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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

A SID 5A form must be completed where a project is paid on a monthly basis or against quarterly invoices. No SID 5A is required where payments are made at milestone points. When a SID 5A is required, no SID 5 form will be accepted without the accompanying SID 5A.

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Project identification

1. Defra Project code LS3306

2. Project title

Increasing dairy cow fertility through the precise control of nutrition

3. Contractororganisation(s)

School of BiosciencesDivision of Agriculture & Environmental SciencesUniversity of NottinghamSutton Bonington CampusLoughborough, LeicsLE12 5RD

54. Total Defra project costs £ 2,020,977

5. Project: start date................ 01 January 2001

end date................. 31 March 2006

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

Sub-fertility in dairy cows is one of the most important problems facing the UK dairy industry. Calving rate to first service is only approximately 40% and this has been declining at about 0.75-1% per annum. Reduced fertility also has major implications for the economic sustainability of individual dairy herds and the economic competitiveness of the national herd, which is approaching the point where it is not sustainable. In addition, total methane and ammonia emissions from dairy herds could be reduced by 20% if fertility was improved to levels achieved by the best herds. Indeed modelling suggests there is a threefold difference in methane emissions per unit output within the range of dairy systems found in the UK today. Hence the The objective of this work was to determine the specific nutrients that have direct effects on the reproductive system, particularly follicle growth, oocyte quality and embryo survival in dairy cattle. This would then enable the formulation of diets for improved reproduction, rather than just for increased milk production. The work described encompassed twelve major in vivo studies, with 50% of the work carried out in lactating dairy cows. It also included an additional six in vitro studies, with associated in vitro approaches linked to the in vivo studies, including the development of new methodology (e.g. differential staining of blastocysts).

A number of significant conclusions have come out of this programme of work:

Carbohydrate source can affect oocyte and embryo quality, resulting in changes in cell number and rate of blastocyst production.

Increased levels of dietary starch can have a detrimental effect on the developmental potential of oocytes in the high-yielding dairy cow.

Feeding starch based diets increased plasma insulin concentrations whereas leucine was accompanied by an increase in plasma glucagon, but not insulin concentrations.

Fatty acid supplementation improved oocyte quality resulting in higher blastocyst production.

Source of dietary fats influenced oocyte developmental potential by affecting both the cleavage rate of oocytes and the quality of the embryos.

Higher milk yields were associated with reduced developmental potential of oocytes, but

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a high-fat diet buffered oocytes against these effects.

Fibre based diets appeared to be associated with more developmentally competent embryos.

The effect of level of feeding on oocyte quality was dependent on body condition.

Importantly the effect of level of feeding on oocyte quality was demonstrated to be cumulative over time.

Fatter animals on a higher level of feeding can be hyperinsulinaemic, and it was demonstrated for the first time in ruminants that this condition is associated with impaired oocyte quality and blastocyst development.

A change in diet can induce a rapid alteration in peripheral metabolic hormones, such as insulin, IGF-1 and leptin, within 2-3 days and these are associated with changes in ovarian function.

Diet appears to impact directly on early luteal function in that dietary induction of higher insulin secretion was associated with a decrease in serum progesterone in lactating dairy cows.

Modifying the feeding regime successfully produced large diurnal fluctuations in hormone concentrations, but there appeared to be no associated effect on oocyte quality in heifers.

Leptin can act directly on bovine granulosa and theca cells to inhibit steroidogenesis.

In conclusion, this integrated approach using both lactating dairy cows and dairy heifers, and incorporating both in vivo and in vitro approaches, has resulted in a significant advance in our understanding of how dietary factors can influence oocyte quality and hence embryo development. These results have also demonstrated how quickly diet can have its effect on peripheral metabolites and hormones, even in a ruminant species. The findings have also shown that these effects are cumulative over time, are closely integrated with body condition and have a significant impact on both the follicle and oocyte at all stages of development. Interestingly, the results demonstrated that diets optimum for the initiation of oestrous cycle activity may not be suitable for the production of good quality embryos.

These findings were utilised in the associated Link Project (LK0646) to develop similar dietary regimes which resulted in a significant increase in pregnancy rate from 27% to 60% at 120 days of lactation (see final Link Project report). Hence using this dietary strategy we demonstrated that dairy cow fertility can be enhanced without compromising milk production. In this regard, this combined programme of work demonstrated that the fertility of 10,000 litre cows could be altered to the equivalent fertility of 6,000 litre cows, namely fertility was restored to levels seen 20 years ago. Significantly this was achieved whilst still maintaining milk yields of 9-10,000 litres. Importantly, such an improvement would also have a marked positive impact on reducing the environmental footprint of dairy cows. Possible reductions have been estimated as 24% of national methane emissions and 17% of ammonia emissions. Indeed this possible reduction in emissions will not be achieved unless the current problem of infertility in dairy cows is overcome. In summary, the increased understanding which has emanated from this strategic work could have a significant impact on the nutritional and reproductive management of lactating dairy cows. In addition, this improvement in fertility, if implemented after further confirmatory studies, would also have a beneficial environmental impact.

Future work: The progress made in the previous programme of work has provided both a sound basis and the confidence that further significant progress can be made. A parallel three pronged approach combining nutritional, genetic and oestrus studies needs to be continued as all these aspects impact on dairy cow fertility. This work should include (i) improved dietary strategies for increased reproductive efficiency, (ii) identification of specific genetic markers for selecting more fertile cattle and (iii) improve both oestrus detection rates as well as increasing the proportion of cows exhibiting standing oestrus.

This programme of work would utilize the modern high-yielding Holstein dairy cow, since this is the important breed in the UK numerically, and has the greatest problem with fertility. All projects would also be informed by the need to assess the environmental impacts of any proposed changes in dairy cow management, with a view to minimizing undesirable environmental

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outputs. In this regard, this integrated programme will also provide novel data on methane and nitrogen emissions in response to diet composition from individual cows kept under commercial conditions. It would also allow the development of strategic plans for reducing methane and nitrogen emissions by UK livestock.

Hence this integrated approach would not only result in an improvement in dairy cow fertility, a key requirement if environmental targets are to be met, but would reduce environmental impact by dietary improvements, whilst maintaining economic sustainability. An impact on economic sustainability would help to ensure the uptake of new management strategies, whilst helping to safeguard UK milk production.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

Scientific Objectives:Sub-fertility in dairy cows is one of the most important problems facing the UK dairy industry. Calving rate to first service averages 40%. Work at Nottingham has demonstrated that it has been declining at about 0.75-1% per annum over the past 30 years, while milk yield has almost doubled (Royal et al. 2000). Reduced fertility therefore has major implications for the economic sustainability of individual dairy herds and the economic competitiveness of the national herd, which is approaching the point where it is not sustainable (Lucy 2001). In addition, Garnsworthy (2004) showed that total methane and ammonia emissions from dairy herds could be reduced by 20% if fertility was improved to levels achieved by the best herds. The model estimates that there is a threefold difference in methane emissions per unit output within the range of dairy systems found in the UK today. Hence the aim of the work was to identify the means of counteracting this decline in UK dairy cattle fertility. Indeed an improvement in conception rates of approximately 10% per year would benefit the UK industry by at least £300 million per annum.The objective of this work was to determine the specific nutrients that have direct effects on the reproductive system, namely follicle growth, oocyte quality and embryo survival, in dairy cattle. This would then enable us to formulate diets for improved reproduction, rather than just for increased milk production. These improved diets should ensure that the appropriate patterns and levels of gene expression, required to sustain the key homeorhetic processes, are present, thereby resulting in increased conception rates and hence reversing the current decline in dairy cow fertility. The experimental approach involved feeding cattle on diets that were precisely formulated to alter key metabolic factors known to influence reproduction, monitoring follicular changes in these cattle by ultrasound scanning and carrying out in vitro culture of oocytes and blastocysts.In summary, this work is central to the challenge of improving the efficiency and welfare of the UK dairy industry while reducing the environmental impact, through a more scientific approach to nutrition. The overall objective was to move from empiricism to prediction by determining the principles underlying the dietary effects on fertility and so help to reverse the current decline in fertility in UK dairy cows. The specific objectives of the work were:Objective 1: To determine the effect of diet on oocyte quality and the developmental potential of the oocyte

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Objective 1.1: Effect of diet on oocyte quality and embryo development in lactating dairy cows.Objective 1.2: To determine the effect of specific nutrients on oocyte development.

Objective 2: Identification of specific nutritional factors that influence oocyte maturation and blastocyst development.Objective 3: To investigate whether diets fed for shorter periods (3-4 days) can have significant effects on reproductive functionObjective 3.1: To determine the effect of short-term feeding on metabolic hormones, oocyte quality and follicular function.Objective 3.2: To determine the effect of short-term feeding on corpus luteum function.Objective 4: To evaluate the role of leptin in controlling ovarian function in cattleObjective 4.1 To determine the effect of leptin on cultured bovine granulosa and luteal cells.Objective 4.2: To examine the effect of short-term diets, inducing high or low circulating insulin concentrations, on response of granulosa and thecal cells to leptin in vitro.Objective 4.3: To investigate the effect of diets, inducing high or low circulating insulin concentrations, on early luteal function.Methods, Results and Specific ConclusionsThe objective of this work was to determine the specific nutrients that have direct effects on the reproductive system, namely follicle growth, oocyte quality and embryo survival in dairy cattle. This would then enable the formulation of diets for improved reproduction, rather than just for increased milk production. The work described has encompassed twelve major in vivo studies, with 50% of the work carried out in lactating dairy cows. It has also involved an additional six in vitro studies, with associated in vitro approaches linked to the in vivo studies, including the development of new methodology (e.g. differential staining of blastocysts). All experiments were conducted in accordance with the requirements of the Home Office Animals (Scientific Procedures) Act 1986 and incorporated a full ethical review of each study at each of the collaborating organisations.Objective 1: To determine the effect of diet on oocyte quality and the developmental potential of the oocyteEstablish the technique of ultrasound guided oocyte ovum pickup (OPU) in high yielding dairy cowsThis objective included the validation of a number of methods and novel techniques. The source of oocytes for in vitro embryo production is of paramount importance for two main reasons. Firstly, oocyte quality will determine the rate of success in the maturation and the fertilisation of oocytes in vitro. When oocytes are recovered from ovaries the quality of the oocytes is affected by the metabolic and endocrine status of the cattle prior to slaughter. Secondly, the genetic background of the oocytes obtained from an abattoir is sometimes unknown, and in vitro embryo production from this material could result in embryos with undesired characteristics. An alternative was to control both the endocrine and metabolic milieu under which the oocytes develop and the genetic background of the material. This was achieved by collecting oocytes directly from the ovaries of selected cattle in vivo and this was carried out through transvaginal aspiration of follicles using an ultrasound scanner. Another advantage of in vivo collection of oocytes was that different treatments could be applied to a cow so temporal changes could be investigated with the same animal acting as its own control. The aim was first to establish the technique of ultrasound guided oocyte ovum pickup (OPU) for the recovery of oocytes and second to evaluate the effect of progesterone, FSH and the interval between oocyte collection on the quality and number of oocytes recovered. A total of 1228 follicles were aspirated during the study, yielding a total of 707 oocytes (57.7% recovery) of which 304 oocytes were of good quality (43%; see Figure 1). The number of follicles (>3mm) present in the ovaries of the heifers was 14.7 0.5. Heifers treated with progesterone implants had significantly fewer follicles (12.8 0.8) than non-treated heifers (15.7 0.7). There was no effect of FSH or interval between collections on the number of follicles in the ovary. The number of follicles aspirated (13.3 0.5) was positively correlated (P<0.01) with the total number of follicles (>3mm). Neither FSH, nor interval between collections affected the number of aspirated follicles. However, progesterone treatment did decrease the number of aspirated follicles (with CIDR=12.0 0.7 versus without CIDR=14.0 0.6). The total number of oocytes recovered was significantly affected by the interval between collections (3 days: 6.8 0.6 vs. 4 days: 8.5 0.6), but not by progesterone or FSH treatment. The overall proportion of oocytes recovered was 55 2%, although a 4 day interval between collections showed a better recovery rate than 3 days (3 days =49.3 3.0% vs. 4 days = 61 2.0%). The proportion of good quality oocytes was not affected by any of the variables studied and

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had an overall mean of 41 2%. Finally, the diameter of the dominant follicle did not differ between treatments and its presence in the ovary did not affect the number of follicles aspirated or the number and quality of the oocytes recovered. Figure 1. Morphological assessment and classification of bovine oocytes. A: Grade 1. Compact cumulus (> 4-5 layers) with a homogeneous ooplasm, B: Grade 2. Compact cumulus of one or two layers with homogeneous ooplasm having a coarse appearance, C: Grade 3. Less compact cumulus (slightly expanded cumulus) with irregular ooplasm containing dark clusters, D: Grade 4. Denuded oocyte or expanded cumulus, irregular ooplasm. (see Fouladi-Nashta et al. 2007).

