Abstract
Feeding a high-concentrate diet to heifers during the juvenile period, resulting in increased body weight (BW) gain and adiposity, leads to early-onset puberty. In this study, we tested the hypothesis that the increase in GnRH/LH release during nutritional acceleration of puberty is accompanied by reciprocal changes in circulating leptin and central release of neuropeptide Y (NPY). The heifers were weaned at 3.5 months of age and fed to gain either 0.5 (Low-gain; LG) or 1.0 kg/day (High-gain; HG) for 30 weeks. A subgroup of heifers was fitted surgically with third ventricle guide cannulas and was subjected to intensive cerebrospinal fluid (CSF) and blood sampling at 8 and 9 months of age. Mean BW was greater in HG than in LG heifers at week 6 of the experiment and remained greater thereafter. Starting at 9 months of age, the percentage of pubertal HG heifers was greater than that of LG heifers, although a replicate effect was observed. During the 6-h period in which CSF and blood were collected simultaneously, all LH pulses coincided with or shortly followed a GnRH pulse. At 8 months of age, the frequency of LH pulses was greater in the HG than in the LG group. Beginning at 6 months of age, concentrations of leptin were greater in HG than in LG heifers. At 9 months of age, concentrations of NPY in the CSF were lesser in HG heifers. These observations indicate that increased BW gain during juvenile development accelerates puberty in heifers, coincident with reciprocal changes in circulating concentrations of leptin and hypothalamic NPY release.
Introduction
The pubertal initiation of high-frequency, episodic secretion of gonadotropin-releasing hormone (GnRH) is largely dependent upon metabolic cues throughout prepubertal development. Growth restriction during the juvenile period has been demonstrated to delay puberty (Kennedy & Mitra 1963, Kennedy 1969, Foster & Olster 1985, Kile et al. 1991), whereas increased adiposity accelerates reproductive maturation in mammals (Frisch & McArthur 1974, Lee et al. 2007, Castellano et al. 2011). In cattle, feeding a high-concentrate diet to heifers during the juvenile period, resulting in increased body weight (BW) gain and adiposity, leads to early maturation of the reproductive neuroendocrine system and earlier puberty (Gasser et al. 2006a,b, Cardoso et al. 2014). Although the mechanisms involved in this process have not been completely elucidated, leptin and leptin-sensitive cells in the CNS appear to play a critical role (Garcia et al. 2003, Maciel et al. 2004a, Zieba et al. 2005). Leptin, a hormone secreted mainly by adipocytes and positively correlated with body fat mass (Ahren et al. 1997), does not acutely affect the secretion of luteinizing hormone (LH) in adequately fed ewes (Henry et al. 1999) and cows (Amstalden et al. 2002). Nevertheless, leptin prevents a fasting-induced reduction in LH pulsatility in prepubertal heifers (Maciel et al. 2004b) and increasing prepubertal secretion of leptin appears to play a permissive yet critical role in timing of the onset of puberty (Garcia et al. 2002, Cardoso et al. 2014). As several studies have demonstrated that leptin does not act directly on GnRH neurons (Finn et al. 1998, Hakansson et al. 1998, Quennell et al. 2009), leptin's influence on the metabolic control of reproductive maturation is probably relayed by intermediate pathways.
Neuropeptide Y (NPY) neurons in the arcuate nucleus (ARC) contain the leptin receptor (Baskin et al. 1999), play an orexigenic role in energy homeostasis (Wang et al. 1997), and are responsive to changes in nutritional status (Kalra & Kalra 2003). In ovariectomized ewes, i.c.v. infusion of leptin for 3 days reduces the expression of NPY mRNA in the ARC (Henry et al. 1999). Furthermore, NPY has been proposed to mediate the inhibitory effects of undernutrition on reproductive function (Kalra & Crowley 1984). Contrary to observations in rats in which NPY can have either stimulatory or inhibitory effects on LH release dependent on the gonadal steroid milieu (Sahu et al. 1987), NPY has predominantly inhibitory actions on the release of LH in ruminants irrespective of steroidal influences (Barker-Gibb et al. 1995, Gazal et al. 1998, Estrada et al. 2003, Morrison et al. 2003). These effects of NPY are largely due to inhibition of GnRH release (Gazal et al. 1998) and may be mediated by a direct action on GnRH neurons (Klenke et al. 2010). In addition, central infusion of NPY in prepubertal rats is followed by a delay of pubertal maturation similar to that observed in food-restricted animals (Pralong et al. 2000). Thus, we reasoned that increased leptin secretion may facilitate pubertal maturation of the hypothalamic–adenohypophyseal–gonadal axis by suppressing hypothalamic NPY release. To test this hypothesis, we examined whether feeding a high-concentrate diet during the juvenile period, a strategy known to accelerate BW gain, increase circulating concentrations of leptin and advance the onset of puberty (Gasser et al. 2006a,b, Allen et al. 2012, Cardoso et al. 2014), would result in lower concentrations of NPY in third-ventricle (3V) cerebrospinal fluid (CSF) concomitantly with increased secretion of GnRH and LH.
