Metabolic changes in follicular fluid of the dominant follicle in high-yielding dairy cows early post partum

Characteristics of the intrafollicular environment to which the preovulatory oocyte is exposed may be one of the major factors determining subsequent fertility. The aim of our study was to examine to what extent metabolic changes that occur in early post partum high-yielding dairy cows are reflected in the follicular fluid (FF) of the dominant follicle (>8 mm). Nine blood samples were taken per cow from nine high-yielding dairy cows between 7 days before and 46 days after parturition. From Day 14 post partum on and together with blood sampling, FF samples of the largest follicle were collected from the same cows by means of transvaginal follicle aspiration. Serum and FF samples were analyzed using commercial clinical and photometric chemistry assays for glucose, beta-hydroxybutyrate (beta-OHB), urea, total protein (TP), triglycerides (TG), non-esterified fatty acids (NEFA) and total cholesterol (TC). All cows lost body condition during the experimental period (0.94+/-0.09 points) illustrating a negative energy balance during the experimental period. In FF, glucose concentrations were significantly higher and the TP, TG, NEFA and TC concentrations were significantly lower than in serum (P<0.05). The concentrations of glucose, beta-OHB, urea and TC in serum and in FF changed significantly over time (P<0.05). Throughout the study, changes of all metabolites in serum were reflected by similar changes in FF. Especially for glucose, beta-OHB and urea the correlations were remarkably high. The results from the present study confirm that the typical metabolic adaptations which can be found in serum of high-yielding dairy cows shortly post partum, are reflected in follicular fluid and, therefore, may affect the quality of both the oocyte and the granulosa cells.     

A study relating the composition of follicular fluid and blood plasma from individual Holstein dairy cows to the in vitro developmental competence of pooled abattoir-derived oocytes

The fertility of high-performance (high milk yield) dairy breeds such as the Holstein within the Australian dairy herd has been on the decline for the past two decades. The 12-month calving interval for pasture-based farming practices results in oocyte maturation coinciding with peak lactation, periods of negative energy balance, and energy partitioning for lactation, causing energy deficiency in some organ systems, including the reproductive system. Oocyte developmental competence (the ability to undergo successful fertilization, embryo development, and establishment of pregnancy) is intrinsically linked with the composition of follicular fluid (FF). The aim of this study was to determine if there was a relationship between the fat and carbohydrate levels in plasma and FF and the ability to support in vitro oocyte maturation (IVM). Plasma and FF were collected in vivo from eight Holstein cows between 52 and 151 days post-partum. Plasma glucose trended (P = 0.072) higher and triglyceride levels were significantly higher than in FF (P < 0.05), but there were no relationships between FF and plasma composition. Glucose FF concentration was negatively related to follicular lactate and nonesterified fatty acid (NEFA) levels and days post-partum. Conversely, FF triglyceride concentrations were positively related to FF NEFA levels and negatively related to milk fat and protein composition. Abattoir-derived cumulus–oocyte complexes were cultured in either 50% FF (FF-IVM) or 50% plasma (plasma-IVM), with on-time embryo development then assessed. Although there were no differences between animals, the blastocyst rates after FF-IVM were negatively related to plasma glucose and days post-partum and positively related to body condition score and plasma NEFA levels. In comparison to the previous studies, total NEFA levels in FF were not related to animal parameters and did not influence oocyte developmental competence in vitro. Results from this study suggest that days post-partum and body condition score influence carbohydrate metabolism within the follicular environment, and this may be attributed to the pasture-based feed system applied in the Australian dairy industry.     