In conclusion, these results demonstrated that OPU had been successfully developed at Nottingham and could be used in high yielding dairy cows. These results also confirmed that it was not necessary to treat lactating cattle with either progesterone implants or FSH, thereby allowing the direct effect of diet on oocyte quality and embryo development to be investigated. Development of a fast and efficient method of differential staining of bovine blastocysts.Previously, techniques of TUNEL labelling (see Zeuner et al. 2003) for the detection of apoptotic (dying) cells and differential staining for counting the ratio of inner cell mass (ICM, cells that develop into the foetus) to trophectoderm (TE, cells that develop in to the placenta) cells are used separately for assessment of embryo quality in different species (see Figure 2). The majority of these techniques are antibody based, time consuming and give inconsistent results. Within the current studies we developed a simple and fast method for simultaneous differential staining and TUNEL labelling of bovine embryos (see Fouladi-Nashta 2005). The methodology that was developed involved taking cleaved embryos, produced from in vitro matured and fertilised oocytes, and culturing to the blastocyst stage in synthetic oviductal fluid (SOF) culture medium supplemented with 4 mg/ml BSA and 5% Foetal calf serum (FCS). Embryos were partially permeabilised in 0.5% Triton X-100 solution containing 2 mM/ml TOTO-3 dye (Molecular Probes) for 30. TOTO-3 is a cell impermeant nucleic acid dye, thus only permeabilized cells are stained red. The embryos were then quickly washed in PBS containing 3 mg/ml (PVA), fixed for 15 min at RT in 4% paraformaldehyde containing 10mg/ml-1 Hoescht and TUNEL labelled using a Cell Death Kit (Roche) for 30 min at 37°C in a humid chamber. The embryos were then treated with RNase A (50 U/ml) for 30 min at 37°C, washed and mounted in a small drop of glycerol on a glass slide. RQ1-DNase (3 U/ml) treated embryos were used as a positive control. After three dimensional re-construction using a Leica TCS SP2 confocal microscope, the number of inner cell mass cells (ICM), that develop into the foetus (blue), trophectodem (TE) cells, that develop in to the placenta (red), and apoptotic nuclei (green) were determined(see Figure 2).The results demonstrated that only the peripheral cells of blastocysts were labelled red, indicating that TE cells were permeabilized by the short exposure to the detergent Triton. ICM cells were consistenly stained blue by the cell permanent dye Hoechst. Apoptotic nuclei were found in both types of cells. More consistent differential staining was observed in hatched blastocysts (n=30) than zona-enclosed blastocysts (n=35), also more apoptotic nuclei were observed. No differences were found in the consistency of the technique for embryos grown with or without FCS. When compared to dual staining without TUNEL, no differences in cell number (74 ± 22), and ICM/TE ratio of 0.28 ± 0.06 were detected, indicating that the TUNEL procedure does not affect the labelling of DNA. The technique of differential staining allowed total cell number and allocation of cells to the TE and the ICM to be determined in blastocyst-stage embryos in subsequent experiments. All three of these parameters, in addition to the ratio of ICM/trophoblast cell numbers, provide valuable information on embryo development and was used in a number of the subsequent studies. In addition the number of apoptotic (dying) cells also gives a measure of embryo health.In conclusion, these initial observations demonstrated that this method has been validated for bovine embryos and although not discussed here, was also successfully applied to porcine and ovine embryos. This technique has the advantage of being fast and provides a simple but robust method for assessment of embryo quality. It can also be used to determine the time and origin of ICM and TE cell

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A B C D

differentiation whilst monitoring the degree of apoptosis in different culture systems and in different species.

Figure 2. Example of a blastocyst triple stained for the inner cell mass (ICM: blue), the trophectoderm (TE: red) and apoptotic nuclei (green). See Fouladi-Nashta et al. 2005.

Objective 1.1: Effect of diet on oocyte quality and embryo development in lactating dairy cowsThe aim was to determine the specific nutrients that have direct effects on the reproductive system, namely follicle growth, oocyte quality and embryo survival in high yielding lactating dairy cattle (see Armstrong et al. 2003; Webb et al. 2004). This would then enable the formulation of diets for improved reproduction, rather than just for increased milk production. The experimental approach involved feeding cattle on diets that were precisely formulated to alter key metabolic factors known to influence reproduction. This extensive series of experiments was run in parallel with a series of six experiments carried out within the Link project entitled the ‘Nutritional improvement of fertility in dairy cows’ (LK0646) to determine the influence of precise changes in diet on metabolic function in high yielding dairy cows. Hence the metabolic information from the Link project was used in part in the formulation of diets in the studies described. In addition, the impact of diet on ovarian function and oocyte quality in these current studies was utilised in the final experiments outlined in the Link project which investigated the impact of diet on pregnancy rate. In the series of studies described below the effect of diet on reproductive function was assessed (i) by measuring ovarian follicular changes by ovarian ultrasound scanning and using ovum pickup (as described above) and (ii) followed by in vitro culture of oocytes to the blastocyst stage as a measure of quality and developmental capacity. Effects of dietary carbohydrate source on oocyte /embryo quality and development in high yielding lactating dairy cattleLactating dairy cows suffer from negative energy balance after calving, causing reduced fertility (Beam & Butler, 1999). We have demonstrated that altering dietary energy sources, such as increasing starch, can partially alleviate the effects of negative energy balance by enhancing ovarian activity and initiation of oestrous cycles (Gong et al. 2002). The objective of this study was to examine effects of carbohydrates on oocyte quality and embryo development. Oestrous cycles were synchronized in cows on isoenergetic diets containing either Low Starch (9%) or High Starch (23%). Each cow underwent 7 sessions of ultrasound guided ovum pick up (OPU) and oocytes were collected, graded, matured and fertilized in vitro. Blastocyst quality was assessed using differential staining combined with TUNEL labelling. In addition, serum collected from OPU cows, was used to culture abattoir-derived oocytes (see Objective 2). Total cell number, the ratio of ICM to TE cells and the percentage of apoptotic in the two cellular compartments were measured in all blastocysts as described above.Cleavage rate of oocytes was the same for both dietary groups (Low Starch: 65.8 ± 4.5% versus High Starch: 60.5 ± 4.7%). However the High Starch diet adversely affected blastocyst numbers as a percentage of fertilized oocytes (Low Starch 27.0 ± 4.2% versus High Starch: 15.6 ± 3.5%; P<0.05) and cleaved embryos (Low Starch: 41.1 ± 5.7% versus High Starch: 25.8 ± 5.3%; P<0.01). The High Starch diet also produced a higher proportion of grade 4 (poor quality, as above) oocytes (Low Starch: 12.5% versus High Starch: 27.5%). The Low Starch diet produced better quality blastocysts when comparing ICM/TE ratio (Low Starch: 0.44 ± 0.06 versus High Starch: 0.29 ± 0.06; p<0.01).In conclusion, the results of this study demonstrated clearly that carbohydrate source can affect oocyte quality and hence embryo development, resulting in changes in cell number and rate of blastocyst

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TE

ICM

Apoptotic

production. The specific role of dietary starch on the structure and function of oocytes, and the possible mechanisms through which the developmental potential of oocytes are improved, require further investigation. Importantly however, this study demonstrated the possible detrimental effects of increased levels of dietary starch on the developmental potential of oocytes in the high-yielding dairy cow. This has direct implications for fertility in high yielding lactating dairy cows.Effects of dietary fatty acids on oocyte quality and development in lactating dairy cowsAs discussed in high genetic merit lactating dairy cows negative energy balance prolongs post-partum anoestrus (see Beam and Butler 1999). Dietary fats might improve reproductive function by increasing energy balance of the cow, increasing the total number of follicles, or stimulating growth and size of the preovulatory follicle (see Staples & Thatcher 2005). Availability of fatty acid precursors can also result in increased steroid and eicosanoid secretion, which can alter ovarian and uterine function. The purpose of this study was to examine the effects of level of rumen inert fatty acids on developmental competence of oocytes in lactating dairy cows.The effects of two levels of a calcium soap of fatty acids, on oocyte development in lactating dairy cows was investigated. Oestrous cycles were synchronized in 22 cows on a silage-based diet supplemented with either Low (200g/day) or High (800 g/day) fat (Megalac). A total of 1051 oocytes were collected by ultrasound-guided ovum pickup (OPU) in seven sessions per cow at 3–4 day intervals. Oocytes were matured, fertilized, and cultured to the blastocyst stage in vitro (see Fouladi-Nashta et al. 2007). Embryo quality was assessed by differential staining of Day 8 blastocysts.The results demonstrated that there was no effect on the quality of oocytes (grades 1–4) or cleavage rate. However, high fat significantly improved blastocyst production from matured (P<0.005) and cleaved (P<0.05) oocytes. Blastocysts from the high-fat group had significantly more total, inner cell mass and trophectoderm cells than the low-fat group (P<0.05). Regression analysis confirmed the negative effects of milk yield (P<0.001), dry matter intake (P<0.001), metabolizable energy intake (P<0.005), and starch intake (P<0.001) on blastocyst production in the Low-fat group, but not in the High-fat group. Within the Low-fat group, blastocyst production was negatively related to growth hormone (P<0.05) and positively related to leptin (P<0.05). The Low-fat group had higher non-esterified fatty acids than the High-fat group (P<0.05). In conclusion, supplementation of fatty acids improved oocyte quality resulting in higher blastocyst production. Higher milk yields were associated with reduced developmental potential of oocytes in cows given a low-fat diet. Provision of a high-fat diet buffered oocytes against these effects, resulting in significantly improved developmental potential. As in the previous study this has significant implications for improving fertility in lactating dairy cows. Effects of source of dietary fatty acids on oocyte quality and embryo developmentSupplementary dietary fat as a source of energy for feeding high yielding dairy cows in negative energy balance can influence fertility by changing concentrations of hormones and metabolites and impacting at various points in the neuroendocrine-reproductive axis (see Staples & Thatcher 2005). The previous study demonstrated the beneficial influence of adding fatty acids to diets on oocyte quality (see above; Fouladi-Nashta et al., 2007). The aim of this study was to determine the effect of short-term feeding of three dietary sources of fatty acids on the developmental potential of oocytes in high yielding dairy cows.Twelve Holstein dairy cows were allocated to three groups. Each group received a commercial total mixed ration (TMR) based diet containing either rumen inert fat, Soyabean or Linseed as the main fatty acid source for 3 periods of 3.6 weeks (25 days). Within each period cows were fed the experimental diets for 2 weeks before the start of ovum pick up (OPU), and this was repeated a further 3 times with 3-4 day intervals between each OPU.Significant differences were observed in the profiles of fatty acids in plasma and milk reflecting differences in the dietary sources of the fatty acids. Significant differences were observed in the cleavage rate of the oocytes which was highest in the rumen inert fat group (P<0.05). In addition, blastocysts produced from the rumen inert fat group had a higher ratio of inner cell mass to trophectoderm cells (P<0.05) and a lower proportion of apoptotic cells in the trophectoderm. In conclusion, short-term feeding of different dietary fat sources changed the fatty acid profiles corresponding to the dietary sources of the fatty acids. It appears that saturated and monounsaturated fatty acid sources improve oocyte developmental potential by increasing both the cleavage rate of oocytes and the quality of embryos compared with polyunsaturated fatty acid sources. Further studies to examine fatty acid composition of oocytes, ovarian follicular cells and blastocysts are required to determine the uptake of the different fatty acids.