Materials and methods
All animal-related procedures used in this study were approved by the Institutional Agricultural Animal Care and Use Committee (IAACUC) of the Texas A&M University System (AUP# 2009-151).
Animals and nutritional model
Twenty-five crossbred heifers (½ Angus, ¼ Hereford, ¼ Brahman) were utilized in two replicates over a 2-year period (one replicate/year). Within each year, heifers were weaned at approximately 3.5 months of age (age at weaning=106±3 days), stratified by date of birth, and assigned randomly to be fed individually until 11 months of age either to achieve a relatively low rate of BW gain (Low-gain (LG), 0.5 kg/day; n=6/replicate) or a high rate (High-gain (HG), 1.0 kg/day; replicate 1, n=6; replicate 2, n=7). Diets were balanced using the level 2 solution of the Large Ruminant Nutrition System (LRNS; http://nutritionmodels.tamu.edu/lrns.htm), which is based on the Cornell Net Carbohydrate and Protein System as described by Fox et al. (2004). Targeted average daily gain was attained by adjustments in the dry matter intake based on BW gain determined every 2 weeks. Ingredients and diet chemical composition are given in Table 1.
Ingredients and chemical composition of diets fed to prepubertal heifers in the current study. Diet A was provided to heifers during the first 10 weeks of the experiment and diet B was fed to heifers from week 11 until the end of the study (30 weeks)
| Item | Diet Aa | Diet Ba |
|---|---|---|
| Ingredientsb | ||
| Alfalfa hay (%) | 22.42 | 17.88 |
| Cottonseed hulls (%) | 6.71 | 10.67 |
| Rolled corn (%) | 40.37 | 53.68 |
| Cane molasses (%) | 4.48 | 3.58 |
| Cottonseed meal (%) | 6.71 | 7.14 |
| Corn gluten feed (%) | 17.91 | 5.29 |
| Urea (%) | – | 0.58 |
| Producers 12:12 premix (%) | 0.46 | 0.44 |
| Calcium carbonate (%) | 0.94 | 0.74 |
| Chemical compositionb | ||
| Metabolizable energy (Mcal/kg) | 2.59 | 2.62 |
| Crude protein (%) | 15.00 | 14.8 |
| Digestible intake protein (%) | 10.65 | 10.80 |
Diets were balanced using the Large Ruminant Nutrition System (LRNS).
Dry matter basis.
Heifers were allocated individually and fed an acclimation diet for 2 weeks post-weaning. After the acclimation period, heifers were fed solely diet A until 6.5 months of age, afterwards they received diet B until the end of the experiment (11 months of age). Blood samples from the coccygeal vasculature were collected twice a month for the duration of the experiment for determination of serum concentrations of leptin. Starting at 7.5 months of age, blood samples were collected twice weekly until the end of the experiment or until puberty was determined (at least three consecutive samples with concentrations of progesterone ≥1 ng/ml), whichever occurred first. Serum was collected from blood samples by centrifugation (2200 g for 20 min at 4 °C) and stored at −20 °C until assessment for hormone concentration.
