was seen in cows resuming their ovarian cyclicity within 35 days whereas in those with delayed onset of cyclicity the leptin level remained unchanged. Up to week 5 this tendency resulted in exactly the same leptin values as seen in Exp. 1 with significant differences between the group means in both of the weeks 5 and 10. It was concluded that the plasma leptin concentration in postpartum dairy cows may interfere with resumption of cyclic ovarian function, but its influence may be only permissive (e.g. a minimal concentration above a supposed threshold seems to be the prerequisite of the onset of cyclicity)

The associations between occurrence of diseases, milk yield, and body condition score on conception risk after first artificial insemination were analysed in an observational study of 43 farms participating in a herd health program. Data were taken from 9369 lactations, from 4382 cows inseminated between 20 and 180 days in milk in the period 1990 to 1996. Two logistic regression models, one containing data from all lactations and a subset containing data from 1762 lactations with body condition scoring were used to determine pregnancy risk at first AI. A 1-point loss in body condition score during the 100 days after calving resulted in a 20 percent reduction in the risk of conception. Relative within herd kg milk yield had a significant and linear negative association with fertility. Increases and decreases in the percentage milk fat (or milk fat to protein ratios) from the monthly test days near to AI date are significantly (P < 0.01) predictive of AI outcome. Metritis, cystic ovarian disease, lameness, and mastitis gave odds ratios of for pregnancy risk ranging from 0.35 to 1.15, largely dependent on the interval in days from final disease occurrence to first AI. The advantages of coding diseases for linear or non-linear time-dependency were examined. Other diseases with significant effects associations to pregnancy risk were displaced abomasum, milk fever and retained foetal membranes. These diseases showed little relationship between fertility and the number of days since last occurrence. Results of this study confirm the negative effects of milk yield, body score condition loss, and disease on dairy cow fertility. The effects of some diseases on first service conception are strongly dependent on the interval since last disease occurrence. This is especially valid for clinical mastitis which has an extremely weak effect on conception if occurring prior to AI and is associated with > 50 percent reduction in pregnancy risk if occurring in the 3 wk period directly after AI.

A prospective cohort study was designed to compare the effects of two different intrauterine antibiotic treatments for endometritis on fertility in a multi-farm trial during a 4 year-period. Product A contained 1.5 million I.E. of Procaine Penicilline-G and 500 mg of Neomycine sulfate in 20 ml of an oil in water suspension with a gas-driven applicator (Ubrocelan®, Boehringer-Ingelheim, Germany) and product B (positive control) contained 2g of Oxytetracycline and 500.000 I.E. Colistine in a 20 ml oil in water suspension (Colicycline® , A. U. V., Cuyk, The Netherlands). Diagnosis was by field veterinarians during routine monthly visits. Pyometra, vaginitis, toxic endometritis and post-insemination uterine treatments were excluded from the study. Only cases of non-toxic endometritis prior to first insemination in the period from 1- 150 days after calving were included. Cows were allocated to treatment group based on ear tag number (even/uneven) at the time of diagnosis by the veterinarian. Cows received only a single treatment. Cases were coded as censored at 150 days if not yet inseminated and at 180 days if not yet pregnant. A total of 623 cases of endometritis in first or later parity cows on 13 farms were used in the analysis. The distribution of treatments among farms was unequal ( chi-square = 0.167) , but no farm had more than 70% of one treatment and farm was included as a correction factorin all models. A larger number of controls than cases also had no effect on study outcome. The effect oftreament on the interval (days) from from treatment to conception or censoring was analyzed with a Cox proportional hazards model. Other variables included in the models were season, parity, herd and time of treatment (late/early) with respect to calving. There was no effect of treatment on the number of days to breeding or days open. Early/late ( cutoff = 40 days) endometritis was a significant factor for the interval from treatment to first insemination and conception rate to first breeding. Early/late metritis, however, was not a factor for the interval from treatment to pregnancy. This study suggests that there is an equal treatment efficacy for intrauterine therapy of endometritis between the two products tested. The authors discuss the growing trend away from intrauterine treatments and the suitability of survival analysis techniques for multi-farm field studies of this type.