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Objective 1.2: To determine the effect of specific nutrients on oocyte developmentAs discussed, metabolic and nutrient status are believed to be central to the decline in fertility as cows have been selected for increased milk yield. An understanding of how nutrition affects follicular growth and oocyte development would help to address this decline leading to improvements in fertility among both dairy and beef cows (see O’Callaghan et al. 1999; Webb et al. 2003), so leading to improved financial performance in these herds. In this context, a retrospective analysis of data from an earlier experiment, (see Bridge Link Grant LS0204, Defra 2001) conducted by the current research consortium, demonstrated a relationship between embryo development in vitro and plasma insulin concentrations in oocyte donor heifers. This relationship implied an optimum level of plasma insulin beyond which embryo development was compromised. High concentrations of plasma insulin can arise when animals are fed high levels of a starchy diet, particularly when they are in good body condition. By utilising a dairy heifer model, this series of experiments enabled the effects of alterations in body composition and dietary intake on plasma insulin and embryo development to be investigated. These experiments, as with the previous studies, were complementary to the in vitro studies where the effects of insulin and other growth factors on oocyte and embryo development were assessed directly (see Objective 2 below).The cumulative effects of changes in body composition in relation to plane of nutrition on oocyte quality and embryo developmentThere is compelling evidence to indicate that alterations in dietary nutrient supply during antral follicle development can significantly affect the developmental potential of oocytes, but the underlying mechanisms are poorly understood (Sinclair et al. 2003). However, a retrospective analysis of the data from the Bridge-Link Experiment (LS0204) suggested a threshold level of blood insulin in cattle beyond which oocyte viability was compromised. The current experiment was designed to confirm this initial observation and to formally test the hypothesis that hyperinsulinaemia can compromise oocyte viablity and early embryo development in cattle. The experiment further sought to assess the long-term accumulative effects of alterations in dietary composition on the post-fertilisation developmental competence of oocytes during a nine-week period equivalent in duration to that between calving and mating in lactating dairy cows.The experiment was a 2 x 2 factorial design with 6 replicates in which the factors were body condition score (BCS; Low [2.0 units] vs High [3.5 units]) and level of feeding (Maintenance and Twice Maintenance). Oocytes were recovered weekly from Simmental x Holstein heifers on 12 separate occasions by transvaginal ultrasound guided follicular aspiration (Ovum Pick-Up; OPU) and matured (IVM), fertilised (IVF) and cultured (IVC) in vitro. These 12 occasions were arranged as six two-week sessions, separated by a one week period during which animals were permitted to ovulate naturally, and ovarian folliclular development was recorded by transrectal ovarian ultrasonography. After three two-week sessions (i.e. the first 9 weeks of experimentation), the animals were rested and nutritionally re-aligned so that they regained the levels of body condition attained at the beginning of the experiment. A further period of three two-week sessions of OPU, separated by one week of ovarian ultrasonography, then commenced. At the end of this period, and in order to quantify systemic insulin resistance, indwelling jugular catheters were inserted and a glucose tolerance test conducted. Mean body condition scores differed (P<0.001) between the Low and High Groups at the start of the experimental period. Both body condition and level of feeding significantly altered plasma insulin concentrations. There was a two-fold increase in plasma insulin concentrations between the Low Group on the maintenance diet and the Low Group on the twice maintenance diet and the High Group on the maintence diet; and a further two fold increase between the High Group fed twice maintenance and the Low Group fed twice maintenance and the High Group fed the maintence diet (see Adamiak et al. 2005). A glucose tolerance test confirmed that the High body condition score heifers, on the twice maintenance treatment, exhibited signs of insulin resistance. Follicle size was greater for heifers in High than Low body condition (12.8 versus 11.9 mm: SED = 0.31; P<0.05) and for heifers on twice maintenance than on a maintenance level of feeding (13.0 versus 11.8 mm; SED = 0.31; P<0.01). Heifers on the twice maintenance level of feeding had more medium-sized (4-8 mm) and large (>8 mm) follicles than heifers on the maintence level of feeding (P<0.05). A number of High body condition score heifers fed twice maintenance, had large cystic follicles, consistent with the effects of hyperinsulinaemia. Cleavage rates following IVM and IVF were unaffected by dietary treatment. However, mean blastocyst yields were greater (P=0.07) for twice maintenance than maintenance fed heifers, although there was an indication that yields were reduced in the High animals fed twice maintenance. Indeed, blastocyst yields significantly (P<0.05) deteriorated during the nine-week experimental period for heifers on this experimental treatment (see Figure 3). In contrast, blastocyst

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yields improved over time for low body condition score heifers fed at twice maintenance. In contrast, blastocyst yields were unchanged during the nine weeks of the experiment for both the Low and High Groups fed maintenance rations.In conclusion, the results demonstrated that the effect of feeding level on oocyte quality is dependent on the body condition of the animals; the higher level of feeding being beneficial to oocytes from animals in low body condition, but detrimental to oocytes from animals of moderately-high body condition. Importantly, the effects of high levels of feeding on oocyte quality were shown to be cumulative, with blastocyst yields for relatively fat heifers on twice maintenance deteriorating with time on study relative to thinner heifers on the same level of feeding (see Figure 3). Finally, a significant proportion of the moderately fat animals on the high level of feeding were hyperinsulinaemic, and this demonstrated for the first time in ruminants that this condition is associated with impaired oocyte quality and blastocyst development. Figure 3. Post-fertilization development in vitro (a) Mean ( ± SEM) blastocyst yields (%) and (b) change in blastocyst yields (change in logit proportion with time (weeks) ( ± SEM)) over six sessions of OPU spanning seven weeks. Note that - L: Low BCS; H: High BCS; M: maintenance diet; 2M: twice maintenance diet. (see Adamiak et al. 2005)

Carbohydrate and fatty acid metabolism in bovine oocyte donors determines post-fertilization development in vitroThe aim of this study was to assess the interactive effects of carbohydrate type (fibre versus starch) and fatty acid (FA) supplementation (0 versus 6 % calcium soaps of palm oil fatty acids) on the post-fertilisation development of oocytes recovered from moderately lean and fat heifers. A secondary objective was to compare the FA composition of plasma to that of granulosa cells (GCs) and cumulus-oocyte complexes (COCs) from these animals, and to relate these findings to the developmental potential of oocytes. Thirty-two beef x dairy heifers of approximately 20 months of age and with an average initial weight (mean SEM) of 396.9 ± 4.1kg were used. All animals were accommodated in individual pens on slatted floors. Immediately prior to the experiment the body-condition scores (BCS; six-point scale of Lowman et al. (1976), where 0 = lean and 5 = obese) of these animals were determined and they were allocated accordingly to either a Low (2.5 ± 0.09 units) or Moderate (3.0 ± 0.06 units) body condition score group. Animals within each BCS group were then ranked again by BCS and allocated to one of four dietary treatments: High Fibre (F) either with or without FA supplementation (six percent Megalac;®

calcium soaps of palm oil fatty acids, 440 g/kg palmitic acid (C16:0), 400 g/kg oleic acid (C18:1, n-9), 95 g/kg linoleic acid (C18:2, n-6), 50 g/kg stearic acid (C18:0), and 15 g/kg myristic acid (C14:0); Volac International Ltd, Royston, Herts, UK) or High Starch (S), again either with or without FA supplementation (six percent Megalac®). This resulted in a 2 x 2 factorial design with diets containing predominantly one of two types of carbohydrate (Fibre vs Starch), either with or without FA, replicated four times within each of the Low and Moderate BCS groups (see Adamiak et al 2006). Alterations to the source of fermentable carbohydrate and the lipid content of the diets inevitably led to differences in energy density (MJ metabolisable energy (ME)/kg dry matter). Concentrate levels for each of the four dietary treatments were adjusted to ensure that each animal received 1000 KJ ME/kg liveweight0.75/day, equivalent to approximately twice the maintenance energy requirements for this type

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0

5

10

15

20

25

Blas

tocy

st y

ield

, %

-0.5-0.4-0.3-0.2-0.100.10.20.3 Change in blastocyst yield

LM L2M HM H2M LM L2M HM H2M

a b

of animal (AFRC, 1993), offered as two meals (at 08.00 and 16.00 h). Plasma, granulosa cells and COCs were recovered on Day 5 of a synchronised oestrous cycle for fatty acid analyses. Oocytes were recovered again on Days 10 and 15 of the same cycle following short-term ovarian stimulation (FSH plus GnRH) and matured, fertilised and cultured to the blastocyst stage in vitro.The results demonstrated that high levels of dietary starch and/or fatty acids reduced (P<0.05) Day 8 blastocyst yields in lean, but not in moderately fat heifers. Dietary induced alterations to the FA content of plasma were less apparent in granulosa cells and COCs. Although dietary lipid increased the FA content of COCs, the selective uptake of saturated FAs at the expense of mainly polyunsaturated FAs within the follicular compartment ensured that the FA composition of COCs was largely unaffected by diet (see Adamiak et al. 2006 for more detail).In conclusion, this study confirmed and extended our previous findings that the effects of diet on oocyte quality are dependent on animal body composition. Dietary mediated alterations in plasma concentrations of key reproductive and metabolic hormones, in addition to oocyte quality, were detectable only in Low BCS animals. The underlying mechanisms for such effects remain elusive, although the inclusion of high levels of starch and fatty acids (mainly saturated fatty acids) in the diet of relatively lean oocyte donors reduces post-fertilisation development. The follicular compartment is selective for saturated fatty acids from plasma and, for the range of diets offered in the present study, was largely effective in ensuring oocytes of uniform fatty acid composition. The concentration of saturated fatty acids within COCs, however, is inherently high and it is possible that further increases in the content of these fatty acids, brought about by dietary lipid supplementation, impairs post-fertilisation development, perhaps by compromising membrane fluidity and function in ways that are not fully understood. These data also established a robust nutritional framework for more detailed studies into the mechanistic effects of dietary composition on the post-fertilisation developmental potential of oocytes.The effect of changing dietary amino acid composition on plasma metabolite and hormone concentrations and on oocyte quality in Holstein heifersThe previous results (see above; Adamiak et al. 2005; 2006; Fouladi-Nashta et al 2007) demonstrated that the quality of oocytes recovered by follicular aspiration (OPU) from dairy cows and heifers is influenced by the composition of the diet and body condition. In particular, increasing the amount of starch in the diet is correlated with decreased development to the blastocyst stage and is associated with high plasma insulin concentrations (Gong et al. 2002); this effect was dependant on body condition. Branched chain amino acids, particularly leucine, stimulate insulin release from a variety of pancreatic models. Therefore manipulation of plasma insulin concentrations by increasing the supply of dietary leucine could provide an alternative approach, than feeding diets containing large proportions of starch, to improve the developmental potential of the oocyte. The present study therefore, investigated the effects on oocyte quality of manipulating diet composition to increase plasma leucine concentrations. The diets were chosen so that the interactive effects of dietary leucine and carbohydrate (starch- or fibre-based diets) supply could be assessed.Holstein heifers weighing 522 37 kg, and with a body condition score of 2.2 0.5 units, were assigned (8 per treatment) to either a Fibre- (F; 355 g neutral detergent fibre (NDF) and 149 g starch / kg dry matter (DM)) or Starch-based (S; 148 g NDF and 420 g starch /kg DM) concentrate diet containing either Low (L, 8.3 g/kgDM) or High (H, 10.3 g/kg DM) dietary leucine; giving a 2 x 2 factorial arrangement of treatments (FL, FH, SL, SH). Oestrus was synchronised by progesterone (CIDR) and OPU began 7 days after CIDR removal. Each heifer underwent 6 sessions of OPU (2 per week for 3 weeks). Recovered oocytes were matured, fertilised and cultured in synthetic oviductal fluid (SOF) in vitro to the blastocyst stage. Individual animal identity of oocytes was maintained throughout. Blood samples were obtained from each heifer, before and 1.5h after feeding, weekly for 3 weeks. Plasma hormone concentrations were determined by radio-immunoassay and free amino acids by HPLC of ο-phthaldialdedyde derivatives. Feeding diets containing high leucine did achieve significantly higher plasma leucine (LvH; 13.1v16.4 μg/ml; P<0.001) and tyrosine (LvH; 6.1 v 6.6; P=0.035) concentrations as planned. Post-feeding insulin and glucagon concentrations were greater (P<0.001) than prior to feeding, but there were no interaction between sample time and treatment. Feeding starch increased mean plasma insulin concentrations (see Table 1) whereas increasing dietary leucine increased the mean plasma glucagon concentrations and the insulin:glucagon ratio.