Metabolite and ionic composition of follicular fluid from different-sized follicles and their relationship to serum concentrations in dairy cows

Metabolic changes in blood serum may be reflected in the biochemical composition of follicular fluid and could indirectly influence oocyte quality. The purpose of this study was to examine the biochemical composition of follicular fluid harvested from different-sized follicles and its relationship with that of blood serum in dairy cattle. Following slaughter, blood samples were collected from dairy cows (n=30) and follicular fluid aspirated from three size classes of non-atretic follicles (<4 mm, 6–8 mm and >10 mm diameter). Samples remained independent between cows and between size classes within cows. Serum and follicular fluid samples were assayed using commercial clinical and photometric chemistry assays for ions (sodium, potassium and chloride) and metabolites (glucose, β-hydroxybutyrate (β-OHB), lactate, urea, total protein, triglycerides, non-esterified fatty acids (NEFA) and total cholesterol). Results showed that follicular fluid concentrations of glucose, β-OHB and total cholesterol increased from small to large follicles and decreased for potassium, chloride, lactate, urea and triglycerides. There was a significant concentration gradient for all variables between their levels in serum and follicular fluid (P<0.05). Significant correlations were observed for chloride (r=0.40), glucose (r=0.56), β-OHB (r=0.85), urea (r=0.95) and total protein (r=0.60) for all three follicle size classes and for triglycerides (r=0.43), NEFA (r=0.50) and total cholesterol (r=0.42) for large follicles (P<0.05). The results from the present study suggest that the oocyte and the granulosa cells of dairy cows grow and mature in a biochemical environment that changes from small to large follicles. Furthermore, the significant correlation between the composition of serum and follicular fluid for the above-mentioned metabolites suggests that metabolic changes in serum levels will be reflected in the follicular fluid and, therefore, may affect the quality of both the oocyte and the granulosa cells.     

Milk fatty acids as indicators of negative energy balance of dairy cows in early lactation

Most dairy cows experience negative energy balance (NEB) in early lactation because energy demand for milk synthesis is not met by energy intake. Excessive NEB may lead to metabolic disorders and impaired fertility. To optimize herd management, it is useful to detect cows in NEB in early lactation, but direct calculation of NEB is not feasible in commercial herds. Alternative methods rely on fat-to-protein ratio in milk or on concentrations of non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHB) in blood. Here, we considered methods to assess energy balance (EB) of dairy cows based on the fatty acid (FA) composition in milk. Short- and medium-chain FAs (primarily, C14:0) are typically synthesized de novo in the mammary gland and their proportions in milk fat decrease during NEB. Long-chain FAs C18:0 and C18:1 cis-9 are typically released from body fat depots during NEB, and their proportions increase. In this study, these FAs were routinely determined by Fourier-transform infrared spectroscopy (FTIR) of individual milk samples. We performed an experiment on 85 dairy cows in early lactation, fed the same concentrate ration of up to 5 kg per day and forage ad libitum. Daily milk yield and feed intake were automatically recorded. During lactation weeks 2, 4, and 6 after calving, two milk samples were collected for FTIR spectroscopy, Tuesday evening and Wednesday morning, blood plasma samples were collected Thursday morning. Net energy content in feed and net energy required for maintenance and lactation were estimated to derive EB, which was used to compare alternative indicators of severe NEB. Linear univariate models for EB based on NEFA concentration (deviance explained = 0.13) and other metabolites in blood plasma were outperformed by models based on concentrations of metabolites in milk: fat (0.27), fat-to-protein ratio (0.18), BHB (0.20), and especially C18:0 (0.28) and C18:1 cis-9 (0.39). Analysis of generalized additive models (GAM) revealed that models based on milk variables performed better than those based on blood plasma (deviance explained 0.46 vs. 0.21). C18:0 and C18:1 cis-9 also performed better in severe NEB prediction for EB cut-off values ranging from −50 to 0 MJ NEL/d. Overall, concentrations of C18:0 and C18:1 cis-9 in milk, milk fat, and milk BHB were the best variables for early detection of cows in severe NEB. Thus, milk FA concentrations in whole milk can be useful to identify NEB in early-lactation cows.     