Infertility is one of the manifestations of clinical copper deficiency in dairy cattle that results from high dietary molybdenum intakes. This trial investigated the effects of slow release glass copper, cobalt and selenium boluses (Cosecure) on the number of inseminations required for confirmed pregnancy, calving interval and trace element status in dairy cattle. Sixty Holstein Friesian dairy cattle were allocated to one of two treatment groups, 30 cattle were treated with two soluble glass trace element boluses (Cosecure®,Telsol Ltd.) on day 0 and 169 while the remaining 30 cattle were left untreated (Controls). The cattle were grazed at pasture (day 0 to 165) which has been shown to contain high levels of molybdenum (range from 2.3 to 3.1 mg/kg DM). Blood samples were taken between days 0 and 280 to measure copper and selenium status and the number of inseminations and calving intervals were recorded. The control cattle required significantly more inseminations to confirmed pregnancy compared with the Cosecure treated cattle (2.5 v 1.7 SEmean 0.16, p<0.01) and had a significantly longer calving interval (397 v 372 days, SEmean 9.2, p<0.05). Plasma copper concentrations were significantly higher in the Cosecure treated cattle on days 30, 78 and 135 (p<0.01) compared with the controls, although the controls were all in the normal range (> 12 μmol/l). Cosecure treated cattle had significantly higher glutathione peroxidase (GSHPx) activities on day 246 (P.0.05). However, on all sample dates, the GSHPx activities were in the normal range for both treatment groups (>40units/ml PCV). The dietary cobalt intakes of the cows were measured (1.5 to 2.1 mg/kg DM in grass and 2.1 to 2.7 mg/Kg DM in compound) and found to be well in excess of the requirement of 0.2 mg/Kg DM. It was concluded that treatment with slow release glass copper, cobalt and selenium boluses (Cosecure) reduced the number of inseminations required for conception in the dairy cows due to the slowly released copper.

Signs of clinical copper deficiency in ruminant animals include infertility. Blood copper concentrations alone are not an adequate indicator of copper status, as they take no account of the thiomolybdate challenge to the animal. The use of the ratio of caeruloplasmin activity to plasma copper concentration should provide a better indicator of molybdenum induced copper problems. The objectives of this study were to obtain veterinary feedback on the University of Leeds Trace Element Laboratory's (ULTEL) assessment of copper status and responses to any recommended supplementation. Blood samples from 713 female cattle from 114 herds were assessed for copper status, including plasma copper concentrations, serum caeruloplasmin activities and the ratio between the caeruloplasmin and plasma copper (CP/PlCu ratio). On the basis of only plasma copper analysis 95.0 % of the cattle were classified as being of normal copper status using Veterinary Laboratory Agency reference ranges. However, when the CP/PlCu ratio was also considered only 35.6 % of the cattle would be classed as having normal copper status. Appropriate copper supplementation would be required by 30.7 % of the cattle and a further 27.8 % could also benefit from additional copper. Veterinary responses to a questionnaire (80 % returned) indicated that infertility was a major problem in their clients’ herds. Where copper supplementation was recommended and administered an increase infertility was observed. In herds showing poor or lack ofoestrous behaviour/ later embryo loss (signs of clinical copper deficiency), 91.7 % reported that these signs were reduced by copper supplementation. The relevance of the CP/PlCu ratio as a diagnostic aid in the identification of copper responsive infertility has been supported by the responses of the veterinary surgeons.

The aim of the study was to evaluate differences between cows in the “quality” of their oocytes defined as their ability to support embryonic development. Ten cows from the same herd, all primiparous and non-pregnant were submitted for oocyte collection by Ovum-Pick Up (OPU). The oocytes were matured in vitro and fertilised with semen from the same bull. In vitro embryo development, both quantitatively (percentages showing cleavage and forming blastocysts) and qualitatively (differential cell counting in blastocysts) was determined at the blastocyst stage (Day 7). The number of oocytes collected, the number of blastocysts obtained and the blastocyst formation rate varied between cows (P<0.001). The mean percentage of inner cell mass cells tended to higher for embryos derived from one cow. These results provide evidence that the quality of the oocytes was influenced by their maternal origin. Follicular growth also varied between cows.