There was no significant interaction between dietary carbohydrate and leucine. There were also no significant differences between treatments in the numbers or quality of oocytes recovered by OPU.

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Oocyte cleavage (mean ± SEM; F versus S, 0.57 ± 0.04 versus 0.52 ± 0.03; NS) was greater for fibre-based diets. There was however, a significant interaction (P=0.033) between dietary carbohydrate and leucine for blastocyst development such that leucine had no effect on fibre-based diets (FL versus FH; 0.29 ± 0.06 versus 0.31 ± 0.05), but improved blastocyst development when animals were fed starch-based diets (SL versus SH, 0.18 ± 0.045 versus 0.30 ± 0.052). Overall, blastocyst development from matured oocytes tended to be greater when a fibre-based diet was fed (F versus S; 0.19 ± 0.03 versus 0.12 ± 0.02; P=0.10).

Table 1. Mean (+ SED) plasma insulin and glucagon concentrations (ng/ml). Note that F: Fibre; S: Starch; L: low leucine; H: high leucine.

Diet FL FH SL SH SED F v S L V H

Insulin 1.48 1.39 1.89 1.81 0.255 P=0.028 NS

Glucagon 0.13 0.16 0.13 0.16 0.017 NS P=0.011

Insulin:glucagon 11.9 9.3 14.8 11.2 1.78 NS P=0.021

SED for 48 observations.

In confirmation of previous results, feeding starch based diets increased plasma insulin concentrations and tended to adversely affect oocyte development. Increasing dietary leucine did achieve an increase in plasma leucine concentrations, as planned, and this was accompanied by an increase in plasma glucagon, rather than insulin concentrations, resulting in a decrease in the insulin:glucagon ratio for both diets. In conclusion, the positive effect of dietary leucine on embryo development when starch-based diets were fed may be related to reductions in insulin:glucagon ratio. Effect of diet and feeding frequency on bovine oocyte qualityAs demonstrated previously (see above; Adamiak et al. 2005; 2006; Fouladi-Nashta et al 2007), quality of oocytes recovered by OPU is influenced by diet and diet-induced changes in hormones. It is not known if diurnal changes in hormones induced by frequency of feeding affect oocyte quality. Hence the aim of this experiment was to determine if diurnal changes in hormone concentrations, induced by changing the frequency of feeding (see also Gutierrez et al. 1997) influences oocyte quality and hence blastocyst development.Using a 2 x 2 factorial design, Holstein heifers (six per treatment) were fed either fibre- (F) or starch-based (S) diets containing either 189 or 478 g starch/kg dry matter. The diets were offered in either two or four equal meals per day and supplied twice the maintenance energy requirement (see above for details). Blood samples were obtained both at weekly intervals (3 samples per heifer, collected before feeding) during the experiment and throughout an entire 24 h period (16 samples per heifer). Each heifer underwent six sessions of OPU (twice weekly). Oocyte quality was assessed by development to the blastocyst stage in SOF in vitro following fertilization. Mean weekly plasma insulin concentrations did not differ between diets, but plasma glucagon concentrations were greatest when heifers were fed the starch-based diet twice daily compared with the other diets. When heifers were offered four meals per day, there was no meal-related change in hormone concentrations. However, when heifers were offered two meals per day, plasma insulin concentration increased after feeding the starch-based, but not the fibre-based diet. Mean plasma insulin concentrations (μIU/ml SED 7.5) were greater (P=0.04) when the Starch-based diet was fed (52 versus 40) and with 2 times daily feeding (P=0.02; 53 versus 39), as a result of increased post-feeding concentrations (SED 17.4; H versus L, 68 versus 41, P<0.02; 2 times versus 4 times, 72 versus 37, P=0.017). Results for glucagon were similar to insulin. Treatment did not influence ovarian follicle size distribution or oocyte recovery (of aspirated; 0.53 ± 0.231) by OPU. Although oocyte cleavage (of matured) was numerically greater when the starch-based diet was fed (P>0.05, 0.63 ± 0.05 versus 0.53 ± 0.049), blastocyst yield (of cleaved) was similar for all treatments (0.27 ± 0.023). In conclusion, this experiment extended the findings of previous studies both on the changes in hormone secretion (Gutierrez et al. 1997) and on the effect of diet on oocyte quality. Modifying the feeding regime successfully produced large diurnal fluctuations in hormone concentrations, but there was no associated effect on oocyte quality, suggesting that the oocyte may be insensitive to short-term meal-related fluctuations in plasma hormone concentrations. Furthermore, the results of the present experiment are consistent with the previous observations that plasma insulin concentrations and oocyte

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quality are less sensitive to diet composition in heifers fed at twice maintenance than in heifers fed at maintenance. Objective 2: Identification of specific nutritional factors that influence oocyte maturation and blastocyst development.The overall objective was to identify nutritionally-modulated factors that have either a positive or negative effect on blastocyst development and hence subsequent pregnancy rate. Therefore a bioassay approach, as used in our previous studies (see above; Adamiak et al. 2005; 2006; Fouladi-Nashta et al 2007) and found to be highly effective in assessing nutritional influences on the oocyte and early embryo development was utilised. These in vitro studies were therefore, complementary to the in vivo studies described in Objective 1 above and the final report of the LK0646 Link Project. In order to investigate the influence of diet, serum was collected from both lactating dairy cows (Nottingham) and dairy heifers (SAC, Aberdeen) fed on the different diets, (as described in Objective 1) and the effects on in vitro oocyte development to the blastocyst stage tested. Effects of bovine serum, from lactating dairy cows fed different diets, on oocyte development.

(i) Comparison of starch versus fibre based dietsThe effect of either a starch or a fibre based diet (see Objective 1.1 above) on developmental competence of oocytes collected from slaughterhouse derived ovaries was investigated. Bovine oocytes (n = 745) were matured in the presence of either 10% OPU sera from individual animals or foetal calf serum (FCS). Following in vitro fertilisation, all cleaved embryos were cultured with same source of serum up to the blastocyst stage. No differences were observed in the cleavage rate and development to the blastocyst stage between these treatments. However, serum from animals fed on a fibre based diet as opposed to starch based diets produced more hatched blastocysts indicating that this diet might produce more developmentally competent embryos.Concentrations of amino acids in serum samples collected from individual animals in both dietary groups were analysed. Higher concentrations of total amino acids and lower urea were measured in the Fibre dietary group. Concentrations of glutamic acid, leucine and lysine were higher in the Fibre group, and higher concentrations of glutamine, alanine and valine were detected in the Starch dietary group.Changes in the progesterone concentration in the circulation were also assessed by radio-immunoassay in plasma samples collected from individual animals at the time of OPU (see Objective 1.1). Animals in the Starch group had significantly higher peripheral progesterone concentrations (P<0.05) than animals in the Fibre group.In conclusion, the use of OPU serum for in vitro culture of abattoir derived oocytes did not show any differences in the developmental competence of the oocytes and quality of the blastocysts. It seems that the presence of cow serum, regardless of dietary treatment, improves the developmental potential of oocytes. However, serum from the Fibre group produced more hatched blastocysts indicating that this diet may produce more developmentally competent embryos and could explain the positive influence of fibre on reproductive performance. It is possible that these effects are modulated via changes in circulating amino acids, but this needs to be investigated further including whether higher concentrations of amino acids reflect either the different amino acid content of the dietary components or are the products of microbial action on the high fibre based diet.

(ii) Comparison of high and low fat dietsSerum was processed from blood samples collected two weeks after the start of feeding animals on each dietary treatment (see Objective 1.1). The serum samples from each dietary group were pooled and used during oocyte maturation and/or embryo culture of abattoir derived ovaries. The treatments included: foetal calf serum (FCS) as control; 2. Low Fat serum (10%) added during oocyte maturation and during embryo culture (10%); 3. High Fat serum (10%) added during oocyte maturation and during embryo culture (10%); 4. FCS (10%) added during oocyte maturation and Low Fat serum during embryo culture (10%); 5. FCS (10%) added during oocyte maturation and High Fat serum during embryo culture (10%).A total of 1714 oocytes were cultured and fertilised. The overall cleavage rate of fertilised oocytes was 74%. No differences were found in the cleavage rates of oocytes matured in the presence of either FCS or each one of Low or High Fat OPU serum. Development to the blastocyst stage was higher in oocytes that were matured in the presence of either of OPU sera than in the FCS (~50% versus 36.5%). This difference was maintained when either Low or High Fat OPU serum was added to the FCS (46.4%). Overall there were no differences between the Low and High fat serum collected from lactating dairy cows in the developmental potential of oocytes in vitro.

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Peripheral serum concentrations of known essential amino acids were also measured, some of which have already been shown to be involved in oocyte maturation (see previous study). There were no marked differences between cows fed either High versus Low Fat diets. However, total amino acid concentrations in the peripheral circulation, and the concentrations of glutamine, glycine and alanine in High Fat diet was higher than in serum, collected from the Low Fat dietary groups.In conclusion, the addition of OPU serum to abattoir derived oocytes showed that serum can have a beneficial effect on the developmental potential of oocytes matured in the presence of FCS. It improved the rate of blastocyst production, although there was no major difference between serum from either the Low or High fat groups. Interestingly the addition of both Low Fat and High Fat serum in the IVF culture resulted in a high percentage (~50%) of blastocyst development.Effects of bovine serum, from dairy heifers fed different diets, on oocyte developmentThe aim was to investigate the effects of alterations in dietary carbohydrate source and lipid supply on the post-fertilisation developmental potential of oocytes from maiden dairy heifers (see Objective 1.2 above). In this experiment specially formulated diets were offered to a group of 32 heifers for a 6-week period commencing 16 days prior to prostaglandin (PG) F2a administration. These animals were stimulated with gonadotrophins as described earlier, to create intra-follicular conditions similar to that of a pre-ovulatory follicle in order to enhance the post-fertilisation developmental potential of oocytes. Sera were harvested from these animals and used to supplement the media for in vitro embryo culture. Oocytes were harvested from ovaries recovered post-mortem from a local abattoir and the bovine zygotes cultured to the blastocyst stage in vitro in the presence of these sera, similar to that described above.Sera from Thin and Fat heifers offered a High Fibre (F) or Starch (S) diet alone (0) or with 6% w/w (6) protected fat (Ca soaps of fatty acids, Megalac) were added to 20 ul drops of SOF at 10% v/v. Bovine zygotes (n = 1,480), from oocytes matured and fertilised using standard procedures, were cultured in these drops at 38.8C under oil in a humidified atmosphere of 5% CO2 and 5% O2. Pyruvate metabolism, total cell counts and TUNEL analysis were conducted on Day 8 blastocysts.Plasma fatty acids for F0, F6, S0 and S6 were 0.75, 1.82, 0.50 and 1.39 ug/ml; SED=0.07; P<0.001. Cleavage rates averaged 70.8% and were unaffected by serum source. Blastocyst yields averaged 24.0 ± 2.1, 21.0 ± 1.9, 18.2 ± 2.4 and 23.1 ± 3.0% for F0, F6, S0 and S6 (P=0.09) respectively, and were higher (P<0.05) with serum from Thin than from Fat animals (24.2 ± 1.9 versus 18.9 ± 1.2%). Total cell number/embryo (109.0 ± 5.0) did not differ between treatments. Pyruvate metabolism (pmol/embryo/3h; 16.5 ± 6.5, 26.1 ± 2.9, 26.1 ± 3.1 and 19.2 ± 2.5; P<0.05) and TUNEL positive cells (3.8 ± 0.4, 6.9 ± 0.9, 6.4 ± 0.7 and 5.3 ± 0.5% for F0, F6, S0 and S6, respectively; P<0.01) were greater for F6 and S0 diets. In conclusion, from our earlier in vivo study with the sera donating heifers, it was demonstrated that the fatty acid content of the diet can alter the lipid content of oocytes. In the present study, fatty acids altered early embryo viability by modifying energy metabolism and the incidence of apoptosis. Taken collectively, the data indicate that response to alterations in dietary carbohydrate and lipid supply very much depends on the body composition of the animal (see also Objective 1.2). Those heifers in moderately poor condition responded to these dietary treatments in a similar way to dairy cows offered similar high fibre and starch diets (see Objective 1.1 above), with or without lipid supplementation. Overall conclusionsSera from dairy cows: Sera were collected from a number of lactating animals on a range of different diets (see Objective 1.1), and used for in vitro culture to examine their effect on the developmental competence of oocytes. Following IVF, all cleaved embryos were cultured with the same source of serum up to the blastocyst stage. No difference was observed in cleavage rate or development to the blastocyst stage. Developmental competence of oocytes and quality of the blastocysts were not affected by the range of sera obtained from cows fed the different diets. The use of cow serum did however improve the developmental potential of oocytes regardless of dietary treatment. Interestingly, serum from the Fibre group produced more hatched blastocysts suggesting that this diet might produce more developmentally competent embryos.Sera from dairy heifers: Sera were collected from dairy heifers that differed in body condition, and which were offered diets that differed in their carbohydrate source (fibre or starch) and level of rumen-protected fatty acids, for use in embryo culture. The data revealed complex interactions between these dietary factors on embryo development in vitro. In general, sera from fatter heifers were less effective in supporting early embryo development. Sera from heifers on both fibre plus fat diets and high starch diets increased metabolic stress and programmed cell death in embryos. Sera from heifers offered a

