W. Ronald Butler

Early-lactating dairy cows are vulnerable to metabolic dysfunctions and diseases and the consequent negative effects of these disturbances on appetite. The exaggerated response of early-lactating cows to metabolic disturbances is illustrated by their response to a hyperinsulinemic-euglycemic clamp (HEC) performed over a 48-h period. Initiation of HEC on d 7 of early lactation (EL) reduced feed intake by 33% but had no effect when performed in late pregnancy (LP, 31 d prepartum). We asked whether growth differentiation factor 15 (GDF15), a novel hormone inhibiting feed intake via activation of neuronal centers, could explain the effect of HEC on appetite in EL. This focus was prompted by increased GDF15 production in primates and rodents experiencing metabolic stressors. In plasma samples collected immediately before and after a 48-h period of HEC in EL and LP, HEC increased plasma GDF15 exclusively in EL, and this increase was positively associated with the reduction in feed intake. The major site of GDF15 production in EL was identified as liver on the basis of 7.7- to 87-fold higher expression than adipose tissue or muscle and increased hepatic GDF15 mRNA abundance during HEC. The HEC stimulated hepatic expression of the GDF15 transcriptional activators ATF4 and CHOP across physiological states and expression of the indicator of amino acid deficiency ASNS in EL but not LP. We next asked whether plasma GDF15 is also regulated by a subset of metabolic disturbances prevailing in EL cows (elevated plasma fatty acids, excessive liver triglyceride, and ketosis). The effect of increased plasma fatty acids was evaluated by i.v. infusion of a lipid emulsion (intralipid) in nonpregnant, nonlactating cows in the absence or presence of glucagon treatment. Intralipid caused a progressive rise in plasma GDF15; this stimulatory effect was increased in the presence of glucagon, resulting in a 2.4-fold increase over control after 13 h of infusion. Positive effects of intralipid were associated with increased hepatic expression of GDF15 and its transcriptional activators ATF4 and sXBP1; presence of glucagon increased GDF15 mRNA further but had no additional effect on ATF4 or sXBP1. Indices of GDF15 production were examined in healthy cows segregated on d 7 of lactation as having low (11.5%) liver triglyceride; neither plasma GDF15 nor hepatic GDF15 mRNA differed between the 2 groups. Finally, plasma GDF15 was measured in early-lactating dairy cows remaining healthy or diagnosed with clinical ketosis. Plasma GDF15 was 1.8-fold higher in ketotic than healthy cows. Overall, these data show upregulation of GDF15 production by a subset of metabolic factors and raise the possibility that GDF15 could contribute to the lower appetitive drive of early-lactating cows experiencing metabolic disturbances.

In transition dairy cows, plasma levels of the insulin-sensitizing hormone adiponectin fall to a nadir at parturition and recover in early lactation. The transition period is also characterized by rapid changes in metabolic and hormonal factors implicated in other species as positive regulators of adiponectin production (i.e., negative energy balance, lipid mobilization) and others as negative regulators (i.e., reduced leptin and insulin and increased growth hormone and plasma fatty acids). To assess the role of onset of negative energy balance and lipid mobilization after parturition, dairy cows were either milked thrice daily (lactating) or never milked (nonlactating) for up to 4 wk after parturition. Plasma adiponectin was 21% higher across time in nonlactating than lactating cows. Moreover, nonlactating cows recovered plasma adiponectin at similar rates as lactating cows even though they failed to lose body condition. Next, we assessed the ability of individual hormones to alter plasma adiponectin in transition dairy cows. In the first experiment, dairy cows received a constant 96-h intravenous infusion of either saline or recombinant human leptin starting on d 8 of lactation. In the second experiment, dairy cows were studied in late pregnancy (LP, starting on prepartum d -31) and again in early lactation (EL, starting on d 7 postpartum) during a 66-h period of basal sampling followed by 48 h of hyperinsulinemic-euglycemia. In the third experiment, cows were studied either in LP (starting on d -40 prepartum) or EL (starting on d 7 postpartum) during a 3-h period of basal sampling followed by 5 d of bovine somatotropin treatment. Plasma adiponectin was reduced by an average of 21% in EL relative to LP in these experiments, but neither leptin, insulin, or growth hormone treatment affected adiponectin in LP or EL. Finally, the possibility that plasma fatty acids repress plasma adiponectin was evaluated by intravenous infusion of a lipid emulsion in nonpregnant, nonlactating cows in the absence or presence of glucagon for 16 consecutive hours. The intralipid infusion increased plasma fatty acid concentration from 102 to over 570 µM within 3 h but had no effect on plasma adiponectin irrespective of presence or absence of glucagon. Overall, these data suggest that energy balance around parturition may regulate plasma adiponectin but do not support roles for lipid mobilization or sustained changes in the plasma concentration of leptin, insulin, growth hormone, or fatty acids.