Surgical cannulation of the 3V and intensive sampling
Starting at 6 months of age, heifers were acclimated to handling to minimize stress effects on the variables measured. At 7.5 months of age (age=228±3 days), a subgroup of heifers (n=3 per group per replicate) was selected randomly to be surgically fitted with 3V cannulas according to our laboratory procedure, as described previously (Gazal et al. 1998). This well-established experimental paradigm has been utilized to measure the release of hypothalamic neuropeptides in several species, including rats (Szczepanska-Sadowska et al. 1983), sheep (Skinner et al. 1995), cattle (Gazal et al. 1998), and nonhuman primates (Van Vugt et al. 1985). The location and function of the cannulas were verified by radiography and continuous flow of CSF. A period of at least 2 weeks was allowed for heifers to recover from surgery before CSF was sampled.
At approximately 8 months of age (age=248±5 days), heifers were fitted with jugular catheters on the day before the intensive sampling. At the same time, the animals were treated prophylactically with antibiotics as reported previously (Gazal et al. 1998). On the day of sampling, heifers were placed in stanchions and 20 cm of polyethylene tubing (0.58 mm i.d.×0.96 mm o.d.; Intramedic Clay Adams Brand, Becton Dickinson, Sparks, MD, USA) were inserted using aseptic techniques through the guide cannula. The position of the tubing was adjusted until CSF withdrawal was easily accomplished using a blunt 22-gauge needle and tuberculin syringe. The end of the tubing was connected to another 60-cm section of polyethylene tubing. The collection end of the tubing was secured approximately 40–50 cm away from the heifer's head to facilitate semi-remote sampling. The blood samples (6 ml) were collected via a remotely secured extension connected to the jugular catheter, simultaneously with CSF samples (600 μl) at 15- (replicate 1: n=3/group) or 10-min intervals (replicate 2: n=3/group) for 6 h, and intensive blood sampling continued for an additional 6 h. The basis of the slight difference in intensive sampling intervals between the two replicates was to ensure accurate detection of GnRH and LH pulses; however, the sensitivity of pulse detection between replicates did not differ. In all cases, void volumes of CSF and blood created by the indwelling tubing and the extensions were discarded before every sample collection. The blood samples were dispensed in tubes containing 100 μl of heparin solution (10 000 IU/ml) and 5% EDTA, and placed on ice immediately. Plasma was separated by centrifugation (2200 g for 20 min at 4 °C) and stored at −20 °C until LH determination. The CSF samples were placed on ice immediately and, within 30 min, stored at −20 °C until NPY analysis. Intensive blood and CSF sampling procedures were repeated at approximately 9 months of age (age=276±3 days) in heifers that had not achieved puberty yet (LG, n=6; HG, n=4). One HG heifer did not have a functional 3V cannula for CSF sampling at 9 months of age; thus only jugular blood samples for LH determination were collected at this time point.
RIA
To confirm the pubertal status of heifers, circulating concentrations of progesterone were determined using a commercial RIA kit (Coat-A-Count, Siemens Healthcare, Malvern, PA, USA) as reported previously (Fajersson et al. 1999). Sensitivity was 0.1 ng/ml with intra- and inter-assay coefficients of variation (CV) of 5.5 and 13% respectively. Circulating concentrations of leptin were determined in triplicates, in a single assay, using a highly specific ovine leptin RIA validated for use in bovine serum (Delavaud et al. 2000). The sensitivity of the assay was 0.1 ng/ml with an intraassay CV of 6%. The plasma concentrations of LH were determined in duplicate 200-μl aliquots with a validated RIA (McVey & Williams 1991). The sensitivity of the assay averaged 0.1 ng/ml, and average intra- and inter-assay CV were 5 and 9.5%, respectively. Concentrations of GnRH were determined in duplicate 200-μl CSF samples as described by Ellinwood et al. (1985). Antiserum BDS-037 (Dr Alain Caraty, INRA Centre de Tours, Nouzilly, France) was used as the source of primary antibody at a working dilution of 1:50 000. The sensitivity of the assay was 0.5 pg/ml, and average intra- and inter-assay CV were 5 and 16% respectively. Finally, concentrations of NPY were determined directly in duplicate 100-μl CSF samples using a commercial bovine RIA kit (Peninsula Laboratories, Belmont, CA, USA). No improvements in the detection of NPY in CSF samples were observed when methanol or C18 Sep-Column extraction methods were tested. Recovery of added mass and parallelism between serial dilutions of samples with the standard curve were performed to validate the use of the RIA kit directly on bovine CSF samples. The sensitivity of the assay averaged 0.25 ng/ml, with intra- and inter-assay CV of 4.5 and 11.5% respectively.