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high fibre diet, similar to dairy cow serum, without fat supplementation or a high starch diet with fat supplementation supported the highest yields of metabolically normal embryos.Taken together the in vitro results demonstrate similar responses particularly with regard to the effects of fibre, high starch and fat supplementation. They also confirm a number of the findings from the in vivo studies, both in lactating dairy cows and in dairy heifers.Objective 3: To investigate whether diets fed for shorter periods (3-4 days) can have significant effects on reproductive functionPrevious work has shown that feeding diets to increase systemic concentrations of insulin in heifers can alter circulating concentrations of IGF-I and leptin, and increase the size of preovulatory follicles and circulating concentrations of progesterone during the early luteal phase (see Armstrong et al. 2003). However, it is not clear whether this reflects a direct effect on the corpus luteum or a carry-over effect of the diet via preovulatory follicle development. In addition, the speed with which ruminants respond to changes in diet and subsequent effects on ovarian function is also not known in detail. Hence the aims of this study were to address these questions in lactating dairy cows by determining if diet acts either on the preovulatory follicle, early CL function or both. Because of the temporal nature of follicle development and subsequent corpus luteum development it was decided to incorporate Objectives 3.1 and 3.2 into a single large experiment investigating the effects of short-term effect on follicular development and subsequently CL function. In addition, the experiment was designed to enable the investigation of: (i) the duration of dietary treatment required, (ii) whether diet has an effect on pulsatile secretion of LH and (iii) the response of LH to exogenous GnRH challenge during the early luteal phase of the oestrous cycle.Forty multiparous lactating dairy cows were used for this study (n = 10 per group). Cows were fed diets with either high starch (High Starch diet; 18% starch, 30% NDF), high fibre (High Fibre diet; 9% starch; 35% NDF), or a combination of both diets (see Figure 4; also Objective 1). Serum samples were collected weekly. At 25 ± 5 days postpartum, cows were subjected to an oestrous synchronization treatment with a standard protocol using progestagen (CIDR) and PGF2a. Seven days after ovulation PGF2a was given to induce a second synchronized oestrus. Seven days after the second ovulation all the cows were challenged, i.m., with 200µg GnRH. Figure 4. Schematic representation of the experimental protocol

Cows were blocked by calving date and milk yield and allocated to 4 groups (see Figure 4 above):1. Group 1 was fed the High Starch diet until one day after removal of the CIDR. These cows were

then fed the High Fibre diet until the day of the second PGF2a injection. Animals were then changed back to the High Starch diet and remained on this diet until the end of the experiment.

2. Group 2 was fed the High Starch diet throughout the experimental period.3. The ration for Group 3 was changed to the High Fibre diet on the day of CIDR insertion and

animals remained on this diet until the end of the experiment.4. The ration for Group 4 was changed to the High Fibre diet on the day of CIDR insertion and

animals remained on this diet until one day after the removal of the CIDR. The ration was then changed back to the High Starch diet until the day of the second PGF2a injection, and then changed again to the High Fibre diet until the end of the experiment.

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All diets were fed as complete rations and at each change over in diet cows were given 3 days to adapt to the new diet. Starting from the day of first PG2a injection, serum samples were collected daily and cows were scanned regularly to determine the size of preovulatory follicles, the day of ovulation and diameter of the corpora lutea. Throughout the experimental period, daily milk yield and weekly body weight and body condition score were recorded for all cows. Five days after both synchronized ovulations, plasma samples were collected every 15 minutes for 6 hours, via a jugular cannula, for measurement of peripheral LH concentrations. To determine LH response to GnRH challenge, seven days after the second synchronized ovulation, serial serum samples were collected at 30 minute intervals for 3 hours.The data from Groups 1 and 2 showed (see Figure 5) that peripheral insulin concentrations increased gradually during early lactation and displayed an acute increase coincident with oestrus. As expected, the High Fibre diet significantly (P<0.05) decreased serum insulin concentrations. It is particularly interesting that the response of insulin to the change in diet was rapid (see Figure 5). Compared with the High Starch group, cows fed the High Fibre diet had significantly (P<0.01) higher progesterone concentrations. The size of the preovulatory follicle (High Starch:18.1 + 0.4mm; High Fibre:17.9 + 0.3mm) on the day before ovulation and size of the CL

Figure 5. Mean (± SEM) peripheral insulin concentrations for Groups 1 and 2.

(High Starch:14.3 + 0.3 mm, High Fibre:14.3 + 0.2mm) after ovulation, as determined by ultrasound scanning, did not differ between groups. LH pulsatile profiles on Day 5 after ovulation, including LH pulse frequency, pulse amplitude and basal concentrations, did not differ between the two groups. Also diet did not affect LH response to exogenous GnRH determined on day 7 after ovulationThe results demonstrated that cows fed a High Starch diet during the early luteal phase had higher insulin, but lower progesterone concentrations in the peripheral circulation, when compared with cows on the High Fibre diet. However, diet did not affect size of the CL, LH pulsatile secretion or LH response to GnRH. Therefore it is concluded from this part of the study that a diet that induces higher insulin secretion can cause a decrease in serum progesterone in lactating dairy cows. Importantly, the diet seemed to act directly on early luteal function, and whilst LH secretion is unlikely to be involved, the exact mechanism requires to be investigated further. The data from Groups 3 and 4 demonstrated that the patterns of progesterone secretion were similar and were not different between Group 2 and Group 3. It also confirmed that the switch over in diets was accompanied by a a significant change in peripheral insulin concentrations within 2 to 3 days. However, the effect of switching the diet on progesterone secretion appeared more complicated. A switch from a fibre based diet to a starch based diet tended to lead to a decrease in progesterone secretion, whereas the effect was opposite when the switch in was from a starch to a fibre based diet.In conclusion, the results have demonstrated that an alteration in diet can alter circulating concentrations of metabolic hormones rapidly and that the changes in these peripheral hormones are accompanied by changes in ovarian function. However, the same diet may have different effects on preovulatory follicle development compared with corpus luteum function. Importantly this suggests that a single diet may not be suitable for the differing physiological state observed during the post- partum period in lactating dairy cows (see later results for further discussion).

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Objective 4: To evaluate the role of leptin in controlling ovarian function in cattleOur previous studies have demonstrated that dietary induced changes in circulating insulin and IGF-I concentrations affect follicle dynamics and ovarian steroidogenesis (Gutierrez et al. 1997; Armstrong et al. 2001; see Objectives 1 and 3 above). We have also provided evidence indicating that the local ovarian growth factor system is one mechanism through which the effects of these metabolic hormones on follicle growth and steroid production are mediated (Armstrong et al. 2001; 2003; Webb et al. 2004). Leptin is also likely to be involved in this process and it has been proposed as a metabolic signal for reproduction (Armstrong et al. 2003). For example, reduced nutrient intake, as well as reducing concentrations of insulin and IGF-I, results in decreased peripheral leptin concentrations (Amstalden et al. 2000).4.1: To determine the effect of leptin on cultured bovine granulosa and luteal cells.The aim of this objective was to investigate the effect of leptin on the proliferation and differentiation of bovine granulosa cells. The experiments were designed to determine the optimal concentrations of leptin required to modulate FSH stimulated steroidogenesis by granulosa cells in vitro. Three different leptin preparations were tested. The cells were challenged with different doses of recombinant leptin; one ovine leptin (Preparation 1; gift from Dr Kiesler, University of Missouri) and two bovine leptin (Preparations 2 and 3, were gifts from Drs D’Ochio, University of Queensland, and Ghertler, University of Nebraska respectively) preparations in combination with physiological concentrations of FSH and insulin. Granulosa cells were collected from small (<4mm), medium-sized (4-8mm) and large (>8mm) bovine follicles and cultured according to procedures developed at the Roslin Institute (see Webb et al. 2003 for references). Briefly, this involved suspending cells from the different sized follicles in culture medium consisting of McCoy 5A modified medium supplemented with 1% PenStrep solution, 10ng/ml bovine insulin and Long R3-IGF-I, 3 mM glutamine, 20mM Hepes, 5mg/ml apo-transferrin, 4ng/ml sodium selenite, 0.1% BSA and 10-7 M testosterone.

Cells were counted and seeded into individual wells in 96-well culture plates (50-75 x103 viable cells per 50l culture medium) and made up to a final volume of 250l culture medium containing the appropriate concentration of FSH and leptin. After 2 and 4 days of culture, 80% (200l) of the culture medium was removed (taking care not to disturb the loosely adherent cells) and replaced with fresh medium. After 6 days of culture, granulosa cell number was estimated and the granulosa cell conditioned culture medium was stored at –20oC. Oestrogen and progesterone were measured by previously validated in-house time-delayed fluorescence immunoassay procedures. Three separate cultures were performed and the results analysed by a split plot ANOVA after log transformation of the steroid production data. Validation of culture conditions: The effect of incubation time on the production of oestradiol and progesterone by granulosa cells cultured in the presence of 1ng/ml FSH was investigated. Both oestradiol and progesterone production by granulosa cells from small and medium-sized follicles increased during the culture period. In contrast oestradiol production by granulosa cells from large follicles remained constant while progesterone production increased.Comparison of the effect of leptin on steroidogenesis in granulosa cellsGranulosa cells were cultured in the presence of 1ng/ml FSH and 0, 5, 10 and 20ng/ml leptin (Preparation 1). Oestradiol and progesterone were measured after 2, 4 and 6 days of culture. Steroid production was corrected for the number of viable cell after culture for 144 hours. The results demonstrated that leptin inhibited the production of oestradiol by granulosa cells isolated from small bovine follicles. In contrast, leptin inhibited progesterone production by granulosa cell from large follicles and did not affect progesterone production by granulosa cells from either small or medium-sized follicles.The effect of leptin Preparations 1 and 2 on FSH-stimulated oestradiol production by small follicles. As expected, ovine leptin (Preparation 1) was more potent than bovine leptin (Preparation 2). Leptin Preparation 3 had no effect on oestradiol or progesterone production by granulosa cells at the concentrations used in this experiment and it appears there may be some query over the three dimensional structure of this recombinant preparation (Clarke, personal communication). There was no effect of leptin, at the concentrations used in these experiments, on the number of granulosa cells counted after 144h culture. In conclusion, the results demonstrated that leptin can inhibit FSH-stimulated oestradiol production by bovine granulosa cells in vitro. Leptin can also inhibit progesterone production by bovine granulosa

