Reproductive performance of pasture-fed dairy cows supplemented with monopropylene glycol.

AIM: To determine the effects of monopropylene glycol (MPG) on resumption of oestrous cycles and pregnancy rates in mixed-aged cows in commercial herds, when administered for 6 weeks before the planned start of mating (PSM). METHODS: A trial was conducted using 1,814 pasture-fed predominantly Holstein-Friesian cows on four spring-calving dairy farms in the Waikato region of New Zealand. Each farm contained >500 cows, >60% of which had a body condition score (BCS) < or =4.5 two weeks before the planned start of calving. Within each farm, cows that had calved but not been seen in oestrus were allocated at random to one of three treatment groups, viz Control cows receiving no MPG (n=580), a second group receiving a single dose of 200 ml MPG after the morning milking (n=622; MPGx1), and a third group receiving 200 ml MPG after each morning and afternoon milking (n=612; MPGx2). Administration of MPG commenced 6 weeks prior to the PSM. Observations for oestrous behaviour and removal of tail paint was carried out at least daily. One week prior to the PSM, all cows with no recorded oestrus were examined to determine whether they had ovulated or not, and anovulatory cows were treated to induce oestrus and ovulation. Cows were artificially inseminated ~6 weeks from the PSM, then bulls were introduced for up to 12 weeks. Pregnancy diagnosis was carried out using transrectal ultrasonography at 12 and 16 weeks after the PSM. Milk production was determined from one herd test during the treatment period, and a second 2-3 weeks after the end of the treatment. RESULTS: There were no effects of treatment with MPG on resumption of oestrous cycles. Pregnancy rates at 12 and 16 weeks after the PSM were greater for cows in the MPGx1 group compared with those in the MPGx2 and Control groups (p<0.05). The yield of milk protein at the fi rst herd test was greater in cows in the MPGx1 and MPGx2 groups than in Control cows (p=0.013). CONCLUSION: This study showed that MPG given to cows with a low BCS for 6 weeks prior to mating had limited benefits on reproductive outcomes.

Insulin resistance in divergent strains of Holstein-Friesian dairy cows offered fresh pasture and increasing amounts of concentrate in early lactation.

The objective of this study was to determine whether the physiological response to an intravenous glucose challenge would be affected by genetic strain or concentrate supplementation in grazing Holstein-Friesian cows in early lactation. North American (NA; n = 30) or New Zealand (NZ; n = 30) cows were randomly allocated to 1 of 3 feeding treatments. All cows were offered a generous pasture allowance, and 4 of the 6 groups received either 3 or 6 kg of dry matter (DM)/cow per day of concentrates. During wk 5 of lactation, all cows underwent an intravenous glucose challenge. Cows of NA origin produced more milk than NZ cows, but there was no significant strain effect on milk fat or protein yield. Milk yield and the yield of individual components increased with increasing level of concentrate eaten, but there were no significant strain x diet interactions. During wk 1 to 6, mean body weight and body condition score decreased in all treatments. Average body weight was greater in NA cows, but body condition score was greater for NZ cows. There was no strain or diet effect on the length of the postpartum anovulatory interval, with cows ovulating before 40 d postpartum on average. Glucose fractional turnover rate was greater in NZ cows compared with those of NA origin and in all cows receiving 6 kg of DM concentrates, indicating a less severe insulin resistance in those treatments. Consistent with this, the time taken to dispose of half the peak glucose concentration was less when 6 kg of DM concentrate was fed, and tended to be less in NZ than in NA cows. There was no effect of genetic strain on glucose area under the curve (AUC) at 60 or 120 min, but AUC at both time points was less in cows receiving 6 kg of DM concentrates per day. Neither genetic strain nor nutrition affected basal or peak insulin concentrations, insulin increment, or insulin AUC, and there were no strain x diet interactions for any of the glucose challenge response variables measured. In conclusion, differences in milk production between NA and NZ cows in early lactation can, at least in part, be explained by the greater degree of insulin resistance in the NA cows, and this insulin resistance can be overcome by supplementing grazing cows with 6 kg of DM concentrates.

Long-term infusions of ghrelin and obestatin in early lactation dairy cows.

