Abstract
The objective of this study was to examine the effect of nutrition during the first 18 weeks of life on the physiological and transcriptional functionality of the hypothalamic (arcuate nucleus region), anterior pituitary and testes in Holstein–Friesian bull calves. Holstein–Friesian bull calves with a mean (±s.d.) age and bodyweight of 19 (±8.2) days and 47.5 (±5.3) kg, respectively, were assigned to either a HIGH (n = 10) or LOW (n = 10) plane of nutrition, to achieve an overall target growth rate of 1.2 or 0.5 kg/day, respectively. At 126 ± 1.1 days of age, all calves were euthanised. Animal performance (weekly) and systemic concentrations of metabolic (monthly) and reproductive hormones (fortnightly) were assessed. Testicular histology, targeted gene and protein expression of the arcuate nucleus region, anterior pituitary and testes were also assessed using qPCR and immunohistochemistry, respectively. The expression of candidate genes in testicular tissue from post pubertal 19-month-old Holstein–Friesian bulls (n = 10) was compared to that of the 18-week-old calves. Metabolite and metabolic hormone profiles generally reflected the improved metabolic status of the calves on the HIGH (P < 0.001). Calves offered a HIGH plane of nutrition were heavier at slaughter (P < 0.001), had larger testes (P < 0.001), larger seminiferous tubule diameter (P < 0.001), more mature spermatogenic cells (P < 0.001) and more Sertoli cells (P < 0.05) in accordance with both morphological and transcriptional data. Overall, testicular gene expression profiles suggested a more mature stage of development in HIGH compared with LOW and were more closely aligned to that of mature bulls. Ghrelin receptor was the only differentially expressed gene between LOW and HIGH calves in either the anterior pituitary (P < 0.05) or arcuate nucleus region of the hypothalamus (P < 0.10) and was upregulated in LOW for both tissues. This study indicates that an enhanced plane of nutrition during early calfhood favourably alters the biochemical regulation of the hypothalamus–anterior pituitary–testicular axis, advancing testicular development and hastening spermatogenesis.
Introduction
Following the inception of genomically assisted selection, young genetically elite bulls are now being identified within weeks of birth as potential sires for use in artificial insemination programmes. However, these bulls cannot produce ejaculates of sufficient quality for cryopreservation until they are greater than 1 year of age (Murphy et al. 2018) and demand for their semen often far exceeds supply. Hastening the onset of puberty and subsequent sexual maturation would therefore benefit the industry and make high-quality semen available at a younger age (Harstine et al. 2015).
The hypothalamic–anterior pituitary–testicular axis (HPT) is a cohesive biological system that controls the secretion of male hormones and in turn spermatogenesis (Ramaswamy & Weinbauer 2014). The hypothalamus is acknowledged as the homeostatic regulator of the body (Morton et al. 2014). Signals from hormones including leptin, ghrelin, insulin and insulin-like growth factor 1 (IGF-1) are received by hypothalamic neurons located in the arcuate nucleus region (Amstalden et al. 2011) and signal nutritional status to the preoptic nuclei region that are largely involved in the regulation of reproduction (Lechan & Toni 2000). The gonadotropin-releasing hormone (GnRH) nerve projections, originating from the preoptic nuclei region, attach to the median eminence, where they secrete GnRH into the hypophyseal portal system leading to the anterior pituitary (Fujioka et al. 2007).
Bull calves experience a transient rise in anterior pituitary-derived systemic luteinising hormone (LH) from approximately 8 to 20 weeks of age (Evans et al. 1996). Furthermore, studies have shown that bull calves offered a high plane of nutrition during the first 6 months of life had an earlier and greater rise of LH (Thundathil et al. 2016). Enhanced nutrition during this critical period has a direct effect on hypothalamic GnRH pulsatility, ultimately leading to enhanced LH pulsatility as well as testosterone synthesis and release, leading in turn to advanced testicular development and pubertal onset (Dance et al. 2015, Byrne et al. 2018). Additionally any delay in the timing of puberty as a consequence of undernutrition during calfhood cannot be mitigated by dietary augmentation thereafter (Brito et al. 2007a , Byrne et al. 2018).
While the reproductive axis has the capacity to respond to changing metabolic signals (Hill et al. 2008), and it is clear that a high plane of nutrition in early calfhood can advance puberty in both male and female cattle (Kenny et al. 2018), the precise molecular mechanisms by which this is mediated have not been elucidated to date in the bull. Thus, the objective of this study was to characterise the effects of a high compared to a low plane of nutrition during the first 18 weeks of life on aspects of physiological and transcriptional functionality of the hypothalamic (arcuate nucleus region), anterior pituitary and testes in Holstein–Friesian bull calves.