Elevated non-esterified fatty acid concentrations during bovine oocyte maturation compromise early embryo physiology

Elevated concentrations of serum non-esterified fatty acids (NEFA), associated with maternal disorders such as obesity and type II diabetes, alter the ovarian follicular micro-environment and have been associated with subfertility arising from reduced oocyte developmental competence. We have asked whether elevated NEFA concentrations during oocyte maturation affect the development and physiology of zygotes formed from such oocytes, using the cow as a model. The zygotes were grown to blastocysts, which were evaluated for their quality in terms of cell number, apoptosis, expression of key genes, amino acid turnover and oxidative metabolism. Oocyte maturation under elevated NEFA concentrations resulted in blastocysts with significantly lower cell number, increased apoptotic cell ratio and altered mRNA abundance of DNMT3A, IGF2R and SLC2A1. In addition, the blastocysts displayed reduced oxygen, pyruvate and glucose consumption, up-regulated lactate consumption and higher amino acid metabolism. These data indicate that exposure of maturing oocytes to elevated NEFA concentrations has a negative impact on fertility not only through a reduction in oocyte developmental capacity but through compromised early embryo quality, viability and metabolism.     

Effect of non-esterified fatty acids on bovine granulosa cell steroidogenesis and proliferation in vitro

In high-yielding dairy cows, the negative energy balance (NEB) during the first weeks post partum may influence dominant follicle growth and steroidogenesis. Since non-esterified fatty acid (NEFA) concentrations are elevated during NEB and are shown to be toxic for several cell types, we investigated the individual and combined effects of the three main NEFA's on granulosa cell proliferation and steroidogenesis in vitro. Granulosa cells from large follicles were cultured for two days in serum free medium in the presence of palmitic (C16:0) (PA), stearic (C18:0) (SA) and/or oleic acid (C18:1) (OA). Addition of 150, 300 or 500 microM of PA and SA inhibited cell proliferation (P<0.05) while OA only elicited such an effect at 500 microM (P<0.01). In the combination treatment (150 microM of each fatty acid), cell numbers were also reduced (P<0.01). These inhibitory effects on cell number are partly due to the induction of apoptosis by these NEFA's, as was demonstrated by annexin V-FITC/propidium iodide staining of the granulosa cells. Oestradiol-17beta production was stimulated by all doses of PA, by 300 and 500 microM of SA and by 500 microM of OA (P<0.05). Combined treatment with 150 microM of each fatty acid also stimulated oestradiol-17beta production per 10(4) cells (P<0.05). We can conclude that PA, SA and to a lesser degree OA modulate granulosa cell proliferation and steroidogenesis in vitro. These effects may be involved in the occurrence of ovarian dysfunction during the postpartum period in high-yielding dairy cows.     

Evaluation of the Change of Serum Copper and Zinc Concentrations of Dairy Cows with Subclinical Ketosis

Ketosis in dairy cows can lead to poor reproductive success and decreased milk production. Since the serum concentrations of copper (Cu) and zinc (Zn) are closely associated with the health status of cows, we investigated whether serum concentrations of Cu and Zn differed in dairy cows with subclinical ketosis and healthy dairy cows. Blood samples of 19 healthy dairy cows and 15 subclinically ketotic dairy cows were collected from three farms, and the concentrations of β-hydroxybutyrate (BHBA), glucose, non-esterified fatty acids (NEFA), Cu, and Zn were determined. Subclinically ketotic dairy cows had significantly higher BHBA and NEFA levels (p?<?0.01) and lower glucose (p?<?0.01) than healthy dairy cows. Likewise, serum concentrations of Zn were significantly decreased (p?<?0.05) in dairy cows with subclinical ketosis. There was no significant difference observed for serum Cu concentration between healthy and subclinically ketotic dairy cows. This study suggests that a decreased serum Zn concentration could be a cause of decreased reproductive performance in subclinically ketotic dairy cows.     