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor and a potential orexigenic agent in monogastrics and ruminants. Obestatin has been reported to have the opposite (anorexigenic) effect. Fifty one multiparous cows were randomly allocated to 1 of 3 groups (n = 17): a control group and 2 groups with cows continuously infused with 0.74 mumol/d of ghrelin (GHR group) or obestatin (OBE group) subcutaneously. Infusions began 21 d in milk, and treatments continued for 8 wk. Generalized linear models were used to determine the treatment effect on average daily and cumulative milk production and composition, and plasma ghrelin, growth hormone, insulin-like growth factor (IGF)-1, leptin, nonesterified fatty acids, and glucose. Mixed models, with cow included as a repeated effect, were used to determine if treatment effects differed by week postcalving for milk production, body weight, and body condition score (BCS; scale 1 to 10). Parity, breed, week of the year at calving, treatment, week postcalving, and the 2 wk preexperimental average of each measure (covariate) were included as fixed effects. Treatment did not affect dry matter intake. Cows infused with GHR lost more BCS (-0.71 units) over the 8-wk study period than the control (-0.23 BCS units) cows, and on average were thinner than cows in either of the other 2 treatments (0.2 BCS units). Consistent with the extra BCS loss in GHR cows, plasma IGF-1, glucose, and leptin concentrations were reduced and plasma nonesterified fatty acid concentrations were greater in GHR cows. Despite a numerical tendency for GHR cows to produce more milk (1,779 kg) than control (1,681 kg) or OBE (1,714 kg) cows during the 8-wk period, milk production differences were not statistically different. However, the timing of the numerical separation of the lactation curves coincided with the significant changes in BCS, IGF-1, and leptin. Results indicate a positive effect of ghrelin infusion on lipolysis. Further research is required to determine if the numerical increase in milk production, which coincides with the increased negative energy balance, is real.

Effect of restricted feeding and monopropylene glycol postpartum on metabolic hormones and postpartum anestrus in grazing dairy heifers.

This study was designed to determine the effects of feed restriction and monopropylene glycol (MPG) supplementation on the reproductive, milk production, and somatotropic axes in dairy heifers postpartum. At calving, 49 Holstein-Friesian heifers were allowed either unrestricted (UNR; n = 18) or restricted access to pasture with (RES+MPG; n = 13) or without (RES; n = 18) MPG supplementation (250 mL drenched twice daily for 150 d). The average body condition score (BCS) of the heifers was 5.3 +/- 0.2 on a scale from 1 to 10 (where 1 = emaciated and 10 = obese). Body condition score and body weight were similar among the groups at calving and decreased after calving for all groups. However, body weight loss was around 10% greater for the RES and RES+MPG groups from wk 3 to 12 compared with UNR group. The length of the postpartum anestrous interval was similar for all groups (47, 51, and 45 +/- 5 d for the UNR, RES, and RES+MPG, respectively). Average milk production, protein, fat, and lactose yields during the first 12 wk postpartum were greater in the UNR group than in the RES and RES+MPG groups. Feed restriction affected plasma concentrations of insulin, with lower concentrations in the RES group compared with the UNR group. There were no differences in plasma concentrations of insulin between the RES+MPG group and the UNR or RES groups. An effect of feed restriction was observed on insulin-like growth factor-I concentrations and also a treatment by time interaction with a changing pattern through time as concentrations in the UNR group increased relative to the RES and RES+MPG groups. There were no differences in growth hormone concentrations among the groups. Glucose concentrations were lower in the RES group when compared with RES+MPG and UNR groups and this difference lessened over time. Plasma concentrations of nonesterified fatty acids were greater in the RES group compared with the RES+MPG and UNR groups. Leptin concentrations in the UNR group were greater than in the RES and RES+MPG groups. Hepatic growth hormone receptor 1A, total growth hormone receptor, and insulin-like growth factor-I relative mRNA expressions decreased postpartum with no effect of feed restriction, MPG supplementation, or interaction between time and treatment. During a challenge with MPG, insulin secretion was stimulated but no effect on postpartum anestrous interval in the treatment groups was observed. It was concluded that restricted pasture availability postpartum in dairy heifers calving in optimal BCS had no effect on the postpartum anestrous interval. It did however decrease milk production; thus, we can infer that monopropylene glycol supplementation does not act to prevent loss of milk yield.

Short communication: change in plasma ghrelin in dairy cows following an intravenous glucose challenge.