Materials and methods
All procedures involving animals were approved by the Teagasc Animal Ethics Committee (TAEC30/2013); licensed by the Irish Health Products Regulatory Authority (licence number AE19132/P013) in accordance with the European Union Directive 2010/36/EU.
Experimental design and animal management
Twenty Holstein–Friesian bull calves with a mean (±s.d.) age and bodyweight of 19 (±8.2) days and 47.5 (±5.3) kg respectively, were sourced from three high health status commercial dairy farms blocked on age, sire (n = 4), liveweight and farm of origin. After a 5-day acclimatisation during which the calves feed allowance was increased daily to reach their target growth rate, calves were assigned to either a HIGH or LOW plane of nutrition. Calves were individually fed milk replacer and concentrates (Tables 1 and 2) using an electronic feeding system (Forster-Tecknik Vario; Engen, Germany). Calves on HIGH received 1200 g of milk replacer in 8 L of water daily, together with concentrate ad libitum. Calves on LOW were allocated 500 g of milk replacer in 4 L of water plus a maximum of 1 kg of concentrates daily. Diets were designed based on National Research Council guidelines (NRC 2001). Calves were weaned when consuming a minimum of 1 kg of concentrate for 3 consecutive days, at a mean age (±s.d.) of 82 (±3.9) days. Following weaning, HIGH were offered concentrates ad libitum, while LOW calves received 1 kg of concentrate daily. All calves had daily access to fresh water and approximately 0.5 kg of hay. Calves were weighed weekly. At a mean age (±s.d.) of 126 (±1.1) days of age, all calves were euthanised following intravenous administration of sodium pentobarbitone (1 mL/1.4 kg bodyweight; Euthatal, Merial S.A.S, Toulouse, France). Death was confirmed by lack of ocular response and was followed by exsanguination and decapitation.
Chemical composition of milk replacer offered to Holstein–Friesian bull calves from 2 to 12 weeks of age.
| Chemical composition (g/kg) | Values |
|---|---|
| ADF | 12.0 ± 1.98 |
| Crude ash | 65.7 ± 2.22 |
| CP | 216.3 ± 1.24 |
| DM (%) | 96.7 ± 0.15 |
| NDF | 5.1 ± 1.00 |
| Oil B | 235.0 ± 44.10 |
ADF, acid detergent fibre; CP, crude protein; DM, dry matter; NDF, neutral detergent fibre; Oil B, acid hydrolysis.
Diet and chemical composition of concentrate diet offered to Holstein–Friesian bull calves from 2 to 18 weeks of age.
| Concentrate | Values |
|---|---|
| Diet composition (%) | |
| Rolled Barley | 26.5 |
| Soya bean meal | 25 |
| Maize | 15 |
| Beet pulp | 12.5 |
| Soya hulls | 12.5 |
| Molasses | 5 |
| Mineral and vitamins | 2.5a |
| Vegetable oil | 1 |
| Chemical composition (g/kg) | |
| ADF | 103.1 ± 6.76 |
| Crude ash | 68.8 ± 0.91 |
| CP | 167.9 ± 1.86 |
| DM (%) | 88.9 ± 0.66 |
| NDF | 204.3 ± 18.2 |
| Oil B | 30.8 ± 0.72 |
Pre-weaning: 2–12 weeks of age; post-weaning: 12–18 weeks of age.
aMineral and vitamin composition: vitamin A (10 IU/kg), vitamin D3, (2 IU/kg), vitamin E (40 mg/kg), iodine (8 mg/kg), cobalt (40 mg/kg), copper (88 mg/kg), manganese (81 mg/kg), zinc (139 mg/kg) and selenium (11 mg/kg).
Oil B, acid hydrolysis.
The head was removed from the carcass and the skullcap was opened within 10 min of death. The brain was then removed from the skull by severing the infundibulum, optic nerves and brain stem. The region of the hypothalamus containing the arcuate nucleus tissue was dissected according to Komatsu et al. (2012). Two small triangular sections were harvested from either side of the bottom of the third ventricle of the hypothalamus, which contains the arcuate nucleus. The pituitary gland was removed from the sella turcica following which anterior and posterior sections of the pituitary gland were separated. The testes were also excised, the tunica albuginea, epididymides and any excess connective tissue removed and the testes were weighed. Two portions of the testicular parenchyma were dissected from each testis. One section of each tissue was fixed in 10% neutral buffered formalin and then prepared for histological sectioning. The second section was snap-frozen in liquid nitrogen, and subsequently stored at −80°C for long-term storage pending further processing.