Pulse detection and statistical analysis
BW gain, concentrations of leptin and NPY, and mean concentrations, amplitude, and frequency of GnRH and LH pulses were analyzed by mixed-model analyses for repeated measures using the MIXED procedure of SAS (version 9.3; SAS Institute, Inc., Cary, NC, USA). The sources of variation were treatment, replicate, time and their interactions. Time was used as the repeated variable, and heifer was used as the subject. The frequency and amplitude of LH pulses were determined using a pulse-detection algorithm, Pulsefit 1.2 (Kushler & Brown 1991). Temporal coincidences between GnRH and LH pulses within heifers were determined as defined by Gazal et al. (1998). The percentage of pubertal heifers was analyzed using the CATMOD procedure of SAS (SAS Institute, Inc.). Main effects were considered significant when P≤0.05.
Results
BW gain and circulating concentrations of leptin
As no replicate effect was observed for BW gain and circulating concentrations of leptin, data were pooled by treatment. Mean (±s.e.m.) BW at the beginning of the experiment did not differ between groups (140.9±3.2 kg). BW increased linearly in both dietary groups (Fig. 1). As expected based on the experimental design, HG heifers had greater (P<0.05) BW than LG heifers starting at week 6 of the experiment and continuing throughout the study (P<0.05). Beginning at week 8 of the experiment, circulating concentrations of leptin were greater (P<0.05) in HG than that in LG heifers and remained greater (P<0.05) throughout the study (Fig. 2). Body condition score, assessed according to a scale from 1 to 9 as described by Herd & Sprott (1986), was greater (P<0.05) in HG (6.0±0.2) than that in LG heifers (4.9±0.1) at week 16 of the experiment (approximately 8 months of age).
Mean (±s.e.m.) BW of heifers weaned at approximately 3.5 months of age and fed for 30 weeks to achieve either a low rate of BW gain (LG, n=12 (0–30 weeks)) or a relatively high rate of BW gain (HG, n=13 (0–16 weeks), 12a (17–20 weeks), 9a (21–24 weeks), 8a (25–28 weeks), 6a (29–30 weeks)). BW of HG heifers was greater (P<0.05) than that in LG heifers beginning at week 6 and continuing throughout the study. aHeifers were excluded from the study as they reached puberty.
Citation: Journal of Endocrinology 223, 3; 10.1530/JOE-14-0504
Mean (±s.e.m.) circulating concentrations of leptin in heifers weaned at approximately 3.5 months of age and fed for 30 weeks to achieve either a low rate of BW gain (LG, n=12 (0–30 weeks)) or a relatively high rate of BW gain (HG, n=13 (0–16 weeks), 12a (17–20 weeks), 9a (21–24 weeks), 8a (25–28 weeks), 6a (29–30 weeks)). The concentrations of leptin were greater (P<0.05) in HG heifers than that in LG heifers beginning at week 8 of the experiment and remained greater (P<0.05) until the end of the study. aHeifers were excluded from the study as they reached puberty.
Citation: Journal of Endocrinology 223, 3; 10.1530/JOE-14-0504
Pulsatile patterns of GnRH and LH release, and central concentrations of NPY relative to peripheral circulating leptin
No replicate effect was observed for the mean concentrations or pattern of secretion of GnRH, LH, and NPY; therefore, data were pooled by treatment. At 8 and 9 months of age, circulating concentrations of leptin were greater (P<0.05) in HG than that in LG heifers subjected to CSF sampling (Fig. 3A). The concentrations of NPY in the CSF did not exhibit a pulsatile pattern of secretion during the 6-h sampling period; thus, overall mean concentrations were used to compare the effects of dietary treatments. At 8 months of age, the concentrations of NPY in the 3V CSF tended to be less (P=0.17) in HG than that in LG heifers (Fig. 3B). At 9 months of age, the concentrations of NPY in the 3V CSF were lower (P≤0.02) in HG prepubertal heifers than that in LG heifers (Fig. 3B).