The reported post transfer survival rate to term of cloned embryos derived from both undifferentiated blastomeres and differentiated foetal and adult cells is very low (typically less than 20%). Furthermore, it is acknowledged that there are many technical issues that remain to be resolved to improve the efficiency of nuclear transfer before the technique will find widespread, practical and cost-effective use in multiplying valuable livestock in agriculture. The purpose of this study was to compare early embryo morphology following embryo transfer of nuclear transfer blastocysts derived from somatic cells of an adult Friesian cow with that of standard in vitro-produced embryos. In the present study, 150 embryos were transferred in bulk (i.e., 15, 20 or 25 per recipient) to the ipsilateral uterine horn of 8 recipients using standard non-surgical embryo transfer procedures. Embryos were then recovered following necropsy on either Day 14 or Day 23 of pregnancy and developmental traits described. Embryo recovery and elongation rates were similar on Day 14 of pregnancy (Table 1), although cloned conceptuses were longer and narrower (P<0.05). Embryo recovery and viability rates by Day 23 were similar, although many of the developmental traits appeared more advanced in cloned conceptuses. (Table 1). Allantois development was different because of greater widths and the presence of ‘spurs’ that were not observed with in vitro-produced embryos. We conclude that apparently abnormal conceptus development occurs by about 3 weeks of pregnancy following nuclear transfer, but that embryo survival is not compromised at this early stage of development.

Reduction of the number of days open is economically desirable for the dairy producer. A test is currently being marketed which claims to accurately detect the open cow at 3-7 days post breeding via the detection of a unique protein detected in the serum of the pregnant cow. Evaluation of this test revealed a sensitivity of .51 and a specificity of .55. These results indicate that the test, as performed in this study, did not accurately assess the open status of dairy cattle.

There is no information on the metabolism of the cattle embryo during the period from day 8 to 16 a period of greatest embryonic loss. In this study the rate of protein synthesis and phosphorylation was measured in 13 to 15 day old cattle embryos. The rate of glucose utilisation and amino acid uptake/efflux by day 14 to 16 embryos was also measured. Protein synthesis and phosphorylation activity when expressed per unit of protein decreased with increasing embryo size and age. Similarly the rate of glucose utilisation was greatest for the earlier day 14 embryos. Embryos differed in their requirement for different amino acids. The pattern of uptake/efflux was similar to that of the earlier day 7 embryo. This study suggests that the metabolic rate of cattle embryos expressed per unit of protein content tends to decrease with increasing age and size from the initial burst of activity at day 13 around the time that expansion of the embryo begins.

An analysis on the outcome of 124 inseminations monitored using daily milk progesterone concentration data revealed that those cows in which pregnancy did not occur experienced a 1.7day delay in post-ovulatory luteal progesterone rise when compared to the pregnant group (P<0.001). This suggests that luteal progesterone secretion needs to be initiated prior to day 5 post-ovulation. A comparison between the milk progesterone concentration on day 5 post-ovulation and the pregnancy rate for 1451 inseminations showed the maximum pregnancy rate to occur when the milk progesterone concentration was between 3-9ng/ml. Pregnancy rate fell significantly (P<0.01) when progesterone concentrations were below 3ng/ml, identifying cows that may benefit from progesterone supplementation. The supplementation of progesterone in cows with a day 5 milk progesterone concentration of less than 3ng/ml, using an intra-vaginal progesterone releasing device, only provided benefit for those cows with a day 5 progesterone concentration of between 1-2ng/ml, resulting in a doubling of the pregnancy rate. This data shows that a delay in post-ovulatory progesterone secretion has a detrimental effect on pregnancy but that not all cows with a deficient progesterone concentration benefit from progesterone supplementation.