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor and a potent orexigenic (appetite-stimulating) agent in humans and rodents, but little is known about its effect in dairy cows. Ten multiparous dairy cows 35 d in milk were subjected to an i.v. glucose challenge (300 mg of D-glucose/kg of body weight). Before infusion and at regular intervals after infusion, plasma glucose, insulin, nonesterified fatty acids (NEFA), growth hormone, epinephrine, and ghrelin concentrations were monitored. Plasma insulin rose (27.2 mU/L at 10 min) and NEFA, epinephrine, and ghrelin declined (nadir = 0.22 mmol/L, 22.2 microg/L, and 272 microg/L at 31, 13, and 22 min, respectively) after the glucose infusion. Ghrelin declined for 22 min before returning to suprabasal levels at approximately 75 min postinfusion. Sequential changes of the hormones and metabolites suggested a glucose transporter, type 2- and glucose transporter, type 4-mediated disposal of glucose, and an insulin-mediated reduction in NEFA. Ghrelin and epinephrine declined after glucose infusion and before the insulin peak, but the effect of insulin as a controlling factor in the hyperglycemic reduction in these hormones cannot be discounted. The post-nadir surge in ghrelin may be regulated by the decline in circulating concentrations of glucose and NEFA (an energy-deficit signal). The profile of change in plasma ghrelin in lactating dairy cows after a glucose challenge was similar to that in monogastric animals.

Invited review: New perspectives on the roles of nutrition and metabolic priorities in the subfertility of high-producing dairy cows.

Management, nutrition, production, and genetics are the main reasons for the decline in fertility in the modern dairy cow. Selection for the single trait of milk production with little consideration for traits associated with reproduction in the modern dairy cow has produced an antagonistic relationship between milk yield and reproductive performance. The outcome is a multi-factorial syndrome of subfertility during lactation; thus, to achieve a better understanding and derive a solution, it is necessary to integrate a range of disciplines, including genetics, nutrition, immunology, molecular biology, endocrinology, metabolic and reproductive physiology, and animal welfare. The common theme underlying the process is a link between nutritional and metabolic inputs that support complex interactions between the gonadotropic and somatotropic axes. Multiple hormonal and metabolic signals from the liver, pancreas, muscle, and adipose tissues act on brain centers regulating feed intake, energy balance, and metabolism. Among these signals, glucose, fatty acids, insulin-like growth factor-I, insulin, growth hormone, ghrelin, leptin, and perhaps myostatin appear to play key roles. Many of these factors are affected by changes in the somatotropic axis that are a consequence of, or are needed to support, high milk production. Ovarian tissues also respond directly to metabolic inputs, with consequences for folliculogenesis, steroidogenesis, and the development of the oocyte and embryo. Little doubt exists that appropriate nutritional management before and after calving is essential for successful reproduction. Changes in body composition are related to the processes that lead to ovulation, estrus, and conception. However, better indicators of body composition and measures of critical metabolites are required to form precise nutritional management guidelines to optimize reproductive outcomes. The eventual solution to the reduction in fertility will be a new strategic direction for genetic selection that includes fertility-related traits. However, this will take time to be effective, so, in the short term, we need to gain a greater understanding of the interactions between nutrition and fertility to better manage the issue. A greater understanding of the phenomenon will also provide markers for more targeted genetic selection. This review highlights many fruitful directions for research, aimed at the development of strategies for nutritional management of reproduction in the high-producing subfertile dairy cow.

Nutritional inputs into the reproductive neuroendocrine control system--a multidimensional perspective.

Evolution has shaped regulatory systems to improve the chance of reproductive success in a somewhat unpredictable environment. One of the more powerful regulators of reproductive function in both sexes is metabolic status, defined as the availability of nutrients and energy to the tissues. Here, we briefly review the basics of the relationship between metabolic status and the activity of the system that controls pulsatile GnRH and LH secretion. We then reflect on these relationships within the framework of a model that comprises four interdependent 'dimensions': 1) genetic, 2) structural, 3) communicational, and 4) temporal. Using two major examples, the male sheep and the post-partum dairy cow, we illustrate aspects of each dimension that seemed to have evolved to limit the risks associated with 'the decision to reproduce'. The results of recent studies have also led us to include in our model the concepts of 'metabolic memory' and 'nutrient sensing' to help explain some aspects of the temporal dimension. Throughout the review, we propose directions for future research that could shed light on pathways that have evolved to ensure that animals are able to take the least risky 'decision'.