For comparative purposes, Holstein–Friesian bulls (n = 10) finished on a standard diet (17.9 g/kg DM crude protein; 16.5 MJ/kg DM gross energy) were slaughtered at a mean (±s.d.) age and bodyweight of 586 (±50) days and 532 (±48) kg respectively. Sections of testicular parenchyma were snap-frozen in liquid nitrogen at slaughter and subsequently stored at −80°C pending further processing. These mature bulls were of a similar genetic background to that of the calves described earlier and reared under similar conditions, at the same research facility. Their purpose was to act as a ‘positive control’ in which to evaluate the effects of nutritional augmentation on sexual precocity in the bull calf.
Blood sampling
Blood samples were taken at 2, 6, 10, 14 and 18 weeks of age. Blood samples were collected an hour after morning feeding via jugular venepuncture to determine systemic concentrations of IGF-1, insulin, leptin, adiponectin, albumin, urea, total protein, triglycerides, beta hydroxybutyrate (BHB), creatinine, globulin, glucose, non-esterified fatty acids (NEFA) as well as LH, follicle-stimulating hormone (FSH) and testosterone. The protocol for serum and plasma collection processing has been described by Byrne et al. (2017b).
Metabolite assays
Concentrations of albumin, urea, total protein, triglycerides, BHB, creatinine, globulin, glucose and NEFA were analysed using commercial biochemical assay (Olympus Diagnostics and Randox Laboratories LTD, Co. Antrim, Northern Ireland) on a Beckmann Coulter AU 400 clinical analyser (Olympus Diagnostics) as described previously by Lawrence et al. (2011). Intra-assay CVs were as follows for glucose (3.03, 1.88 and 2.48%), urea (5.59, 2.09 and 1.96%), BHB (1.58, 0.88 and 0.72%), triglycerides (0.005, 0.77 and 0.52%), protein (2.34, 1.55 and 2.3%), albumin (2.44, 1.03 and 1.88%) and creatinine (3.02, 2.01 and 2.01%) for low, medium and high standards, respectively. Globulin concentration was calculated as the difference between total protein and albumin concentrations. All samples, within each metabolite, were analysed on a single assay.
Metabolic hormones
Insulin-like growth factor 1 was quantified by radioimmunoassay (RIA) after an acid–ethanol extraction and Tris neutralisation procedure, as described previously by Beltman et al. (2010). The inter- and intra-assay CVs for the low, medium and high IGF-1 were 10.8, 4.36 and 4.05% and 6.7, 1.57 and 2.77% respectively. The sensitivity of IGF-1 assay was 4 ng/mL. Insulin concentrations were measured by immunoradiometric assay (IRMA; INS-Irma, DIAsource ImmunoAssays SA, Louvain-la-Neuve, Belgium) as described by Ochocińska et al. (2016). The inter- and intra-assay CVs for the low, medium and high insulin standards were for 0.09, 7.16 and 2.93% and 6.58, 9.23 and 6.62% respectively. The sensitivity of insulin assays was 1 ng/mL. Leptin concentration was quantified using a competitive enzyme immunoassay as described by Sauerwein et al. (2004). The range of the assay was between 0.6 and 9.0 ng/mL. The intra-assay CV was 5% as all samples were analysed on a single assay. Adiponectin concentrations were analysed using an enzyme immunoassay described by Heinz et al. (2015). The range of the assay was between 0.03 and 7.0 ng/mL and the intra-assay CV was 5%.
Reproductive hormones
Serum samples were analysed for testosterone concentration using an RIA (Testo-RIA-CT, DIAsource ImmunoAssays SA, Louvain-la-Neuve, Belgium) and assays were performed according to the manufacturer’s instructions. The sensitivity of the assay was 0.1 ng/mL. Intra-assay CV for testosterone was 4.71, 8.05 and 4.87% for low, medium and high testosterone quality controls, respectively, as testosterone was analysed on a single assay.