Follicle development occurs in two or three waves during the bovine oestrous cycle. Artificially extending the duration of ovulatory follicle dominance influences pregnancy rates in cattle, as does the interval from emergence to oestrus in dairy cows undergoing spontaneous oestrous cycles. The objectives of the presented study were to determine whether the interval from ovulatory follicle emergence to oestrus might be altered by diet and/or gonadotropin-releasing hormone (GnRH) treatment. Lactating primiparous Holstein/Friesian cows (n=21) were randomly allocated to one of two diets at calving (Diet 1, n=ll, DM 480 g/kg, metabolisable energy 12.0 MJ/kg DM crude protein 178 g/kg DM, oil B 48 g/kg DM, neutral detergent fibre 318 g/kg DM and diet 2, n=10, DM 440 g/kg, metabolisable energy 12.1 MJ/kg DM, crude protein 172 g/kg DM, oil B 40 g/kg DM, neutral detergent fibre 300 g/kg DM). From 10 days after observed oestrus (oestrus 1), ovarian follicular and luteal development was monitored by daily transrectal ultrasonography until the subsequent oestrus and ovulation. A GnRH analogue was injected (i.m.; 10 μg) 12 days after oestrus 1 in 6 cows fed diet 1 and 5 cows fed diet 2. Oestrous cycle length was longer (p<0.05) in control cows fed diet 1 than those fed diet 2. Treatment with GnRH increased (p<0.005) cycle length in cows fed diet 2 but not those fed diet 1. Increases in cycle length observed were associated with longer luteal phase length. Follicular phase length was reduced (p<0.05) by GnRH treatment in cows fed diet 1. Ovulatory follicles emerged later (p<0.05) in control cows fed diet 1 than those fed diet 2. GnRH treatment delayed (p<0.01) the emergence of the ovulatory follicle in cows fed diet 2, this delay was associated with an increase (p<0.05) in the incidence of 3 follicle waves in oestrous cycles following GnRH treatment. The interval from emergence of the ovulatory follicle to the subsequent oestrus was similar among the treatment groups. We conclude that treatment with GnRH during the mid-luteal phase may delay the emergence of the ovulatory follicle. However, the response is dependent on diet fed. Where ovulatory follicle emergence is delayed, the interval from emergence to the subsequent oestrus was unaffected since oestrous cycle length is extended.

The aim of the study was to conduct a meta-analysis of published data on the effect of GnRH analogues given between 11 and 14 days after first insemination, on pregnancy rates of cattle, to ascertain if a consolidated estimate of treatment response could be determined. The results of 18 studies from 13 published papers were analysed. Odds ratios calculated for each study were found to be heterogeneous, i.e. there was significant variation in treatment response among studies, between 0 and 22% improvement in pregnancy rate. Logistic regression analysis showed that five variables affected the outcome of the model, viz., herd type (beef or dairy), age of animal (cow or heifer), oestrus synchronisation and method and time of pregnancy diagnosis. However when all variables were included only 6/13 of the publications could be analysed (2,541 /10,833 animals).

The present study determined the effect of a GnRH and PGF2α treatment on proportion of cows detected in oestrus, the distribution of oestrous activity and subsequent fertility. The study also incorporated the use of ovarian ultrasound and milk progesterone assay to established a better overall assessment of treatments. Holstein-Friesian cows, averaging 9000-kg milk/lactation, and fed according to their requirements were used in the study. Cows were regularly cyclic; at least 60 days postpartum, had had two oestrous cycles and were clinically sound before being considered for either experiment. In Experiment 1, 274 cows were treated at random with an i.m. injection of 10 μg of GnRH (buserelin) followed 7 days later by 0.75 mg oftiaprost based on the ultrasound diagnosis of a corpus luteum. At that time and 3 days later, milk samples were collected and assayed for progesterone. Cows with progesterone concentrations >1 ng/ml were considered to have a corpus luteum, and luteolysis was assumed to have occurred when concentrations 3 days later were <0.8 ng/ml. In Experiment 2, 1431 cows were treated as above. The response to treatment was assessed by presence of a corpus luteum 7 d after GnRH treatment, oestrous detection rate, and distribution of heats and ultimately on conception rate. Conception rate was compared to untreated control cows inseminated by the same inseminator and using semen from the same sires. Artificial breeding was carried out after oestrous detection and pregnancy diagnosis was done by ultrasound. In Experiment 1, luteolysis was induced in 244/274 (89.1%) cows following PGF2α administration, with 198/214 (92.5%) been diagnosed correctly in heat and 16/214 (7.5%) been diagnosed in error based on progesterone profiles. Oestrous activity was most concentrated on day 3 (59.1%), with 20.7% and 16.7% of heats recorded on d 2 and d 4, respectively. When compared to untreated controls, GnRH-PGF2α treated cows had a better conception rate (56.6 vs. 39.5%, P<0.01). In Experiment 2, 1012/1431 (70.7%) had a CL 7 days after GnRH injection; 709/1012 (70.1%) cows were detected in oestrus, and 359/700 (51.3%) cows became pregnant after insemination compared to 182/434 (41.9%) of untreated controls (P<0.05). The utilisation of GnRH and PGF2α seems to be a useful synchronisation scheme based on AI after oestrous detection. However, variations in fertility after treatment demands a better understanding offactors affecting herd fertility under controlled schemes.