Effect of monopropylene glycol on luteinizing hormone, metabolites, and postpartum anovulatory intervals in primiparous dairy cows.

This study examined the effect of monopropylene glycol (MPG) supplementation on LH secretion, postpartum interval to first ovulation, and milk production in heifers calving with poor body condition score (BCS). Forty-seven heifers were allocated to 3 treatments: 1) heifers with high BCS (BCH; n = 13) that calved at a BCS of 3.4 (BCS scale of 1 to 5); 2) heifers with low BCS (BCL; n = 17) that calved at a BCS of 2.8; and 3) heifers with low BCS that calved at a BCS of 2.8 and were assigned to receive MPG supplementation (BCL + MPG; n = 17) and grazed pasture ad libitum. Monopropylene glycol was drenched (250 mL) twice daily for 16 wk after calving. Patterns of change in plasma LH were measured at 2 and 5 wk after calving. Pulsatile release of LH at 2 and 5 wk was greater in BCL + MPG and BCH cows compared with the BCL control cows. The BCL + MPG cows had lower NEFA concentrations than did the BCL cows during wk 1 to 6 after calving. At 12 wk postpartum, the proportion of cows cycling was 77, 82, and 28% for the BCH, BCL + MPG, and BCL treatments, respectively. Mean milk fat yield was greater for the BCH treatment during the first 12 wk postpartum compared with the BCL + MPG or BCL treatments, which did not differ from each other. Results of this study indicate that MPG supplementation reduced the interval from calving to first ovulation in heifers having poor body condition at calving.

Concentrate supplementation reduces postprandial plasma ghrelin in grazing dairy cows: a possible neuroendocrine basis for reduced pasture intake in supplemented cows.

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor, and a potent orexigenic agent in human and rodent studies. We hypothesized that ghrelin may play a role in the reduced grazing time in dairy cows receiving supplementary feeds. Fifty-eight Holstein-Friesian (HF) dairy cows of New Zealand (NZ; n = 28) and North American (NA; n = 30) ancestry were provided with unrestricted access to pasture and randomly allocated at calving to either 0, 3, or 6 kg of dry matter concentrates in a 2 x 3 factorial arrangement. Concentrates were offered in equal amounts at each milking. In peak lactation (75 and 79 +/- 19.7 d in milk), blood was sampled from all cows prior to the a.m. milking (i.e., baseline) and following 2 h of unrestricted access to fresh pasture after the a.m. milking on 2 consecutive weeks. Daily milk yield and fat, protein, and lactose concentrations were measured on the day of blood sampling. North American cows produced more milk and consumed numerically more pasture than did NZ cows, and NA cows had elevated plasma ghrelin concentrations pre- and postfeeding. A negative association between dry matter intake and postprandial ghrelin concentrations indicated that other controlling factors may be involved. Circulating ghrelin concentrations before feeding were not affected by concentrate supplementation, but increasing supplementation was associated with a linear decline in pasture intake and postprandial ghrelin concentrations. This negative association between concentrate supplementation and plasma ghrelin concentrations offers a potential neuroendocrine basis for the reduced pasture intake when supplements are offered to cows in grazing systems.

Short communication: genetic selection for milk production increases plasma ghrelin in dairy cows.

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor and a potent orexigenic agent in human and rodent studies, but there is limited information about its effect in dairy cows. Twelve low genetic merit and 9 high genetic merit Holstein-Friesian dairy cows in peak lactation that were offered unrestricted access to fresh pasture were used to determine whether genetic selection for milk production resulted in an associated increase in plasma ghrelin concentration in grazing dairy cows. Blood samples were taken prior to the a.m. milking (i.e., baseline) and following 2 h of grazing after the a.m. milking on 2 consecutive wk during peak lactation. Milk production and dry matter intake were greater in high genetic merit cows compared with low genetic merit cows. Plasma ghrelin and growth hormone concentrations were elevated in high genetic merit cows pre- and postgrazing, and there was no significant interaction between genetic merit and time of sampling. Genetic merit did not affect the plasma nonesterified fatty acid or glucose concentration, but the plasma concentrations of metabolites and hormones measured were diminished 2 h after feeding. Data indicate an increase in plasma ghrelin associated with genetic selection for milk production, and an associated increase in dry matter intake.