Mean (±s.e.m.) concentrations of leptin in peripheral blood (A) and NPY in 3V CSF (B) of LG and HG heifers at 8 (LG, n=6; HG, n=6) and 9 months of age (LG, n=6; HG, n=3a). The concentrations of leptin were greater in HG heifers than that in LG heifers at 8 and 9 months of age (*P<0.05). Mean concentration of NPY in the 3V CSF was lower in the HG group than that in the LG group at 9 months of age (**P≤0.02). aTwo HG heifers had already reached puberty and one HG heifer did not have a functional 3V cannula at 9 months of age; thus, data from these three animals were not used in the analyses at 9 months.
Citation: Journal of Endocrinology 223, 3; 10.1530/JOE-14-0504
Individual secretion patterns for GnRH (CSF) and LH (plasma) in representative LG (n=2) and HG (n=2) heifers are shown in Fig. 4. GnRH was secreted in the 3V CSF in a pulsatile pattern, consistent with previous reports on mature cows (Gazal et al. 1998, Zieba et al. 2004). High concordance in the patterns of CSF GnRH and plasma LH secretion was observed. During the simultaneous CSF and blood sampling periods (totaling 126 h), 16 LH pulses were identified and 13 (81.3%) occurred in exact temporal synchrony or within one sampling period after a GnRH pulse. Furthermore, all pulses of LH (100%) occurred within two sampling points after the onset of a GnRH pulse. Within the same sampling period, 19 GnRH pulses were detected. Mean GnRH concentrations (8 months of age, 0.9±0.4 pg/ml; 9 months of age, 1.5±0.8 pg/ml), amplitude (8 months of age, 2.8±1.3 pg/ml; 9 months of age, 2.2±1.0 pg/ml), and pulse frequency (8 months of age, 1.1±0.4 pulses/6 h; 9 months of age, 2.0±0.9 pulses/6 h) did not differ between experimental groups within the 6-h sampling period. However, at 8 months of age, mean frequency of LH pulses monitored over 12 h was greater (P<0.01) in the HG (2.8±0.6 pulses/12 h) than that in the LG group (1.0±0.0 pulses/12 h) before puberty occurred in any heifer. At 9 months of age, the frequency of LH pulses in HG (3.8±1.4 pulses/12 h) and LG heifers (2.0±0.8 pulses/12 h) did not differ. No differences in LH pulse amplitude (8 months of age, 0.9±0.3 ng/ml; 9 months of age, 0.8±0.4 ng/ml) or mean concentrations (8 months of age, 0.4±0.1 ng/ml; 9 months of age, 0.3±0.1 ng/ml) were observed between experimental groups at 8 and 9 months of age. It should be noted that two HG heifers reached puberty before 9 months of age; therefore, they were not included in any of the analyses at this time point.
Patterns of secretion of GnRH in 3V CSF and of LH in plasma of representative LG (#568 and #594) and HG heifers (#587 and #600) at 8 and 9 months of age. Pulses of GnRH and LH are denoted by arrows and asterisks respectively. Pulses of LH detected within the 12-h sampling period are enumerated in parentheses.
Citation: Journal of Endocrinology 223, 3; 10.1530/JOE-14-0504
Pubertal onset
Relatively high rates of BW gain during the juvenile period advanced the onset of puberty in the current study; however, a replicate effect and a treatment×replicate interaction (P<0.03) were observed. The percentage of pubertal heifers in the HG group in replicate 1 was greater (P<0.05) than that in the LG group beginning at 9 months of age, with 50% (3/6), 67% (4/6), and 100% (6/6) of HG heifers achieving puberty by 9, 10, and 11 months of age respectively. For replicate 2, only one (age at puberty: 8.8 months of age) of the HG heifers was pubertal by 11 months of age (1/7, 14%) and the percentage of heifers reaching puberty during the experimental period did not differ significantly between dietary groups. None of the LG heifers (0/12) in either replicate had attained puberty by the end of the experiment (11 months of age).