The objective of the study was to evaluate the effect of prepartum and postpartum (PP) supplementation with 2 isomers of conjugated linoleic acid (CLA) on reproductive parameters and some related metabolic factors in dairy cows. High-producing, multiparous Holstein Friesian cows (n = 60) were allotted to 3 treatment groups: the CLA1 group (n = 20) was supplemented with 70 g of lipid-encapsulated CLA providing 7 g each of cis-9,trans-11 and trans-10,cis-12 CLA from d 21 (d 21) before expected calving until d 7 after artificial insemination (AI), that is, until 77 to 91 d PP; the CLA2 group (n = 20) was supplemented with the same amount of CLA beginning at calving until d 7 after AI; and the control group (n = 20) received an isocaloric, isonitrogenous, and isolipidic diet. Blood samples were taken weekly to measure glucose, insulin, insulin-like growth factor-I (IGF-I), and leptin. Liver biopsy was performed in 10 cows per group for growth hormone receptor 1A and IGF-I mRNA analyses. At d 49 to 63 PP, ovulation was synchronized with the Pre-Synch protocol followed by fixed-time AI. Milk progesterone was monitored from calving until d 35 post-AI. Cows returning to estrus following AI were inseminated. Supplementation with CLA before calving improved the recovery of plasma leptin levels in the early PP period (from the day of calving until wk 3 PP; treatment effect). Later PP (wk 5), plasma IGF-I, and leptin remained significantly higher in both CLA1 and CLA2 groups compared with control, although hepatocellular IGF-I mRNA was not different among groups. Plasma IGF-I levels remained higher in both CLA-treated groups on the day of AI. Growth hormone receptor 1A mRNA levels in hepatic tissue decreased in all groups, reaching a nadir in the first week PP. Days to first PP ovulation did not differ between groups; however, both supplemented groups conceived earlier compared with control (d 97 ± 19, d 97 ± 23, and d 113 ± 30 for CLA1, CLA2, and control, respectively). Plasma progesterone concentration was higher in both supplemented groups on d 2 to 5 following the synchronized ovulation than in controls. We concluded that CLA supplementation around calving alters PP metabolic signals as reflected by higher plasma leptin and IGF-I levels. Conjugated linoleic acid stimulated early luteal function and reduced the PP interval to conception.

The objective of this study was to determine the effects of uterine and systemic inflammatory responses to uterine bacterial contamination at calving in dairy cows on the growth and ovulatory outcomes of the first dominant follicle postpartum. Ovulatory capability of the first dominant follicle postpartum was predicted in 53 multiparous cows by using a combination of follicle growth characteristics and circulating estradiol concentrations. Endotoxin levels were assayed in follicular fluid samples that were aspirated the day after ovulatory outcome prediction. Plasma levels of haptoglobin, a proinflammatory acute phase protein, and paraoxonase, a negative acute phase protein were determined. Uterine bacteria and inflammation were evaluated in three uterine fluid samples from each cow collected on the day of calving, the day after follicle aspiration, and at 35 days postpartum. Cows that had a strong initial uterine inflammatory response (robust recruitment of polymorphonuclear leukocytes of ≥ 35% and cows with uterine pH < 8.5 on the day of calving) were more likely to have an ovulatory first dominant follicle. Follicular fluid endotoxin levels were higher in non-ovulatory cows compared with ovulatory cows. Endotoxin levels in circulation were not different between ovulatory groups but were higher prepartum than on day 7 and 14 postpartum. Systemic inflammation characterized by elevated haptoglobin concentrations was higher in non-ovulatory cows despite similar bacterial contamination and circulating endotoxin levels. Paraoxonase activity in follicular fluid was significantly associated with the paraoxonase activity in plasma, however, plasma paraoxonase concentrations were not different between non-ovulatory and ovulatory cows. Cows with a higher uterine bacterial load on the day of calving had slower ovarian follicle growth. In summary, a robust uterine inflammatory response on the day of calving was positively associated with ovarian function while elevated systemic inflammation during the early postpartum period was negatively associated with the ovulatory status of the first dominant follicle postpartum.