Precalving effects on metabolic responses and postpartum anestrus in grazing primiparous dairy cows.

The effect of increased access to pasture feeding during the last 6 wk of gestation on metabolic responses and postpartum anestrous interval was investigated. Heifers with a body condition score (BCS) of 5.0 (BC5+FF; on a 1-to-10 scale, US = 1.5 + 0.32 x New Zealand) were offered unrestricted pasture, and those with BCS 4.0 were fed either pasture unrestricted (BC4+FF) or restricted (BC4+RES) for the last 6 wk of gestation. After calving, all groups were offered unrestricted pasture. Mean BCS at calving for BC5+FF, BC4+FF, and BC4+RES were 4.7 +/- 0.1, 4.3 +/- 0.1, and 3.5 +/- 0.1, respectively. At 35 d postpartum, LH pulse frequency was lower in BC4+RES than in BC4+FF and BC5+FF, which were similar. At 77 d after calving, 8% of BC4+RES cows had ovulated compared with 75% of BC4+FF and 69% of BC5+FF cows. Metabolic hormonal differences between BC4+FF and BC4+RES were not reflected in the differences between BC4+FF and BC5+FF for LH pulse frequency or ovulation. Unrestricted access to pasture during the final 6 wk of gestation for BC4 heifers reduced the risk of prolonged postpartum anestrus. Systemic factors, tissue sensitivity, and critical developmental set points are probably involved in the integrated control of ovulation by body condition.

Effect of dietary intake on steroid feedback on release of luteinizing hormone in ovariectomized cows.

This study tested the hypothesis that the decline in pulsatile release of luteinizing hormone (LH), resulting from steroid negative feedback, is greater in animals fed a low, compared with a high, plane of nutrition. Two-year-old cows were ovariectomized and six days later were fed diets to provide 1.5 x maintenance requirements (n = 6, supplemented) or 0.5 x maintenance requirements (n = 6, restricted) (Round 1). Pulsatile release of LH was measured over a 14-h period on the fifth day of feeding these diets (Day 1); at 6 h, all animals were treated with an intravaginal insert containing 1.38 g progesterone, which remained in place until the end of Day 3. Pulsatile release of LH was again measured for 14 h on Day 3; at 6 h, all animals were injected intramuscularly with oestradiol benzoate (ODB; 1 mg per 500 kg live weight). Three days later, this protocol was repeated, in a cross-over design, with cows that were previously restricted now being supplemented and those cows previously supplemented, now restricted (Round 2). Plasma concentrations of progesterone after intravaginal progesterone treatment were 1.01 ng mL(-1) higher in restricted cows compared with supplemented cows (P < 0.001) and were also higher in Round 1 than in Round 2 and on Day 1 than on Day 3 (P < 0.001). Plasma concentrations of oestradiol following injection with ODB did not differ between supplemented and restricted cows (P > 0.1). Dietary intake did not affect mean concentrations of LH, pulse frequency or amplitude during the 6-h period before steroid treatment or the change in these variables following steroid treatment; however, the slope of the decline in concentrations of LH following progesterone treatment was significantly more negative in cows fed restricted diets compared with those fed supplemented diets. In Round 2, mean concentrations of LH were higher preceding, and decreased more following, progesterone treatment compared with the decrease after ODB treatment. In conclusion, acute dietary restriction resulted in a more rapid decline in the release of LH following treatment with intravaginal progesterone, and was associated with higher concentrations of progesterone in plasma.

Metabolic factors affecting the reproductive axis in male sheep.