Detrimental effects of high plasma urea nitrogen levels on viability of embryos from lactating dairy cows

High plasma urea nitrogen (PUN) concentrations are associated with decreased fertility in lactating dairy cows. Our objective was to evaluate the quality of embryos flushed from superovulated lactating cows having moderate or high PUN concentrations. Subsequent embryo survival was determined after transfer to recipient heifers with either low or high PUN. Lactating Holstein dairy cows (n = 23; 50-120 days in milk) were randomly assigned to one of two diets designed to result in moderate or high PUN concentrations (15.5 +/- 0.7 and 24.4 +/- 1.0 mg/dl, respectively; P < 0.001) and were fed for 30 days before embryo flushing and recovery. Embryos (n = 94) were evaluated morphologically, frozen and subsequently transferred into synchronized virgin heifers that were fed one of two diets designed to result in either low or high PUN concentrations (7.7 +/- 0.9 and 25.2 +/- 1.5 mg/dl, respectively; P < 0.001; 2 x 2 factorial design). The number, quality and stage of development of recovered embryos were similar for cows with moderate or high PUN. Transfer of embryos from moderate PUN donor cows resulted in a higher pregnancy rate (35%; P < 0.02) than the transfer of embryos from high PUN donor cows (11%). Pregnancy rate was not affected by either recipient diet or the interaction of donor and recipient diets (P > 0.05). These results indicate that high PUN concentrations in lactating dairy cows decrease embryo viability through effects exerted on the oocyte or embryo before recovery from the uterus 7 days after insemination.     

Effects of pressed beet pulp silage inclusion in maize-based rations on performance of high-yielding dairy cows and parameters of rumen fermentation

Beet pulp contains high amounts of pectins that can reduce the risk of rumen disorders compared to using feedstuffs high in starch. The objective was to study the effects of inclusion of ensiled pressed beet pulp in total mixed rations (TMR) for high-yielding dairy cows. Two TMR containing no or about 20% (on dry matter (DM) basis) beet pulp silage were used. The beet pulp silage mainly replaced maize silage and corn cob silage. The TMR were intentionally equal in the concentrations of energy and utilisable crude protein (CP) at the duodenum. TMR were fed to 39 and 40 dairy cows, respectively, for 118 days. The average daily milk yield was about 43 kg/day. No significant differences in milk yield and milk fat or milk protein content were detected. DM intake of cows was significantly reduced by the inclusion of beet pulp silage (23.0 v. 24.5 kg/day). However, a digestibility study, separately conducted with sheep, showed a significantly higher organic matter digestibility and metabolisable energy concentration for the TMR that contained beet pulp silage. In vitro gas production kinetics indicated that the intensity of fermentation was lower in the TMR that contained beet pulp silage. In vitro production of short-chain fatty acids, studied using a Rusitec, did not differ between the TMR. However, the inclusion of beet pulp silage in the ration caused a significant reduction in the efficiency of microbial CP synthesis in vitro. The amino acid profile of microbial protein remained unchanged. It was concluded that beet pulp silage has specific effects on ruminal fermentation that may depress feed intake of cows but improve digestibility. An inclusion of beet pulp silage of up to 20% of DM in rations for high-yielding dairy cows is possible without significant effects on milk yield and milk protein or milk fat.     

Effect of non-esterified fatty acids on bovine theca cell steroidogenesis and proliferation in vitro

Elevated serum non-esterified fatty acid (NEFA) levels associated with a negative energy balance (NEB) may affect ovarian function and hence reproductive performance in high-yielding dairy cows. We have investigated the individual and combined effects of the three major NEFAs on bovine theca cell proliferation and steroidogenesis in vitro. Theca cells from healthy large follicles (>8 mm) obtained from slaughterhouse ovaries were cultured in serum free medium in the presence of 0, 50, 150 and 200 microM of palmitic acid (PA; C16:0); 0, 50, 150 and 250 microM of stearic acid (SA; C18:0); and/or 0, 50, 150 and 250 microM of oleic acid (OA; C18:1). Progesterone and androstenedione concentrations were measured in spent medium after 48 h of culture and cell numbers were determined spectrophotometrically per culture well. Cell viability was assessed by annexin-V FITC/propidium iodide staining. Only the treatment with 200 microM of PA inhibited cell proliferation (P<0.001) when tested individually, both of the mixtures tested (M1=100 microM of PA, 130 microM of SA and 140 microM of OA; M2=200 microM PA, 260 microM of SA and 280 microM of OA) reduced cell numbers (P<0.001). Progesterone and androstenedione production, both per well and per 10(4) cells, were not affected by any of the treatments, with the exception of M2. This mixture reduced progesterone production per well and per 10(4) cells (P<0.05). The effects observed were most likely caused by the cytotoxic action of the NEFAs, as demonstrated by the increased percentage of early apoptotic (M1) and late apoptotic/necrotic cells (M1 and M2) in the combination treatments (P<0.05). When combined, elevated physiological concentrations of PA, SA and OA can modulate theca cell proliferation and steroidogenesis in vitro by reducing theca cell viability. These NEFAs may be one of the mediators through which NEB compromises ovarian functioning and thus fertility in high-yielding dairy cows.     

