Geert Opsomer

Reduced Fertility in High-yielding Dairy Cows: Are the Oocyte and Embryo in Danger? Part I The Importance of Negative Energy Balance and Altered Corpus Luteum Function to the Reduction of Oocyte and Embryo Quality in High-yielding Dairy Cows*

Fertility in high yielding dairy cows is declining, and there is increasing evidence to presume that oocyte and embryo quality are major factors in the complex pathogenesis of reproductive failure. In this report we present an overview of possible mechanisms linking negative energy balance (NEB) and deficiencies in oocyte and embryo developmental competence; specifically, in the high producing dairy cow. Changes in follicular growth patterns during a period of NEB can indirectly affect oocyte quality. The endocrine and biochemical changes, which are associated with NEB, are reflected in the microenvironment of the growing and maturing female gamete, and likely result in the ovulation of a developmentally incompetent oocyte. Even after an oocyte is successfully ovulated and fertilized, a full-term pregnancy is still not guaranteed. Inadequate corpus luteum function, associated with reduced progesterone, and probably also low insulin-like growth factor concentrations, can cause a suboptimal microenvironment in the uterus that is incapable of sustaining early embryonic life. This may partly account for the low conception rates and the high incidence of early embryonic mortality in high yielding dairy cows.

Reduced fertility in high-yielding dairy cows: are the oocyte and embryo in danger? Part II. Mechanisms linking nutrition and reduced oocyte and embryo quality in high-yielding dairy cows.

Dairy cow fertility has been declining during since the mid-80s and this has given rise to numerous scientific studies in which important parts of the pathogenesis are elucidated. Reduced oocyte and embryo quality are acknowledged as major factors in the widely described low conception rates and in the high prevalence of early embryonic mortality. Apart from the importance of the negative energy balance (NEB) and the associated endocrine and metabolic consequences, there is a growing attention towards the effect of the milk yield promoting diets which are rich in energy and protein. Starch-rich diets can improve the energy status and thus the ovarian activity in the early postpartum period but the oocyte and embryo quality can suffer from such insulinogenic diets. Supplementation of dietary fat has a similar dual effect with a beneficial stimulation of the ovarian steroid production while the oocyte and the embryo display an altered energy metabolism and excessive lipid accumulation. High-protein diets can elevate the ammonia and urea concentrations in the blood, leading to changed intrafollicular, oviductal and uterine environments. Oocytes and embryos are highly sensitive to such changes in their microenvironment, possibly leading to a disturbed maturation, fertilization or early cleavage. Several nutrition-linked mechanisms, through which oocyte and/or embryo quality can be affected in modern dairy cows, well after the period of NEB, are proposed and comprehensively reviewed in the present report.

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, beta-hydroxybutyrate (beta-OHB), lactate, urea, total protein, triglycerides, non-esterified fatty acids (NEFA) and total cholesterol). Results showed that follicular fluid concentrations of glucose, beta-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), beta-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.

Insulin Resistance in Dairy Cows

Glucose is the molecule that drives milk production, and insulin plays a pivotal role in the glucose metabolism of dairy cows. The effect of insulin on the glucose metabolism is regulated by the secretion of insulin by the pancreas and the insulin sensitivity of the skeletal muscles, the adipose tissue, and the liver. Insulin resistance may develop as part of physiologic (pregnancy and lactation) and pathologic processes, which may manifest as decreased insulin sensitivity or decreased insulin responsiveness. A good knowledge of the normal physiology of insulin is needed to measure the in vivo insulin resistance of dairy cows.

Chemiluminescence of bovine polymorphonuclear leucocytes during the periparturient period and relation with metabolic markers and bovine pregnancy-associated glycoprotein.

The respiratory burst activity of polymorphonuclear leucocytes (PMN) was evaluated in eight Holstein cows from 8 weeks before until 6 weeks after calving by chemiluminescence (CL). The CL response started to decrease 1 week before parturition, reaching a minimum during the first 2 weeks after calving. From week 3 of lactation, CL increased again and returned to original levels by week 6 of lactation. Plasma concentrations of 3-hydroxybutyric acid, total bilirubin and bovine pregnancy-associated glycoprotein started to increase before parturition to reach a maximum during the first or second week of lactation. The concentrations of glutamic-oxaloacetic transaminase, lactate dehydrogenase, non-esterified fatty acids and bilirubin increased after calving, reaching a maximum during the second week. A small decrease in plasma cholesterol during the week before and after calving was followed by an increase. The CL response of the PMN showed significant temporal relationships with the plasma concentrations of 3-hydroxybutyric acid, bovine pregnancy-associated glycoprotein, bilirubin, glutamic-oxaloacetic transaminase, non-esterified fatty acids; that with cholesterol was nearly significant. This means that the change in the CL response with time coincided with the changes in plasma concentrations of these substances with time and that these changes were significantly related with each other. The results of this study show that the decreased respiratory burst activity of bovine PMN around parturition may be related to the extent of the metabolic and hormonal changes. Although the causative relationships are not proven, these results support earlier results suggesting that 3-hydroxybutyric acid and bovine pregnancy-associated glycoprotein may directly affect neutrophil function, whereas non-esterified fatty acids, cholesterol, bilirubin, and liver enzymes may have potential as diagnostic markers of impaired neutrophil function and consequently increased disease risk around parturition.