Changes in food intake affect the reproductive axis in both sexes, and the nutritional signals involved and the sites that receive those signals are now beginning to be unravelled. Our studies have focussed on the mature male sheep, a model in which high food intake stimulates GnRH-LH pulse frequency for only 10-20 days but continues to promote testicular growth over several months. Different signals and different target organs seem to be responsible for these short- and long-term responses. Short-term dietary treatments lead to changes in blood concentrations of glucose, fatty acids, insulin and leptin, and concentrations of glucose, insulin, leptin and some amino acids in cerebrospinal fluid. It seems unlikely that amino acids affect GnRH-LH secretion directly in sheep. Intracerebroventricular infusions of insulin specifically increase LH pulse frequency, but intravenous, intra-abomasal or intracerebroventricular infusions of glucose have no effect, despite their effects on cerebrospinal fluid insulin concentrations. The addition of fatty acids to the diet also increases LH pulse frequency, but does not affect the concentrations of insulin or leptin in the cerebrospinal fluid. It appears that acute responses to changes in nutrition involve a range of alternative pathways, possibly including interactions among insulin, leptin and energy substrates. Effects of long-term dietary treatments on testicular size are only partly dependent on the GnRH-LH system (that is, on brain control) and so must also depend on other, as yet unknown, pathways. Concepts of 'metabolic sensing and integration' are being developed from the basis of existing knowledge of the central control of appetite and reproduction.

Level of nutrition affects leptin concentrations in plasma and cerebrospinal fluid in sheep.

In mature male sheep, the level of nutrition acutely influences the secretion of reproductive hormones. The mechanism involved is not fully understood but findings in humans and laboratory rodents would suggest a major role for leptin that is secreted from adipose tissue and then travels via the circulation to the central nervous system. Before we can begin to test this hypothesis, we need to be able to measure leptin concentrations in blood plasma and cerebrospinal fluid. We have therefore developed a radioimmunoassay using antibodies raised against biologically active recombinant bovine-ovine leptin. Using this assay, we found that plasma concentrations of leptin were highly correlated to back-fat thickness and to the ratio of back-fat thickness to liveweight, in female and castrated male sheep. Plasma concentrations of leptin were higher in female sheep than in castrated or intact male sheep. Serial samples (every 5 min) suggested that the secretion of leptin in male sheep is episodic but it does not appear to show clear pulsatility, increases post-prandially, or a diurnal rhythm. Leptin concentrations in both plasma and cerebrospinal fluid increased within 5 days in male sheep fed a diet with a high content of energy and protein that also stimulates the secretion of LH pulses. These data suggest that in sheep, as in other species, leptin production is correlated with the mass of adipose tissue and that the hormone passes from the circulation to the cerebrospinal fluid and then to hypothalamic sites. There, it may affect appetite and perhaps GnRH secretion. The role of leptin in the link between nutrition and reproduction needs further investigation.

GnRH secretion into CSF in rams treated with a GnRH antagonist.

The equilibrium of the brain-pituitary-testicular axis is controlled by negative feedback exerted primarily through changes in the circulating concentrations of gonadal steroids. This is usually studied in gonadectomised animals treated with single large doses or constant low levels of exogenous steroid. However, the feedback system probably also contains dynamic components, perhaps expressed as delays to changes in GnRH secretion following a change in steroid concentration. These delays must be measured without interference from surgical procedures, including anaesthesia, bias associated with changes in pituitary responsiveness (which affect the efficiency of pulse detection), and chronic side-effects of gonadectomy. We used a GnRH antagonist ['Antarelix': Ac-D-Nal, D-Cpa, D-Pal, Ser, Tyr, D-Hci, Leu, Lys-(iPr), Pro, D-Ala-NH2] to transiently block LH and steroid secretion (in effect, inducing and reversing castration) in mature male sheep, and measured GnRH secretion into cerebrospinal fluid (CSF) in the third cerebral ventricle. The CSF was withdrawn with a peristaltic pump at a rate of 2 ml/h and pooled every 20 min. Jugular plasma was sampled every 20 min and analysed for testosterone and LH pulses. The antagonist (500 microg i.v.) was injected after 6 h of baseline sampling and the study continued for a further 24 h. The pulses of LH and testosterone disappeared shortly after antagonist injection, with delays of 20 +/- 12 min for LH and 80 +/- 29 min for testosterone. This led to an increase in GnRH pulse frequency, starting 300 +/- 54 min after antagonist injection. Secretion of LH and testosterone pulses resumed at 553 +/- 38 and 530 +/- 30 min (after antagonist injection), and GnRH pulse frequency returned to baseline values after 170 +/- 42 min (relative to LH) and 117 +/- 35 min (relative to testosterone). The consistent nature of these responses across the group of animals suggests that this can be used to test the effects of exteroceptive factors on the dynamics of negative feedback.