Nutritional management of the donor cow

Nutrition of the donor cow can influence oocyte and embryo quality, which can affect the success of embryo transfer. Severe undernutrition compromised ovarian follicular development, with implications for superovulatory response and embryo quality. In postpartum lactating cows, undernutrition or inability to consume sufficient nutrients delayed resumption of ovulation, reduced the number of follicles, and compromised oocyte quality. Moderate undernutrition of nonlactating cows was unlikely to affect embryo quality; conversely, nonlactating animals on maintenance diets usually had better superovulatory responses and improved oocyte competence and embryo quality. The negative effects of overfeeding are thought to be mediated by alterations in endocrine cues, such as hyperinsulinemia and increased glucose and IGF-I, which may interfere with glucose transport in the embryo and increase apoptosis. Manipulating energy sources such as carbohydrates and fatty acids (FA) may influence embryo viability, but the effects of FA were not consistent in vitro; increasing concentrations of unsaturated FA in follicular and embryonic cells usually improved embryo viability and resistance to cryopreservation. Excess protein intake and increased urea and ammonia in body fluids can be toxic to embryos, impairing their development; these effects seemed to be associated with alterations in uterine pH and granulosa cell function. Likewise, toxins in feeds (e.g. gossypol), reduced embryo development and increased pregnancy losses. Diet of donor cows should be formulated to optimize the supply of nutrients to meet needs; however, manipulating energy intake, source of FA and protein content of donor diets, particularly moderate underfeeding in nonlactating cows, may further optimize responses.     

Interactions between negative energy balance,metabolic diseases,uterine health and immune response in transition dairy cows

The biological cycles of milk production and reproduction determine dairying profitability thus making management decisions dynamic and time-dependent. Diseases also negatively impact on net earnings of a dairy enterprise. Transition cows in particular face the challenge of negative energy balance (NEB) and/or disproportional energy metabolism (fatty liver, ketosis, subacute, acute ruminal acidosis); disturbed mineral utilization (milk fever, sub-clinical hypocalcemia); and perturbed immune function (retained placenta, metritis, mastitis). Consequently NEB and reduced dry matter intake are aggravated. The combined effects of all these challenges are reduced fertility and milk production resulting in diminishing profits. Risk factors such as NEB, inflammation and impairment of the immune response are highly cause-and-effect related. Thus, managing cows during the transition period should be geared toward reducing NEB or feeding specially formulated diets to improve immunity. Given that all cows experience a reduced feed intake and body condition, infection and inflammation of the uterus after calving, there is a need for further research on the immunology of transition dairy cows. Integrative approaches at the molecular, cellular and animal level may unravel the complex interactions between disturbed metabolism and immune function that predispose cows to periparturient diseases.     