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cells, isolated from large dominant bovine follicles. However, leptin had no effect on granulosa cell proliferation. Inhibition of FSH-stimulated oestradiol was most apparent in granulosa cells isolated from small follicles. Interestingly this population of follicles is also the most responsive to short-term increases in nutrient intake, both in terms of increased numbers of follicles and oestradiol production (see Gutierrez et al 1997; Armstrong 2001 and 2002, Webb et al 2003). Hence the response of follicular development to diet may not only be regulated by GH, IGF-1 and insulin, but also affected by circulating leptin concentrations being secreted from adipocytes.4.2: To investigate the effect of short-term diets, inducing high or low circulating insulin concentrations, on response of granulosa and thecal cells to leptin in vitro.Increasing evidence from a number of species, and the previous study, has implicated leptin as a signal linking nutritional status with reproductive function. The current study investigated this further with the aim of determining whether there is a direct interaction between leptin and ovarian steroidogenesis in cattle. This study also involved an in vivo component as a change of diet would also provide further confirmatory evidence on the possible effects of rapid changes in metabolic hormones, as demonstrated in Objective 3.The oestrous cycles of 16 heifers were synchronized using a standard protocol, with progesterone (CIDR), PGF2a and GnRH (see Objective 3, Figure 5). Heifers of mean body condition score 2.25 were fed either a maintenance (n=8) or a twice maintenance (n=8) ration for two weeks. Plasma samples were collected throughout the experiment and assayed for leptin, since leptin may provide a more precise measure of body fatness. In addition it may demonstrate direct evidence of leptin acting as a link between nutrition and reproduction. The effect of leptin on ovarian steroidogenesis was also examined using primary cultures of bovine theca and granulosa cells in serum-free conditions (see previous Objective 4.1). The results demonstrated that whilst plasma concentrations of leptin in the Maintenance Group remained constant throughout the experiment, they increased significantly in the twice Maintenance Group. This increase occurred within 2 days of the start of increased feeding and was maintained for the duration of the experiment. Similarly, peripheral IGF-1 and insulin concentrations also increased rapidly after the change in diet as seen in Objective 3.1. As seen previously there was also a significant increase in these hormones around the time of oestrus. These results therefore confirmed those seen in Objective 3.Plasma leptin concentrations showed an additional significant increase 2 days after GnRH treatment in the twice Maintenance Group. In vitro, leptin at doses of 20ng/ml significantly inhibited the LH-stimulated, but not basal androstenedione secretion (see Figure 6) by theca cells from small and medium-sized follicles. Leptin also significantly suppressed FSH-stimulated oestradiol production by granulosa cells from small, but not medium-sized follicles. However, the level of dietary intake had no effect on the inhibitory effects of leptin on theca or granulosa cell steroidogenesis.Figure 6: The interaction of leptin (20ng/ml, open circles) and LH on androstenedione production (pg/104cells) in vitro by thecal cells from small and medium-sized follicles. Note that closed circles are the controls with no leptin. The bars show control concentrations with no LH added to the culture, with the closed bars demonstrating the effect of adding leptin (20ng/ml) with no LH.

In conclusion, this study demonstrated that peripheral leptin concentrations respond quickly to changes in diet as shown in Objective 3, for other metabolites. The results also demonstrated that oestrogen and androgen production by the bovine follicle is inhibited by leptin at physiological concentrations. However, these inhibitory effects were not altered by dietary treatment in vivo. In addition, ovarian

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steroids may interact with nutritional status to directly regulate the secretion of leptin from adipocytes. In conclusion, these results have demonstrated that leptin, ovarian steroids and body condition all appear to interact to alter ovarian function in cattle.

The effect of diets, inducing high or low circulating insulin concentrations, on corpus luteum functionCorpora lutea were collected 3 days after ovulation as this is a key time when progesterone biosynthetic capability is being developed. Hence the effects of feeding either a maintenance or a twice maintenance diet on the expression of two key enzymes regulating progesterone secretion, namely steroid side chain cleavage (SSCC) and steroid acute regulatory protein (StAR) were examined during these early stages of luteal development. These are two key enzymes in the progesterone metabolic pathway and could provide clarification of where diet could impact at the molecular level to alter progesterone production.The results demonstrated that there was no significant dietary affect on the levels of mRNA transcripts encoding cholesterol side chain cleavage in luteal tissue. In contrast, increased dietary intake, maintenance versus twice maintenance, decreased both the 3.0 and 1.8kb mRNA transcripts encoding steroidogenic acute regulatory protein (see Figure 7)

Figure 7. Quantitation of 3.0kb (a) and 1.8kb (b) mRNA transcripts encoding StAR in extracts from bovine CL collected 3 days after an induced ovulation from cattle offered either maintenance (1M) or twice maintenance (2M) diets. *P<0.05 between diets.In conclusion, these results demonstrated that diet can have a direct effect at the molecular level on the mRNA expression of an enzyme regulating early luteal progesterone production. This may be of key significance since our previous work has demonstrated that circulating progesterone concentrations can influence embryo development and survival and hence fertility (see Mann et al. 2003). It was decided that this should be examined further since it is not known whether this effect is due to a direct effect on the corpus luteum or a carry-over effect form the developing dominant follicle.4.3: To investigate the effect of diets, inducing high or low circulating insulin concentrations, on early luteal function.Increased dietary intake can affect both the development of ovarian follicles and serum concentrations of progesterone during the early luteal phase (see Objectives 3 and 4.2). However, it is not clear if this represents a carry over effect of the diet either on the development of the preovulatory follicle, or a direct effect of diet on early corpus luteum (CL) function, or an effect on clearance of progesterone from the peripheral circulation. Hence the aim of this study was to determine the site of action of diet particularly because luteal function in early pregnancy is a critical factor in maintaining embryo survival (see Mann et al. 2003). The major aims of this study were: (i) to determine the direct effect of diet on the CL by only allowing an increase in feed intake after ovulation; (ii) to determine the effect of diet on CL function, particularly the effect on progesterone clearance by correlating serum progesterone concentrations to CL tissue content and in vitro secretion of progesterone; and (iii) to investigate the importance of hypothalamic/pituitary function by measuring pulsatile secretion of LH.Development of a serum-free culture system for bovine luteal cellsA serum free culture system was developed using abattoir derived corpora lutea. The tissue was treated with collagenase and DNAse to isolate luteal cells and the viability of luteal cells ranged from 70 to 90%. Cells were cultured for 144 hours in DEME:F12 medium supplemented with BSA, transferrin, selenium, glutamine, and low doses of insulin and IGF-I. The validation results demonstrated that

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progesterone secretion was maintained in the presence of LH and also responded to LH stimulation in a dose- and time-dependent manner. Dietary challenge prior to collection of corpora lutea: in vivo experiment.Experimental design: Similar to Objective 4.2, sixteen heifers, with a body condition score of approximately 2.25, were used. Oestrous cycles were synchronized by the standard protocol using CIDR, PG2a and GnRH treatment (see Objective 3 for details). Animals were divided into two groups and fed a maintenance ration until 2 days prior to the synchronized oestrus. Half the heifers (n=8) continued to be fed the maintenance diet whereas in the remaining heifers (n=8) the diet was increased to 200% over a 3-day period and continued until the end of the experiment. Serum samples were collected for insulin, leptin, progesterone and IGF-I measurements and ovaries scanned to monitor CL development. Serial samples (every 15 min for 6 h) were also collected on day 5 after synchronized oestrus to determine LH pulsatile profiles. At the end of the experiment, 7 days after the synchronized oestrus, animals were then slaughtered and the corpora lutea collected. Luteal cells were isolated from half of the CL and cultured under serum free conditions to measure the secretion of progesterone (with or without LH stimulation). The other half of the CL was frozen in liquid nitrogen for the measurement of progesterone content.The results demonstrated that twice maintenance feeding increased peripheral insulin concentrations rapidly as described previously (Objectives 1 and 3), although peripheral progesterone concentrations were not changed. Both basal and LH-induced progesterone secretion by cultured luteal cells, and CL tissue content were not affected by in vivo feeding. In addition, LH pulsatile profiles were not different between the groups.In conclusion, the previously observed effect of feeding a twice maintenance diet on progesterone secretion appears to be having its major effect on preovulatory follicle development. However, using this experimental approach there was limited evidence for a direct effect on either early corpus luteum development and/or progesterone clearance from the peripheral circulation. However, these results need to be confirmed since previous results in lactating dairy cows suggested that dietary induction of higher insulin secretion was associated with a decrease in serum progesterone suggesting that there may be an affect of diet on early luteal function although an influence on the preovulatory follicle cannot be discounted. Furthermore these differences could be due to the different experimental designs and/or feeding regimes. If it is the case that the most significant impact of nutrition is on developing preovulatory follicles, this has implications both on the formulation of diets and, in particular, on the timing of dietary intervention.Main Implications of the ResultsThe objective of this work was to determine the specific nutrients that have direct effects on the reproductive system, namely follicle growth, oocyte quality and embryo survival, in dairy cattle. This would then enable the formulation of diets for improved reproduction, rather than just for increased milk production. This work encompassed 12 major in vivo experiments, with 50% of these studies carried out in lactating dairy cows. It also involved 6 major in vitro studies with associated in vitro approaches linked to the in vivo work, including the development of new methodology (e.g. differential staining of blastocysts). A number of significant conclusions have come out of this programme of work:

Carbohydrate source can affect oocyte and embryo quality, resulting in changes in cell number and rate of blastocyst production.

Increased levels of dietary starch can have a detrimental effect on the developmental potential of oocytes in the high-yielding dairy cow.

Feeding starch based diets increased plasma insulin concentrations whereas leucine was accompanied by an increase in plasma glucagon, but not insulin concentrations.

Fatty acid supplementation improved oocyte quality resulting in higher blastocyst production. Source of dietary fats influenced oocyte developmental potential by affecting both the cleavage

rate of oocytes and the quality of the embryos. Higher milk yields were associated with reduced developmental potential of oocytes, but a high-

fat diet buffered oocytes against these effects. Fibre based diets appeared to be associated with more developmentally competent embryos. The effect of level of feeding on oocyte quality was dependent on body condition. Importantly the effect of level of feeding on oocyte quality was demonstrated to be cumulative

over time.

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Fatter animals on a higher level of feeding can be hyperinsulinaemic, and it was demonstrated for the first time in ruminants that this condition is associated with impaired oocyte quality and blastocyst development.

A change in diet can induce a rapid alteration in peripheral metabolic hormones, such as insulin, IGF-1 and leptin, within 2-3 days and these are associated with changes in ovarian function.

Diet appears to impact directly on early luteal function in that dietary induction of higher insulin secretion was associated with a decrease in serum progesterone in lactating dairy cows.

Modifying the feeding regime successfully produced large diurnal fluctuations in hormone concentrations, but there appeared to be no associated effect on oocyte quality in heifers.