Heifers infected with coagulase-negative staphylococci in early lactation have fewer cases of clinical mastitis and higher milk production in their first lactation than noninfected heifers.

Intramammary infections (IMI) in recently calved dairy heifers are more common than was formerly believed but their relevance for future performance has been studied only rarely. In the present study, the association between the IMI status of fresh heifers and their subsequent udder health, milk production, and culling in first lactation was explored. Quarter milk samples were collected between 1 and 4 d in milk (DIM) and between 5 and 8 DIM from 191 dairy heifers in 20 dairy herds for bacteriological culturing and somatic cell count (SCC) analysis. Monthly milk recording data including composite milk SCC and test-day milk yield (MY) were obtained for the first 285 DIM or until culling. Farmer-recorded clinical mastitis cases were available. Data were analyzed using mixed models and survival analysis. Approximately 80% of the fresh heifers (79.8%) had at least one culture-positive quarter. Coagulase-negative staphylococci (CNS) were the most frequently isolated pathogens (72%), followed by esculin-positive streptococci (4.6%) and Staphylococcus aureus (3.5%). Overall geometric mean SCC at quarter level decreased between the first and second samplings from 348,000 to 116,000 cells/mL. Heifers infected with CNS had an intermediate average test-day SCC (84,000 cells/mL) during the first 285 DIM compared with noninfected heifers (53,000 cells/mL) and heifers infected with major pathogens (195,000 cells/mL). Heifers infected with major pathogens had a much lower average daily MY (18.3kg) during first lactation compared with noninfected animals (21.3kg). That CNS-infected heifers out-produced their noninfected counterparts could be at least partially explained by their significantly lower incidence of clinical mastitis (incidence risk 3.6 vs. 21.0%) during first lactation compared with noninfected heifers. We conclude that although CNS cause the majority of IMI in heifers around calving, they should not be a reason for serious concern.

Interrelations Between Glucose-Induced Insulin Response, Metabolic Indicators, and Time of First Ovulation in High-Yielding Dairy Cows

High-yielding dairy cows are more susceptible to metabolic and reproductive disorders than low-yielding cows. Insulin plays a pivotal role in the development of both problems. In the present study, we aimed to assess the glucose-induced insulin responses of dairy cows at different time points relative to calving and to relate this to the metabolic status and the time of first ovulation. Twenty-three healthy, multiparous Holstein-Friesian cows with a high genetic merit for milk yield were studied from 14 d prepartum to 42 d postpartum. Intravenous glucose tolerance tests were performed on -14, 14, and 42 d relative to calving to evaluate the plasma insulin and glucose responses to a glucose load, as estimated by the peak concentration, the area under the curve (AUC), and the clearance rates of insulin and glucose. Blood samples were obtained at 3-d intervals and analyzed for glucose, insulin, and nonesterified fatty acids (NEFA). The time of first ovulation was defined by transrectal ultrasonography and plasma progesterone analysis. Glucose-induced insulin AUC and peak concentration decreased and glucose clearance increased during lactation compared with the dry period. Plasma NEFA concentrations were negatively related to insulin AUC and peak concentrations. Fourteen cows ovulated within 42 d postpartum, and the remaining 9 cows suffered from delayed resumption of ovarian function. Survival analysis demonstrated that cows with lower NEFA concentrations during the dry period tended to have earlier resumption of ovarian activity. In conclusion, our data suggest a decreased plasma insulin response to glucose postpartum in high-yielding dairy cows, possibly contributing to metabolic stress during the early postpartum period. It is hypothesized that NEFA impair glucose-induced insulin secretion in dairy cows. Additionally, our results suggest the importance of lipolysis during the transition period as a risk factor for delayed ovulation.

Insulin resistance: the link between metabolic disorders and cystic ovarian disease in high yielding dairy cows?

A preliminary investigation was performed to examine whether insulin resistance is a factor in the pathogenesis of cystic ovarian disease (COD) in high-yielding dairy cows. In total 30 cows, of which 15 were diagnosed as suffering from COD based on the anamnesis and clinical examination, and the other 15 served as matched controls, were subjected to an intravenous glucose tolerance test (IVGTT). The aim of the study was to investigate whether insulin activity was altered in COD cows. Differences in glucose clearance between the COD cows and their controls were analyzed comparing the fractional turnover rate (k), the glucose half-time (T1/2), and the area under the curve (AUC) 60 and 120 min after infusion. Differences in insulin response were analyzed comparing the insulin increment, the insulin peak concentration, and the AUC 60 and 120 min after glucose infusion. Although insulin resistance, attended by a secondary hyperinsulinemia, is stated to directly contribute to the ovarian abnormalities that characterize the polycystic ovary syndrome (PCOS) in human medicine, this was not observed in COD cows. On the contrary, COD cows appeared to have a low insulin response following an intravenous glucose load as compared with their matched controls. This was illustrated by significantly lower insulin increments (P = 0.04) and lower insulin peak concentrations (P = 0.04). As COD cows had a significantly lower insulin response to a standard glucose load, it was concluded that insulin could be a factor in the pathogenesis of COD in dairy cows.