Discussion
Previous studies have revealed that transcriptional and morphological alterations within the hypothalamic NPY network are involved in the sexual maturation of heifers (Alves et al. 2011, Allen et al. 2012). Results from the current study provide further evidence that the hypothalamic NPY system is a key metabolic-sensing system involved in the nutritionally mediated acceleration of puberty in heifers. Herein, it was demonstrated that elevated BW gain, achieved by feeding a high-concentrate diet to heifers during the juvenile period, leads to greater circulating concentrations of leptin and decreased central release of NPY, characterized by lower concentrations of the peptide in 3V CSF. Furthermore, prepubertal heifers that gained BW at greater rates exhibited increase in pulsatile secretion of LH and an overall advancement of pubertal onset. Because NPY has been demonstrated to have inhibitory effects on GnRH/LH release in ruminants (Barker-Gibb et al. 1995, Gazal et al. 1998, Estrada et al. 2003, Morrison et al. 2003), we propose that the acceleration of reproductive maturation seen in this study is mediated, at least in part, by a diminished NPY inhibitory effect on GnRH episodic secretion.
The pubertal transition in females is characterized by an attenuation of estradiol-negative feedback and increased pulsatile release of LH that provides support for final maturation of ovarian follicles and enhanced ovarian steroidogenesis (Gasser et al. 2006a). As anticipated, heifers fed a high-concentrate diet to promote accelerated BW gain during the juvenile period, a strategy known to advance the onset of puberty, presented a greater frequency of LH pulses during the peripubertal period. Mean concentrations of LH did not differ between groups. Similar relationships have been reported in adult cows in which mean concentrations of LH were similar during the early and mid-luteal periods even though the frequency of pulses was markedly greater during the early-luteal phase (Rawlings et al. 1980, Williams et al. 1983). The limited duration (6 h) of CSF sampling in this study did not allow substantial comparison of the frequency of GnRH pulses between dietary groups. However, the very high correlation observed between GnRH and LH pulses during the initial 6 h of sampling, together with increased pulsatile release of LH in HG heifers over a 12-h sampling period, reinforce the notion that the pubertal ‘escape’ from estradiol negative feedback is regulated mainly at the hypothalamic level (I'Anson et al. 2000).
Nutritional and metabolic signals are perceived largely by the hypothalamus (Cunningham et al. 1999, Schneider 2004), and a major limiting factor for increased episodic secretion of LH during the prepubertal period is the lack of appropriate stimulation of the gonadotrophs by GnRH (reviewed by Amstalden et al. (2014)). This concept is supported by observations that ovarian function is stimulated in immature female monkeys treated with GnRH (Wildt et al. 1980) and in lambs treated with LH (Foster et al. 1984). Our group has demonstrated that leptin has a critical permissive role in controlling pubertal maturation in heifers (Garcia et al. 2002, Maciel et al. 2004a), and this regulation occurs mainly as a result of leptin's effects on GnRH secretion (Zieba et al. 2004). Nonetheless, several studies have failed to demonstrate the presence of leptin receptors in GnRH neurons in rodents (Hakansson et al. 1998) and primates (Finn et al. 1998), indicating that leptin's actions on GnRH release are probably mediated by intermediate pathways.
NPY, a potent stimulator of food intake (Clark et al. 1985) found abundantly in the hypothalamus (Allen et al. 1983), has been proposed to be a major mediator of the inhibitory effects of undernutrition on reproductive function (Crown et al. 2007). Chronic feed restriction markedly increases the abundance of NPY mRNA in the ARC and decreases secretion of LH in ewe lambs (McShane et al. 1993). Furthermore, central infusion of NPY suppresses GnRH and LH release in cattle (Gazal et al. 1998) and sheep (Barker-Gibb et al. 1995, Estrada et al. 2003, Morrison et al. 2003). In primates, NPY mRNA and peptide concentrations in the hypothalamus are diminished during the juvenile–pubertal transition (El Majdoubi et al. 2000). Thus, we reasoned that NPY may play an important role in the metabolic programing of puberty in heifers. Confirming our hypothesis, prepubertal heifers in the HG group exhibited greater concentrations of leptin and reduced concentrations of NPY in 3V CSF at 9 months of age compared with those in the LG group. Because at 8 months of age there was only a tendency for reduced concentrations of NPY in CSF in HG heifers, we presume that the nutritionally mediated suppression of hypothalamic NPY release occurs shortly before the onset of puberty. In the current study, among the HG heifers that achieved puberty during the experiment (n=7), the majority (86%; 6/7) reached sexual maturation around or shortly after 9 months of age (7.9–10.2 months of age).