Leptin can act directly on bovine granulosa and theca cells to inhibit steroidogenesis.In conclusion, this integrated programme of work utilising in vivo and in vitro approaches in lactating dairy cows and dairy heifers has resulted in significant advances in our understanding of how dietary factors can influence oocyte quality. These results have also demonstrated how quickly diet can have its effect, even in a ruminant species, and that these effects are cumulative over time and closely integrated with body condition. Importantly these findings were utilised in the associated Link Project (LK0646) to develop similar dietary regimes which resulted in a significant increase in pregnancy rate from 27% to 60% at 120 days of lactation (see final Link Project report). The increased understanding which has resulted from this work should have significant implications in the nutritional and reproductive management of lactating dairy cows leading to an improvement in fertility if implemented after further confirmatory studies.Possible Future WorkThe UK Government target is to reduce carbon dioxide emissions by 20 per cent below 1990 levels by 2010 and, in the long-term, to reduce emissions by 60 per cent by 2050. Methane is the second most important greenhouse gas in the UK after carbon dioxide. Its global warming potential is 23 times that of carbon dioxide. Methane contributed 12 per cent of the UK’s total emissions of greenhouse gases in 1990 or 25.1 MtC. The major sources were landfill waste, agriculture, natural gas distribution and coal mining. Annual emissions fell by about 50 per cent below 1990 levels to 12.5 MtC in 2004. However, although emissions from non-agriculture sources fell by 62 per cent, agriculture emissions fell by only 13 per cent over this period, and agriculture has now become the largest source of methane emissions. Forty-six per cent of UK methane emissions arise from agriculture. 80 per cent of this methane comes from enteric fermentation in the digestive systems of animals, and 20 per cent from animal waste. Enteric fermentation involves the digestion of cellulose by Archaea, which are bacteria-like microorganisms found in the rumen. Enteric emissions, therefore, originate mainly from grass-fed ruminants.Impact of UK dairyingDairy cows and replacement heifers account for 2.4 MtC, which is 19 per cent of total methane emissions and 42 per cent of agricultural emissions. Emissions by dairy herds have decreased by 10 per cent since 1990 because cow numbers decreased by 24 per cent as milk yield per cow increased by 27 per cent while total output remained relatively steady. It is predicted that the trend for increasing milk yield (+88 kg/cow/yr) will continue for the foreseeable future, so cow numbers and methane emissions are expected to continue falling.If current trends in milk yield and cow numbers could be maintained, then methane emissions from dairy herds would be predicted to decrease by 17 percent of 1990 values by 2020 and 25 per cent by 2050, calculated using Intergovernmental Panel on Climate Change methodology. IPCC methodology, however, calculates emissions according to net energy intake and does not allow for changes in emission factors with changes in diet composition. If higher milk yields are to be achieved, diets will need to contain increasing proportions of starchy feeds, rather than grass and grass silage. This would accelerate decreases in methane, so potential reductions would be 23 percent by 2020 and 50 per cent by 2050. Importantly however, this reduction in emissions cannot be achieved due to current problems of infertility in dairy cows.The ChallengeAs discussed, genetic selection for production efficiency over the past 30 years has led to dramatic increases in milk yield of dairy cows. However, because of a detrimental genetic correlation between yield and fertility, there has been a concomitant decline in reproductive efficiency, leading to premature culling and reduced lifetime performance. Since 1980, annual replacement rates have increased from 25% to 33%

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and herd life has decreased from four lactations to three. The current replacement rate is near the limit of biological sustainability, in terms of ability to produce sufficient replacements to maintain the population. In addition to economic losses caused by infertility, the net environmental burden has increased because outputs of methane, nitrogen and phosphorus during the rearing phase are spread over fewer units of animal product. It has been estimated that the decline in fertility has increased methane emissions by 20% in dairy herds over the past 20 years.If the trend for decreasing fertility could be reversed, and fertility restored to levels seen in the 1970s, then reductions in methane emissions of 32% of 1990 emissions (0.85 MtC) could be achieved by 2020 and 62% (1.63 MtC) by 2050. In addition to reductions in methane emissions, improved fertility would lead to decreases in nitrogen excretion (11% in 2020 and 27% in 2050) and ammonia emissions (20% in 2020 and 40% in 2050).Reversing the infertility trend requires identification of strategies for improving fertility, provision of tools that can be applied under commercial conditions, and persuading the industry to adopt these tools and approaches. This last requirement is vital if predicted savings are to be achieved. Importantly, the economic benefits from improving fertility will provide a large incentive for farmers to adopt proven technology.Progress to date:In the current work, we have shown that the quantity and quality of nutrient inputs can have profound effects on reproductive performance. For example, changes in carbohydrates, fatty acids and amino acids alter length of the postpartum anoestrous period and conception rate. Nutrition affects all levels of the reproductive axis, including hypothalamic release of gonadotrophins, ovarian steroid production, follicular growth and developmental competence of oocytes. These effects appear to be mediated through systemic changes in metabolic hormones, such as insulin, growth hormone, insulin-like growth factors and leptin and through local changes in hormone receptors and gene expression.Incorporating the results from the current extensive programme of work with the results from the Link Project (LK0646) showed that nutritional principles can be used to design dietary strategies that improve fertility under controlled commercial conditions. Our best strategy improved pregnancy rate from 27% to 60% at 120 days of lactation. However, we know that there are other factors that affect fertility, and our industrial partners, on the Link Grant have indicated that this initial study needs to be confirmed before the technology will be adopted by more than 5-10% of UK dairy herds.Furthermore, there is clearly a genetic basis to the problem and hence there is the opportunity for a genetic solution. Genetic gains are cumulative and permanent, and through our previous introduction of a UK fertility index for dairy bulls in our complementary genetics programme of work we have provided the tools whereby selection can be practised. Furthermore we have shown that molecular markers can be identified for fertility traits, so offering the opportunity of selection early in life.In addition, oestrus detection rate in the UK, as in many other countries, is currently about 50%. This is exacerbated by a significant increase in the proportion of animals either not exhibiting standing oestrus or for a significantly shorter time (reduced from ~12-15 hours to ~5-7 hours over the last 20-30 years).Coupled with an estimated pregnancy rate of 40%, this means that only 20% of ovulations actually result in the birth of a calf: 80% of opportunities are missed. For example, in one recent study in the USA involving visual observation of oestrus, 62% of ovulations were missed. In the USA the increasing problem of oestrus detection has forced the implementation of complex ovulation synchronisation protocols involving repeated hormone administration. UK dairy genetics are predominantly imported from North America, so it is not surprising that similar problems exist in the UK dairy herd. But a similar solution is clearly outside the UK remit of “sustainable systems”. If sustainable progress is to be made, strategies to optimise expression of oestrus must be developed. A number of factors have been shown to reduce expression of oestrus, including poor energy balance, high milk yield, poor housing, high environmental temperature and disruption of normal routine, all acting on an animal with a high genetic susceptibility to disruption of normal oestrus behaviour. In assessing oestrus expression it is important to consider all these factors. Future Studies:The progress made in the previous programme of work has provided both a sound basis and the confidence that further significant progress can be made. A parallel three pronged approach combining nutritional, genetic and oestrus studies needs to be continued as all these aspects impact on dairy cow fertility. This work will include (i) improved dietary strategies for increased reproductive efficiency, (ii) identification of specific genetic markers for selecting more fertile cattle and (iii) investigation of the reason for the increase in cattle not exhibiting standing increase coupled with new approached to improve oestrus detection rates.

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This programme of work would utilize the modern high-yielding Holstein dairy cow, since this is the important breed in the UK numerically, and has the greatest problem with fertility. All projects would also be informed by the need to assess the environmental impacts of any proposed changes in dairy cow management, with a view to minimizing undesirable environmental outputs. In this regard, this integrated programme will also provide novel data on methane and nitrogen emissions in response to diet composition from individual cows kept under commercial conditions. It would also allow the development of strategic plans for reducing methane and nitrogen emissions by UK livestock. Hence this integrated approach would not only result in an improvement in dairy cow fertility, a key requirement if environmental targets are to be met, but would reduce environmental impact by dietary improvements, whilst maintaining economic sustainability. An impact on economic sustainability would also help ensure the uptake of new management strategies, whilst helping to safeguard UK milk production.Action Resulting from the Work (Knowledge Transfer)As stated the work carried out within this Strategic Grant was carried out in parallel with the Link Project (LK0646). The current work described above has identified some of the key factors influencing follicular development and oocyte quality. This information combined with the more applied programme carried out in the Link Project has proved our original hypothesis that the optimum strategy for maintaining fertility in high-yielding dairy cows is to feed diets to stimulate follicular development, and to feed diets to improve developmental competence of oocytes. Indeed one of the dietary strategies (see Link Project Report) resulted in a very significant increase in pregnancy rates. Hence using this strategy, we have demonstrated that fertility levels can be enhanced without compromising milk production. In this regard this combined work demonstrated that the fertility of 10,000 litre cows could be altered into the equivalent fertility of 6,000 litre cows, namely fertility was restored to levels seen 20 years ago. However, this has been achieved whilst still maintaining milk yields of 10,000 litres. Importantly such an improvement would also have a marked positive impact on reducing the environmental footprint of dairy cows.The results of this combined programme are currently being presented to the dairy industry because it will have a direct economic benefit for dairy farmers. This is being achieved via a number of routes including presentation at conferences (e.g. Nottingham Cattle Fertility Conference, 2007; Liverpool Cattle Fertility Conference, 2007). Furthermore at least two of the commercial partners involved in the Link project are evaluating these results in the commercial scenario and initial results are positive. Indeed some of these results have already been presented to Nutrition Consultants (e.g. Kite, BOCM), to disseminate this information on-farm.Finally, these data are being published in scientific journals after peer review and presented at both national and international conferences. In this regard, there have already been over 40 publications to date, including four peer reviewed papers in quality journals, in addition to a significant number of refereed reviews that have already been published. Furthermore, we expect that a least a further 10 peer reviewed papers will be published from this work during the next 24 months.References cited in the ReportAdamiak SJ, Mackie K, Watt RG, Webb, R and Sinclair, KD (2005) Impact of nutrition on oocyte quality: Cumulative effects of body composition and diet leading to hyperinsulinemia in cattle. Biology of Reproduction 73: 918-926Adamiak, SJ, Powell, K, Rooke, JA, Webb, R and Sinclair, KD (2006) Dietary carbohydrates and fatty acids determine post-fertilisation development of bovine oocytes in vitro. Reproduction 131: 247-258Amstalden M, Garcia MR, Williams SW, Stanko RL, Nizielski SE and Morrison CD (2000) Leptin gene expression, circulating leptin, and luteinizing hormone pulsatility are acutely responsive to short-term fasting in prepubertal heifers: relationships to circulating insulin and insulin-like growth factor-I Biology of Reproduction 63: 127-133Armstrong DG, McEvoy TG, Baxter G, Robinson JJ, Hogg CO, Woad KJ and Webb R (2001) Effect of dietary energy and protein on bovine follicular dynamics and embryo production in vitro: associations with the ovarian insulin-like growth factor system Biology of Reproduction 64: 1624-1632Armstrong DG Gong JG and Webb R (2003) Interactions between nutrition and ovarian activity in cattle: physiological, cellular and molecular mechanisms. Reproduction Supplement 61: 403-414Beam SW and Butler WR (1999) Effects of energy balance on follicular development and first ovulation in postpartum dairy cows Journal of Reproduction and Fertility Supplement 54: 411-424Fouladi-Nashta, AA, Alberio R, Kafi M, Nicholas B, Campbell KHS and Webb R (2005) Differential staining combined with TUNEL labelling to detect apoptosis in preimplantation bovine embryos. Reproduction Biomedicine Online 10: 497-502

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Fouladi-Nashta AA, Gutierrez CG, Gong JG, Garnsworthy PC and Webb (2007) Impact of dietary fatty acids on oocyte quality and development in lactating dairy cows. Biology of Reproduction 77: 9-17Garnsworthy, PC (2004) The environmental impact of fertility in dairy cows: a modelling approach to predict methane and ammonia emissions. Animal Feed Science and Technology, 112: 211-223Gong JG, Lee WJ, Garnsworthy PC and Webb R (2002) Effect of dietary-induced increases in circulating insulin concentrations during the early postpartum period on reproductive function in dairy cows Reproduction 123: 419-427Gutierrez CG, Oldham J, Bramley TA, Gong JG, Campbell BK and Webb R (1997) The recruitment of ovarian follicles is enhanced by increased dietary intake in heifers Journal of Animal Science 75: 1876-1884Lowman BG, Scott NA & Somerville SH 1976 Condition scoring of cattle Rev. ed. Bulletin, East of Scotland College of Agriculture, no. 6O’Callaghan D. and Boland MP (1999) Nutritional effects on ovulation, embryo development and the establishment of pregnancy in ruminants. Animal Science 68: 299-314Lucy MC. (2001) Reproductive loss in high-producing dairy cattle: where will it end? J. Dairy Sci. 84: 1277-1293Mann GE Green MP Sinclair KD Demmers KJ Fray MD Gutierrez CG Garnsworthy PC and Webb R (2003) Effects of circulating progesterone and insulin on embryo development in beef heifers. Anim. Reprod. Sci. 79: 71-79Royal MD, Darwash AO, Flint APF, Webb R, Woolliams JA and Lamming GE (2000) Declining fertility in dairy cattle: changes in traditional and endocrine parameters of fertility Animal Science 70: 487-502Sinclair KD Rooke JA and McEvoy TG (2003) Regulation of nutrient uptake and metabolism in ruminant embryos. Reproduction, Supplement 61: 371-385Staples CR and Thatcher WW (2005) Effects of fatty acids on reproduction of dairy cows. In: Recent Advances in Animal Nutrition, Eds P.C. Garnsworthy & J. Wiseman, Nottingham University Press 2005 pp 229-256Webb R Nicholas B Gong JG Campbell BK Gutierrez CG Garverick HA and Armstrong DG (2003) Mechanism regulating follicular development and selection of the dominant follicle. Reproduction Supplement 61: 71-90Webb R, Garnsworthy PC Gong JG and Armstrong DG (2004) Control of follicular growth: local interactions and nutritional influences. Journal of Animal Science 82: E63–E74Zeuner A, Muller K, Reguszynski K & Jewgenow K. (2003) Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation. Theriogenology 59: 1421-1433

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

Garnsworthy PC, Sinclair KD and Webb R (2007) Integration of physiological mechanisms that influence fertility in dairy cows. Presented as an invited oral presentation at the International BSAS Conference: Fertility in Dairy Cows: Bridging the Gap (Liverpool, August 2007). Also to be published as a review in Animal 2008.