The consequences of metabolic changes in high-yielding dairy cows on oocyte and embryo quality

Unsatisfactory reproductive performance in dairy cows, such as reduced conception rates, in addition to an increased incidence of early embryonic mortality, is reported worldwide and has been associated with a period of negative energy balance (NEB) early post partum. Typically, NEB is associated with biochemical changes such as high non-esterified fatty acid (NEFA), high β-hydroxybutyrate (β-OHB) and low glucose concentrations. The concentrations of these and other metabolites in the follicular fluid (FF) of high-yielding dairy cows during NEB were determined and extensively analyzed, and then were replicated in in vitro maturation models to investigate their effect on oocyte quality. The results showed that typical metabolic changes during NEB are well reflected in the FF of the dominant follicle. However, the oocyte seems to be relatively isolated from extremely elevated NEFA or very low glucose concentrations in the blood. Nevertheless, the in vitro maturation models revealed that NEB-associated high NEFA and low glucose levels in the FF are indeed toxic to the oocyte, resulting in deficient oocyte maturation and developmental competence. Induced apoptosis and necrosis in the cumulus cells was particularly obvious. Furthermore, maturation in saturated free fatty acid-rich media had a carry-over effect on embryo quality, leading to reduced cryotolerance of day 7 embryos. Only β-OHB showed an additive toxic effect in moderately hypoglycemic maturation conditions. These in vitro maturation models, based on in vivo observations, suggest that a period of NEB may hamper the fertility of high-yielding dairy cows through increased NEFA and decreased glucose concentrations in the FF directly affecting oocyte quality. In addition to oocyte quality, these results also demonstrate that embryo quality is reduced following an NEB episode. This important observation may be linked to the typical diet provided to stimulate milk yield, or to physiological adaptations sustaining the high milk production. Research into this phenomenon is ongoing.

Differences in the glucose-induced insulin response and the peripheral insulin responsiveness between neonatal calves of the Belgian Blue, Holstein-Friesian, and East Flemish breeds.

Decreased insulin sensitivity (IS) in dairy cows supports milk yield but increases the risk for metabolic and reproductive disorders. Although several inducers of decreased IS are known, it is unclear to what extent it is congenitally determined. The main aim was to investigate differences in IS between neonatal calves of the Belgian Blue (BB) breed, reared for beef production, and the Holstein-Friesian (HF) breed, reared for milk yield. Additionally, a small number of East Flemish (EF) calves, a local dual-purpose breed, were compared with the 2 other breeds. Ten BB, 12 HF, and 4 EF calves with similar age, ration, and housing were selected. In the intravenous glucose tolerance test, blood samples were taken at regular intervals after an intravenous glucose bolus of 150 mg/kg. Area under the curve (AUC), peak concentration, and elimination rate of insulin and glucose were computed. The quantitative insulin sensitivity check index (QUICKI) and revised QUICKI were computed using basal glucose, insulin, and nonesterified fatty acid concentrations. In the intravenous insulin tolerance test, blood samples were obtained from 4 calves of each breed at regular times after an intravenous insulin challenge of 0.05 IU/kg. Based on the decline in glucose concentrations relative to basal levels, the insulin-stimulated blood glucose response was computed. Basal insulin concentrations were higher in HF (1.58 +/- 0.40 microU/mL) than in BB calves (0.35 +/- 0.09 mmol/L). Compared with BB calves, HF and EF calves had higher basal glucose concentrations (4.40 +/- 0.16 vs. 5.70 +/- 0.35 and 5.81 +/- 0.13 mmol/L, respectively), insulin peak concentrations (4.62 +/- 1.09 vs. 9.70 +/- 1.45 and 16.44 +/- 5.58 microU/mL, respectively), insulin AUC (86.71 +/- 18.81 vs. 222.65 +/- 45.00 and 293.69 +/- 109.22 microU/mL.min, respectively), and glucose AUC (256.22 +/- 17.53 vs. 335.66 +/- 18.74 and 321.03 +/- 10.05 mmol/L min, respectively). Glucose elimination rates were lower in HF (1.37 +/- 0.22%/min) than in BB calves (2.35 +/- 0.25%/min). The QUICKI was lower in HF and EF than in BB calves (0.52 +/- 0.039 and 0.57 +/- 0.068 vs. 0.76 +/- 0.038, respectively), and the revised QUICKI was lower in HF (0.86 +/- 0.11) than in BB calves (1.59 +/- 0.17). The insulin-stimulated blood glucose response did not differ between breeds. Because management differences were negligible, our results suggest breed-specific differences in glucose partitioning and IS. These findings may reflect different rearing purposes of the breeds, although extrapolation of the data to adult animals should be done cautiously.