Conjugated linoleic acid (CLA) isomers can affect the lipid profile and signaling of cells and thereby alter their function. A total of 5,700 bovine oocytes were used in a structured series of experiments to test the effects of CLA cis-9,trans-11 and CLA trans-10,cis-12 in vitro. In experiment 1, high doses of each CLA isomer during in vitro maturation (IVM) were compared with high or low doses during the entire in vitro culture (IVC) of parthenogenetic embryos. High doses of the CLA isomers ranged from 50 to 200 μM and low doses were 15 and 25 μM. In experiment 2, the low doses of each CLA isomer were tested during IVM/IVC on embryos produced by in vitro fertilization (IVF). Experiment 3 compared the effects of 15 μM doses of each CLA isomer during IVM or IVC of IVF embryos. In experiment 4, post-rewarming survival rates and blastomere counts were assessed for embryos supplemented with each CLA isomer during IVM or for 36 h before vitrification. In experiment 1, when either CLA isomer was provided only during IVM, we observed no effects on overall rates of maturation, cleavage, or blastocysts (92.2 ± 1.6%, 78.3 ± 4.1%, and 28.9 ± 5.1%, respectively). However, high doses of each CLA isomer, but not low doses, during the entire embryo culture period decreased blastocyst rates (5-20%) in a dose-dependent manner. Cleavage rates improved with 15 or 50 μM CLA trans-10,cis-12. Progesterone concentrations in maturation media were significantly increased by high doses of each CLA isomer compared with control, but low doses of CLA isomers had no effect. In experiment 2 with IVF embryos, low doses of each CLA isomer did not alter cleavage rates (average 84.9 ± 1.9%) and only 25 μM CLA trans-10,cis-12 during IVC reduced blastocyst rates below those of controls (25.5 ± 2.1 vs. 38.2 ± 2.3%). The lipid content of embryos was increased and relative expression of the BIRC5 (baculoviral IAP repeat containing 5) gene was depressed by CLA trans-10,cis-12. In experiment 3, low doses (15μM) of each CLA isomer during IVC significantly reduced blastocyst rates (20.6 ± 2.4% and 27.7 ± 1.2% vs. 34.18 ± 1.2% for CLA trans-10,cis-12 and CLA cis-9,trans-11 compared with control, respectively) with less effect of each CLA during IVM. In experiment 4, adding 100 μM CLA cis-9,trans-11 during the final 36 h of culture resulted in a high survival rate after rewarming and culture, and the higher embryo blastomere count was comparable to that of control embryos not undergoing vitrification. In conclusion, supplementation with either CLA isomer did not improve embryo production, but inclusion of CLA cis-9,trans-11 before vitrification improved the quality of bovine IVF embryos after rewarming and culture.