Using similar nutritional models, it has been demonstrated previously that the expression of NPY mRNA in the ARC was decreased in prepubertal heifers that gained BW at a relatively high rate (Allen et al. 2012). Furthermore, the proportion of GnRH neurons in close proximity to NPY fibers in the preoptic area and mediobasal hypothalamus is reduced in heifers with increased BW gain (Alves et al. 2011). However, it remains to be determined whether these changes in NPY projections to GnRH neurons involve NPY cells from the ARC in heifers, because in the female sheep there is no significant direct input originating from the ARC to GnRH neurons located in the preoptic area (Pompolo et al. 2001). Taken together, these observations indicate that increased hypothalamic NPY activity in juvenile heifers may play a role in restraining the pulsatile release of GnRH and LH necessary for final reproductive development. As heifers approach a more positive state of body energy reserves, characterized by increased BW and body condition score and greater circulating concentrations of leptin, NPY inhibitory tone is suppressed, culminating in reproductive competence.
Although no replicate effects were observed on BW gain and circulating concentrations of leptin, the proportion of HG heifers that achieved precocious puberty (puberty ≤300 days) differed between replicates. In a recent study (Cardoso et al. 2014), the majority of the heifers subjected to a similar nutritional regimen reached puberty between 11 and 14 months of age. As this study evaluated puberty only through 11 months of age, the number of heifers attaining puberty between 11 and 14 months of age was not determined. Variability in age at puberty in heifers of similar genetic background and fed diets of analogous formulation to promote similar rates of BW gain has been previously observed by our group, and has been reported by others (Gasser et al. 2006a,b,c). Hence, it is possible that factors other than postnatal nutrition and genetics may influence the maturation of the reproductive neuroendocrine system. For example, maternal nutrition during fetal development has been shown to cause adverse changes in brain functions that regulate nutrient intake, energy expenditure, and endocrine physiology. Some of these changes are characterized by alterations in expression of NPY and the number of NPY cells within the ARC (Warnes et al. 1998, Plagemann et al. 2000), and reduced ability of leptin to signal metabolic status postnatally (Guo & Jen 1995, Levin & Dunn-Meynell 2002, Ford et al. 2007). Therefore, further studies are required to determine whether maternal nutrition may influence the responsiveness of the offspring's hypothalamic circuitry to metabolic cues in prepubertal heifers.
Finally, it should also be noted that leptin may influence pubertal activation of GnRH neurons through additional neuronal pathways, such as the hypothalamic proopiomelanocortin system (Amstalden et al. 2014). Thus, it is possible that, despite the observed decrease in central release of NPY in HG heifers in replicate 2, reciprocal changes in other important neuropeptides that control the activity of GnRH neurons (e.g., α-melanocyte-stimulating hormone) may not have been fully activated. Moreover, because NPY neurons located in the ARC appear to regulate GnRH cells via interneuronal pathways, at least in female sheep (Pompolo et al. 2005), changes in the responsiveness to the nutritional acceleration of puberty in heifers may require alterations in these neuronal connections.
In summary, the results of the current study, in conjunction with those described in previous reports, support the hypothesis that the hypothalamic NPY pathway is involved in the nutritional acceleration of puberty in heifers. Favorable metabolic status during early calfhood, mainly characterized by increased circulating concentrations of leptin, resulted in lower hypothalamic NPY release, greater pulsatile release of LH, and hastened onset of puberty. Collectively, these results contribute to a better understanding of the mechanisms by which elevated BW gain advances the onset of puberty in female mammals, and may be valuable for the development of nutritional strategies that can exploit postnatal neuroendocrine plasticity to optimally time the onset of puberty in heifers.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This project was supported by the Agriculture and Food Research Initiative Competitive grant number 2009-65203-05678 from the USDA National Institute of Food and Agriculture.
Acknowledgements
The authors would like to acknowledge Dr A F Parlow (NIDDK National Hormone and Peptide Program, Torrance, USA) for providing LH RIA reagents and Dr Alain Caraty (INRA, Nouzilly, France) for providing the GnRH antiserum. They also acknowledge the careful animal care and assistance of Randle Franke, Reynaldo Rodrigues Jr, and Ray Villareal.
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