Effect of butaphosphan and cyanocobalamin on postpartum metabolism and milk production in dairy cows

The aim of this study was to determine the effect of butaphosphan and cyanocobalamin (BTPC) supplementation on plasma metabolites and milk production in postpartum dairy cows. A total of fifty-two Holstein cows were randomly assigned to receive either: (1) 10 ml of saline (NaCl 0.9%, control group); (2) 1000 mg of butaphosphan and 0.5 mg of cyanocobalamin (BTPC1 group); and (3) 2000 mg of butaphosphan and 1.0 mg of cyanocobalamin (BTPC2 group). All cows received injections every 5 days from calving to 20 days in milk (DIM). Blood samples were collected every 15 days from calving until 75 DIM to determine serum concentration of glucose, non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHB), cholesterol, urea, calcium (Ca), phosphorus (P), magnesium (Mg), aminotransferase aspartate (AST) and γ-glutamyltransferase (GGT). The body condition score (BCS) and milk production were evaluated from calving until 90 DIM. Increasing doses of BTPC caused a linear reduction in plasma concentrations of NEFA and cholesterol. Supplementation of BTPC also reduced concentrations of BHB but it did not differ between the two treatment doses. Milk yield and milk protein had a linear increase with increasing doses of BTPC. A quadratic effect was detected for milk fat and total milk solids according to treatment dose, and BTPC1 had the lowest mean values. Concentrations of glucose, urea, P, Mg, AST, GGT, milk lactose and BCS were not affected by treatment. These results indicate that injections of BTPC during the early postpartum period can reduce NEFA and BHB concentrations and increase milk production in Holstein cows.     

Effect of nutrition on endocrine parameters, ovarian physiology, and oocyte and embryo development

Reproductive efficiency in high yielding dairy cows has decreased over the past 50 years, despite significant gains in genetic selection for increased milk output. One possible reason for this decline has been a change in the nutritional intake to meet the increased energy and protein demands for higher milk production. Excess energy intake in sheep will lead to significant reductions in progesterone concentrations; the effects in cattle are not so clear. Nutrition, unless radically changed, will have little effect on gonadotropin concentrations in ruminants, and this is in contrast to the situation for pigs and for primates, where very short-term nutritional changes manifest themselves in altered gonadotropin secretion. Cattle with reduced energy intake have smaller dominant follicles and more three-wave cycles, compared with animals on higher feed intakes. One of the main areas where nutrition influences reproductive efficiency is at the level of embryo production. Several studies indicate that excess energy intake reduces the response to superovulation and also decrease the yield of embryos and alters expression of some gene constructs within the developing embryo. The mechanism of this effect is not clear but indications are that the quality of the oocytes may be compromised. Indeed recent data indicate that nutritional changes around the time of mating may have detrimental effects on the establishment of pregnancy in heifers. Thus, nutritional balancing is critical for high-yielding dairy cows, in particular. The challenge remains to modify nutritional and management strategies in such cows to maintain the levels of production made possible by genetic selection and still maintain an acceptable level of fertility.     

Effect of a prolonged release formulation of recombinant bovine somatotropin on plasma concentrations of hormones and metabolites, and milk production in dairy cows.

The effect of a prolonged release formulation of recombinant DNA derived bovine somatotropin (rbST) on the plasma concentrations of growth hormone (GH), insulin-like growth factor-I (IGF-I), insulin, glucose, blood urea nitrogen (BUN) and nonesterified fatty acids (NEFA), and milk production in lactating dairy cows was studied. Eight cows were divided into two equal groups. One group was the noninjected control, and cows in the other group received a single subcutaneous injection of 640 mg rbST. Plasma GH levels in the rbST-treated cows were higher than in the control cows for 10 days after the injection. Plasma IGF-I concentrations were significantly higher in the rbST-treated cows than in the control for 14 days after the treatment. In the rbST-treated cows, the plasma concentrations of insulin and glucose tended to be higher than those in the control until 7 days after the injection. Also, plasma NEFA levels were higher in the rbST-treated cows for 10 days. In contrast, plasma BUN levels were significantly lower in the rbST-treated cows for 17 days after the treatment. For 28 days after the injection, the mean daily milk yield in rbST-treated cows was 4.5 kg (21.2%) more than that in the control cows. In the rbST-treated cows, a highly positive correlation was observed between the mean daily milk yield and the mean plasma concentration of IGF-I throughout the postinjection period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic merit for milk production and reproductive success in dairy cows