Rooke JA, Watt RG, Ainslie A, Alink FM, McEvoy TG, Sinclair KD, Garnsworthy, PC and Webb, R (2007) Effect of diet and feeding frequency on bovine oocyte quality. Domestic Animal Reproduction (submitted)

Fouladi-Nashta AA, Gutierrez CG, Gong JG, Garnsworthy PC and Webb R (2007) Impact of Dietary Fatty Acids on Oocyte Quality and Development in Lactating Dairy Cows. Biology of Reproduction 77, 9-17

Chagas LM, Bass JJ, Blache D, Burke CR, Kay JK, Lindsay DR, Lucy MC, Martin GB, Meier S, Rhodes FM, Roche JR, Thatcher WW and Webb R (2007) Invited Review: New Perspectives on the Roles of Nutrition and Metabolic Priorities in the Subfertility of High-Producing Dairy Cows. J. Dairy Sci. 90: 4022-4032

Rooke JA, Watt RG, Ainslie A., Alink FM, McEvoy TG, Sinclair KD, Garnsworthy PC and Webb R. (2007) The effect of changing dietary amino acid composition on plasma metabolite and hormone concentrations and on oocyte quality in Holstein heifers. Presented as a poster presentation at the International BSAS Conference: Fertility in Dairy Cows: Bridging the Gap (Liverpool, August 2007). Will be published as a BSAS Occasional Publication (in press)

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Webb R, Garnsworthy PC, Campbell BK and Hunter MG (2007) Intra-ovarian regulation of follicular development and oocyte competence in farm animals. Theriogenology 68S, S22-S29

Adamiak SJ, Powell K, Rooke JA., Webb R and Sinclair KD (2006) Body composition, dietary carbohydrates and fatty acids determine post-fertilisation development of bovine oocytes in vitro. Reproduction 131, 247-258

Adamiak SJ, Ewen M, Rooke JA, Webb R. and Sinclair KD (2005) Diet and fatty acid composition of bovine plasma, granulosa cells and cumulus-oocyte complexes. Reproduction Fertility and Development 17, 200.

Adamiak SJ, Mackie K, Watt RG, Webb, R and Sinclair, KD (2005) Impact of nutrition on oocyte quality: Cumulative effects of body composition and diet leading to hyperinsulinemia in cattle. Biology of Reproduction 73: 918-926.

Sinclair, K D and Webb, R (2005). Fertility in the modern dairy heifer. In: Calf and heifer rearing. Principles of rearing the modern dairy heifer from calf to calving. Nottingham University Press, Nottingham, pp 277-306.

Sinclair, K D and Webb, R (2005). Reproductive rate in farm animals: Strategies to overcome biological constraints through the use of advanced reproductive technologies. In: Yields in Farm Species. Constraints and Opportunities in the 21st century. Pp 51-87. Eds R Sylvester-Bradley and J Wiseman. Nottingham University Press.

Adamiak, S J; Ewen, M; Powell, K A; Rooke, J A; Webb, R and Sinclair, K D (2004). Inclusion of bovine serum from different dietary backgrounds influences embryo viability in vitro. Reproduction Abstract Series, No. 31: 19.

Adamiak, S J; Mackie, K; Ewen, M; Powell, K A; Watt, R G; Rooke, J A; Webb, R and Sinclair, K D. (2004). Dietary carbohydrates and lipids affect in vitro embryo production following OPU in heifers. Reproduction, Fertility and Development 16: 193-194.

Fouladi-Nashta, A.A., Campbell, K.H.S., Nicholas, B., Alberio, R. and Webb, R. (2004) A simple and fast method for concurrent differential staining and TUNEL labelling of bovine blastocysts. International Embryo Transfer Society, Abstract 151.

Fouladi-Nashta, A.A., Gutierrez, C.G., Russell, H.F., Garnsworthy, P.C. and Webb, R. (2004) Effects of dietary fatty acids on oocyte quality and development in lactating dairy cows. Biology of Reproduction, Special Issue, Abstract 62.

Garnsworthy, P.C. (2004) The environmental cost of infertility in dairy herds. Cattle Practice 12, 47-49.

Garnsworthy, P.C. (2004) The environmental impact of fertility in dairy cows: a modelling approach to predict methane and ammonia emissions. Animal Feed Science and Technology, 112, 211-223.

Pryce, J.E., Royal, M.D., Garnsworthy, P.C. and Mao I.L. (2004) Fertility in the high producing dairy cow. Livestock Production Science, 86:125-135.

Rooke, J A; Ewen, M; Mackie, K; Staines, M E, McEvoy, T G and Sinclair, K D (2004). Effect of ammonium chloride on the growth and metabolism of bovine ovarian granulosa cells and the development of ovine oocytes matured in the presence of bovine granulosa cells previously exposed to ammonium chloride. Animal Reproduction Science, 84: 53-71.

Sinclair, K. D. (2004). Various entries. In: The Encyclopaedia of Farm Animal Nutrition. Ed. M. F. Fuller. CABT Publishing, Oxon, UK.

Webb, R., Garnsworthy, P.C., Gong, J-G. and Armstrong, D. G. (2004) Control of follicular growth: local interactions and nutritional influences. Journal of Animal Science, 82:E63-E74.

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Armstrong D.G., Gong J.G. & Webb R. (2003) Interactions between nutrition and ovarian activity in cattle: physiological, cellular and molecular mechanisms. Reproduction Suppl 61, 403-414 (refereed).

Adamiak, S J; Mackie, K; Powell, K A; Watt, R G; Dolman, D F; Webb, R and Sinclair , K D (2003). Body composition and dietary energy intake affect folliculogenesis, oocyte quality and early embryo development. Reproduction, Abstract Series No. 30: 62-63.

Garnsworthy, P.C. (2003) The impact of dairy cow fertility on methane emissions. Abstracts of the European Association for Animal Production 54th Annual Meeting, Rome. P 283. Wageningen Academic Publishers, The Netherlands.

Garnsworthy, P.C. (2003) Fertility and greenhouse gas emissions in dairy cows. Journal of Animal Science, 81, Suppl. 1/Journal of Dairy Science, 86, Suppl. 1, 169.

Gong JG, Baxter G, Hogg CO, Luo X, Mitchell MRP, Webb R and Armstrong DG (2003) Evidence for direct interaction between leptin and ovarian steroidogenesis in cattle. Reproduction Abstract Series 30 P50.

Mann GE; Green, MP; Sinclair, KD; Demmers, KJ; Fray, MD; Gutierrez, CG; Garnsworthy, PC and Webb, R. (2003). Effects of circulating progesterone and insulin on early embryo development in beef heifers. Animal Reproduction Science, 79: 71-79.

Robinson, R S; Hammond, A J; Sinclair, K D; Adamiak, S J; Hunter, M G and Mann, G E. (2003). Angiogenic factors in the developing bovine corpus luteum. Reproduction, Abstract Series No. 30: 46.

Sinclair, K D; Garnsworthy, P C and Webb, R (2003). Recent advances in reproductive technologies: Implications for livestock production and animal nutrition. In: Recent Advances in Animal Nutrition 2003. Ed: P C Garnsworthy and J Wiseman. Nottingham University Press. pp89-114.

Sinclair, K D; Rooke, J A and McEvoy T G (2003). Regulation of nutrient uptake and metabolism in ruminant embryos. Reproduction, Suppl 61: 371-385.

Webb R, Garnsworthy PC, Gong JG and Armstrong DG (2003) Control of follicular growth: local interactions and nutritional influences. Joint American Society of Animal Science/American Dairy Science Association Meeting "Triennial Reproduction Symposium", 22-26 June, Phoenix, USA.

Webb R., Nicholas B., Gong J.G., Campbell B.K., Gutierrez, C.G., Garverick H.A. & Armstrong D.G. (2003) Mechanisms regulating follicular development and selection of the dominant follicle. Reproduction Suppl. 61, 71-90.

Armstrong DG, Gong JG, Gardner JO, Baxter G, Hogg CO and Webb R (2002) Steroidogenesis in bovine granulosa cells: the effect of short-term changes in dietary intake. Reproduction 123, 371-378.

Dawuda, P M; Scaife, J R; Hutchinson, J S and Sinclair, K D (2002). Mechanisms linking under-nutrition and ovarian function in beef heifers. Animal Reproduction Science 74: 11-26.

Garverick, H.A., Baxter, G., Gong, J. G., Armstrong, D. G.,Campbell, B. K., Gutierrez, C. G. and Webb, R. (2002) Regulation of expression of ovarian mRNA encoding steroidogenic enzymes and gonadotrophin receptors by FSH and GH in hypogonadotrophic cattle. Reproduction 123, 651-661.

Gong JG, Armstrong DG, Baxter G, Hogg CO, Garnsworthy PC and Webb, R (2002). The effect of increased dietary intake on superovulatory response to FSH in heifers. Theriogenology 5`: 1591-1602.

Gong JG, Lee WJ, Garnsworthy PC and Webb R (2002) Effect of dietary-induced increases in circulating insulin concentrations during the early postpartum period on reproductive function in dairy cows. Reproduction 123: 419-427

Pushpakumara P.G.A., Robinson, R.S. Demmers, K.J., Mann, G.E., Sinclair K.D., Webb, R. and Wathes D.C. (2002) Expression of the insulin-like growth factor (IGF) system in the bovine oviduct at

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oestrus and during early pregnancy. Reproduction 123, 859-868

Webb R, Woad KJ and Armstrong DG (2002). Corpus luteum (CL) function: local control mechanisms. Domestic Animal Endocrinology 23: 277-285.

Gong JG (2001) Manipulation of ovarian follicular development by metabolic hormones and nutrition in cattle: practical implications. The 4th International Conference on Farm Animal Endocrinology, 7-10 October, Parma, Italy.

Gong JG, Moghaddam A, Baxter G, Garnsworthy PC, Webb R and Armstrong DG (2001) The effect of feeding a diet to increase circulating insulin concentrations on LH secretion during the early postpartum period in dairy. Journal of Reproduction and Fertility Abstract Series 27, abstract 15.

Gong JG, Troup KD, McCullough E, Garnsworthy PC, Webb R, Armstrong DG (2001) The effect of feeding a diet to increase circulating insulin concentrations on reproductive performance in dairy cows. The 4th International Conference on Farm Animal Endocrinology, 7-10 October, Parma, Italy.

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