The objective was to evaluate the individual and combined effect of anovulation and cytological endometritis (CTE) on the reproductive performance of dairy cows. A total of 1,569 cows from 3 data sets were used. In data set 1, 403 Holstein cows from 5 dairies in New York were used. In data set 2, 750 Holstein cows from 2 dairies, one in Florida and one in California were used. In data set 3, 416 dairy cows, 165 Holsteins, 36 Jerseys, and 215 Holstein-Jersey crossbreeds from a grazing dairy in Florida were used. Cyclicity and CTE was determined at 35±3 (data set 2) or 49±3 d in milk (data sets 1 and 3). A variable (VarCycCTE) containing all 4 possible permutations between cyclicity (cyclic = Cyc; anovular = Anov) and CTE (present = CTE; absent = Healthy) was created. In the combined data set (sets 1, 2, and 3), pregnancy per artificial insemination (P/AI) diagnosed at 30 to 38 d after first AI was affected by VarCycCTE, with AnovCTE cows having decreased P/AI compared with CycHealthy cows (21.3 vs. 46.7%), whereas AnovHealthy (37.9%) and CycCTE cows (36.0%) had intermediate P/AI. Pregnancy per artificial insemination for the individual data sets and for pregnancy diagnosed at 63 to 74 d after artificial insemination followed a similar pattern. Pregnancy loss was not affected by VarCycCTE. Hazard of pregnancy up to 300 d in milk was affected by VarCycCTE in the combined data sets 1 and 2, with AnovCTE [hazard ratio (HR)=0.55], AnovHealthy cows (HR=0.71), and CycCTE (HR=0.8) having decreased hazard of pregnancy compared with CycHealthy cows. Median days open were 200, 159, 145, and 121 for AnovCTE, AnovHealthy, CycCTE, and CycHealthy, respectively. Hazard of pregnancy for the individual data sets followed a similar pattern. In summary, both anovulation and CTE were negatively associated with reproductive performance and, when combined, they had an additive negative effect.

Despite advantages of in vitro embryo production in many species, widespread use of this technology is limited by generally lower developmental competence of in vitro derived embryos compared to in vivo counterparts. Regardless, in vivo or in vitro gametes and embryos face and must adjust to multiple microenvironments especially at preimplantation stages. Moreover, the embryo has to be able to further adapt to environmental cues in utero to result in the birth of live and healthy offspring. Enormous strides have been made in understanding and meeting stage-specific requirements of preimplantation embryos, but interpretation of the data is made difficult due to the complexity of the wide array of culture systems and the remarkable plasticity of developing embryos that seem able to develop under a variety of conditions. Nevertheless, a primary objective remains meeting, as closely as possible, the preimplantation embryo requirements as provided in vivo. In general, oocytes and embryos develop more satisfactorily when cultured in groups. However, optimization of individual culture of oocytes and embryos is an important goal and area of intensive current research for both animal and human clinical application. Successful culture of individual embryos is of primary importance in order to avoid ovarian superstimulation and the associated physiological and psychological disadvantages for patients. This review emphasizes stage specific shifts in embryo metabolism and requirements and research to optimize in vitro embryo culture conditions and supplementation, with a view to optimizing embryo culture in general, and culture of single embryos in particular.

Molecular events that regulate cellular biosynthesis of steroid hormones have been a topic of intense research for more than half a century. It has been established that transport of cholesterol into the mitochondria forms the rate-limiting step in steroid hormone production. In current models, both the steroidogenic acute regulatory protein (StAR) and the translocator protein (TSPO) have been implicated to have a concerted and indispensable effort in this cholesterol transport. Deletion of StAR in mice resulted in a critical failure of steroid hormone production, but deletion of TSPO in mice was found to be embryonic lethal. As a result, the role of TSPO in cholesterol transport has been established only using pharmacologic and genetic tools in vitro. To allow us to explore in more detail the function of TSPO in cell type-specific experimental manipulations in vivo, we generated mice carrying TSPO floxed alleles (TSPOfl/fl). In this study we made conditional knockout mice (TSPOcΔ/Δ) with TSPO deletion in testicular Leydig cells by crossing with an anti-Mullerian hormone receptor type II cre/+ mouse line. Genetic ablation of TSPO in steroidogenic Leydig cells in mice did not affect testosterone production, gametogenesis, and reproduction. Expression of StAR, cytochrome P450 side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase type I, and TSPO2 in TSPOcΔ/Δ testis was unaffected. These results challenge the prevailing dogma that claims an essential role for TSPO in steroid hormone biosynthesis and force reexamination of functional interpretations made for this protein. This is the first study examining conditional TSPO gene deletion in mice. The results show that TSPO function is not essential for steroid hormone biosynthesis.