The effect of genetic merit for milk production traits - fat, protein and milk yield - in dairy cows on milk production, body condition, blood metabolites, reproductive hormones, feed intake and reproductive performance was studied over a period of 2 years. Cows were grouped into two categories, based on calculated pedigree indices using multiple-trait across country evaluation (MACE). Cows of high genetic merit (HGM, n = 48 in year 1 and n = 46 in year 2) had a mean predicted difference +/- standard deviation for milk production of 475 +/- 76kg. The cows of medium genetic merit (MGM, n = 48 in both years) had a mean predicted difference for milk production of 140 +/- 68kg.The cows calved between January and April, and were offered grass silage ad libitum plus 9kg concentrates per cow per day, irrespective genetic merit, from calving to turnout in March, when they were subjected to one of three grazing systems. Cows were available for rebreeding from late April until late July of each year.High genetic merit cows had higher milk production, incurred greater body condition loss between calving and first service and had lower plasma glucose and insulin-like growth factor-1 (IGF-1) concentrations than medium genetic merit cows. Furthermore, HGM cows had lower first and second service and overall conception rates, and required more services per conception than the MGM cows.Cows that did not conceive to first service were retrospectively compared to those that conceived to first service within each genetic merit group. There were no significant differences between the HGM cows that did not conceive to first service and those that conceived to this service in terms of milk production, body condition score change between calving and first service, feed intake at first service, or in plasma concentrations of glucose, non-esterified fatty acids (NEFA) or IGF-1. Medium genetic merit cows that did not conceive to first service lost more body condition between calving and first service than did those that conceived to this service.In the present study, HGM cows had higher milk production and reduced reproductive performance in comparison with MGM cows. However, reproductive performance was not associated with milk production, feed intake or plasma concentrations of glucose, NEFA or IGF-1 between calving and first service, since there were no significant differences in these variates between high or medium genetic merit cows that did not conceive to first service and those that conceived to this service. Therefore, these variates are unlikely to be useful predictors of reproductive performance, under the conditions of the present study.     

Metabolic adaptation and reticuloruminal pH in periparturient dairy cows experiencing different lipolysis early postpartum

Metabolic adaptation includes an array of concerted metabolic and endocrine events that enable dairy cows bridging the period of energy deficit at the onset of lactation. The present study evaluated metabolic, endocrine and reticuloruminal pH changes in 30 (25 Holstein and five Simmental) periparturient dairy cows experiencing variable lipolysis early postpartum. Cows were fed the same close-up and fresh lactation diets and kept in the same management conditions. Blood samples were collected at day 14, and day 4, relative to expected parturition, and at day 2, and day 21 postpartum, and serum metabolites and hormones related to glucose and lipid metabolism, as well as concentrations of several liver enzymes and acute phase proteins were determined. Additionally, reticuloruminal pH was monitored every 10 min over the last 3 days of the observation period. BW and milk yields were recorded and balances of energy and protein were assessed. Based on serum concentration of non-esterified fatty acids (NEFA) postpartum, cows were retrospectively classified into low (n=8), medium (n=11), and high (n=11) lipolysis groups, with NEFA levels of <0.4 mmol/l, between 0.4 and 0.7 mmol/l, and >0.7 mmol/l, respectively. Overall, elevated NEFA concentrations in the High group went along with a higher ratio of NEFA to cholesterol and reduced insulin sensitivity. While serum glucose, energy deficit and BW loss did not differ, cows of the High group exhibited increased lactate concentrations in the serum, compared with the Medium group. No differences in liver enzymes and acute phase proteins were evidenced among fat mobilization groups, whereas concentration of serum billirubin was lowest in the Low group after parturition. Data of milk yield and milk energy output showed no differences among groups, despite divergences in calculated energy balance and BW change postpartum. Cows of the Low group tended to increase dry matter intake but also showed longer time duration of pH below 6.0 in the reticulorumen (on average 299 min/day compared with 99 and 91 min/day for Medium and High groups, respectively). Differences in metabolic, endocrine and reticuloruminal pH responses indicate diverse metabolic adaptation strategies of early-lactation cows to cope with energy deficit postpartum.     

Preovulatory follicular status and diet affect the insulin and glucose content of follicles in high-yielding dairy cows

Insulin and glucose may be limiting factors for ovarian function in dairy cows genetically selected for high milk yield. The effects of nutrition on the intrafollicular content of insulin and glucose were investigated in Israeli Holstein dairy cattle fed a basic total mixed ration and producing 34-39kg of milk daily. In experiment 1, carried out in 11 oestrus-synchronised cows, little variation in insulin concentration was found in plasma sampled during the luteal phase, but high variation was found in plasma sampled during the follicular phase. Therefore, in order to prevent confounding the effects of diet and of phase in cycle in the following experiments, experimental diets were fed during the luteal phase of synchronised oestrus cycles. In experiment 2, designed as Latin-Square, six cows received sequentially diets containing 17.1 (control) or 19.7% of crude protein, using two sources of supplementary protein, i.e. soyabean meal (SBM) and corn gluten meal (CGM), differing in ruminal degradability and leucine content. When dry matter intake was used as covariant, plasma insulin on day 16 was 29.5 and 26.4% higher in cows fed diets containing SBM and CGM than in the control (P<0.05). In experiment 3, 17 cows were individually fed the basic diet and then switched to isoenergetic diets containing SBM (n=5), CGM (n=6) or corn grain (CG, n=6) given from day 10 to 16 of the synchronised oestrus cycle. On the eve of day 16, and in the morning of day 17, they were administered PGF(2alpha) and the content of 26 largest follicles was aspirated by using the transvaginal ovum pick-up technique. Follicles were sorted into two classes (preovulatory and subordinate) according to oestradiol concentration and the progesterone:oestradiol ratio in follicular fluid (FF). Higher concentrations of insulin (0.282 versus 0.127ng/ml, P<0.0001) and of glucose (0.614 versus 0.386g/l, P<0.002), were found in FF from preovulatory follicles. The insulin concentration in the FF of cows fed the CG diet was 26% higher than in their counterparts fed CGM (P<0.04), SBM being intermediate. Dietary effects did not reach significance in subordinate follicles.The finding that preovulatory follicular status is associated with increased intrafollicular insulin and glucose suggests that insulin is involved in follicular maturation. The nutritional effect on intrafollicular glucose and insulin may have practical implications to optimise feeding in dairy cows during phases of the oestrus cycle.     

Acute fasting in heifers as a model for assessing the relationship between plasma and follicular fluid NEFA concentrations

It is known from epidemiological studies that negative energy balance in early lactating dairy cows is related to a depression in reproductive performance. Elevated plasma concentrations of non-esterified fatty acids (NEFA) are a typical metabolic characteristic of these animals and are proposed as the possible link. The suggestion is that NEFA might have a direct effect on the ovary, by affecting development of the oocyte or the granulosa cells. However, no data is available concerning the relationship between the concentration of NEFA in follicular fluid and plasma. Therefore, a cross-over study with 10 heifers around 15 months of age was conducted to analyze this relationship and examine the suggested effects of the negative energy balance on follicular growth. Investigation of these effects was performed on fasted heifers. The experimental treatment consisted of feeding hay with a subsequent period of fasting, to induce elevated plasma NEFA concentrations. Sampling of follicular fluid was performed using transvaginal aspiration of follicles, which were standardized using a synchronization protocol. In addition, concentrations of glucose, insulin, NEFA, and estradiol were measured in plasma. Follicular estradiol and progesterone concentrations were also measured to assess the quality of the dominant follicle. Fasting resulted in significantly lower plasma glucose (P=0.0006) and plasma insulin (P<0.0001) concentrations, higher plasma estradiol (P=0.008) and higher NEFA (P<0.001) concentrations, and smaller follicles (P=0.04) with lower estradiol:progesterone (E/P) ratios (P=0.05). Concentrations of NEFA in follicular fluid and plasma were closely related. Given this close relationship, we concluded that the presence of high plasma NEFA concentrations might link energy metabolism in early lactation with fertility.