LH was quantified by RIA as previously described by Cooke et al. (1997) modified so that the separation step (second antibody) used a polyethylene glycol (PEG) method. Briefly, after assay incubation with primary antibody, 100 µL of 1% normal mouse serum in assay buffer was added to assay tubes. This was followed by 1 mL of goat-anti-mouse antibody diluted (Equitech-Bio, INC, Kerrville, TX, USA) 1:100 in 5% PEG. Assay tubes were incubated for 1 h at room temperature, centrifuged for 20 min at 1600 g , followed by separation of the free fraction by decanting the supernatant. The intra- and inter-assay CV for LH was 14.46% and 8.59% and 1.38% and 0.25% for low and high LH quality controls, respectively. The sensitivity of the assay was 0.05 ng/mL.
FSH was quantified using RIA as described by Crowe et al. (1997). The intra- and inter-assay CV for FSH was 3.3, 5.03 and 9.26% and 4.26, 4.31 and 1.05% for low, medium and high FSH quality controls, respectively. The sensitivity of the assay was 0.05 ng/mL.
Testicular histology
Histology was employed in the testes to assess outer seminiferous tubule diameter, stage of spermatogenesis, lumen development and nuclear volume density of Sertoli cells. Sections (5 µm thick) were stained using the periodic acid-Schiff method (Bancroft 1996). For each section, the outer seminiferous tubule diameters were measured using a calibrated eyepiece micrometer at ×400 magnification using a bright field light microscope. Measurements were made on 20 different round tubules selected at random from each testis, and mean diameter was calculated. The histological sections were also examined to determine the most mature stages of spermatogenesis at ×1000 magnification, using oil immersion. In the cross-sections of 20 seminiferous tubules per testis, the most mature spermatogenic cell type in the course of spermatogenesis was established using the methodology of Curtis and Amann (1981). The progression of seminiferous tubule development was classified using the method of lumen scoring reported by Rode et al. (2015) (Supplementary Table 4, see section on supplementary data given at the end of this article). Measurements were conducted on 20 different tubules selected at random from each testis.
Sections of testes (5 µm thick) were stained using haematoxylin and eosin (H&E). The point-counting method was carried out at ×1000 magnification by bright field light microscope using a 50 point ocular grid (1250 points were evaluated per testes) to determine the nuclear volume density of Sertoli cells as previously described by Johnson and Nguyen (1986) (Supplementary Fig. 1). The evaluator was blind to the treatments.
RNA isolation and purification
Total RNA was extracted using RNeasy Universal plus Kit (Qiagen). The quantity of the RNA isolated was determined by measuring the absorbance at 260 nm using a Nanodrop spectrophotometer ND-1000 (Nanodrop Technologies, Wilmington, Germany). RNA quality was assessed on the Agilent Bioanalyzer 2100 using the RNA 6000 Nano Lab Chip Kit (Agilent Technologies). RNA was purified using the RNA Clean & Concentrator (Zymo Research, Irvine, CA, USA) and RNA integrity values of greater than eight were deemed to be of high quality.
Complementary DNA synthesis
Total RNA (2 µg) was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) using Multiscribe reverse transcriptase according to manufacturer’s instructions. Samples were stored at −20°C pending further analysis.
Primer design and reference gene selection
All primers targeting reference and candidate genes were obtained from a commercial supplier (Sigma-Aldrich Ireland; Supplementary Tables 1, 2 and 3). Primer3 (http://primer3.ut.ee/) software was utilised to design primers (Koressaar & Remm 2007). Primer specificity was established using the Basic Local Alignment Search Tool (BLAST) from the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). The genes examined in the arcuate nucleus region of the hypothalamus included: kisspeptin (KISS1), G protein-coupled receptor (GPR54), gonadotropin-releasing hormone (GnRH), agouti-related protein (AGRP), neuropeptide Y (NPY), pro-opiomelanocortin precursor (POMC), melanocortin 4 receptor (MC4R), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 1 receptor (IGF-1-R), leptin receptor (OBR) and growth hormone secretagogue receptor (ghrelin receptor; GHSR). The genes examined in the anterior pituitary included: luteinising hormone beta (LHβ), follicle-stimulating hormone beta (FSHβ), GHSR, somatotropin precursor/growth hormone (GH1), gonadotropin-releasing hormone receptor (GnRHR), IGF-1R and IGF-1. The genes examined in the testes included: proliferation cell nuclear antigen (PCNA), Thy-1 cell surface antigen (THY1), tight junction protein 1/zonula occludens protein 1 (ZO1), GATA-binding protein 4 (GATA4), androgen receptor (AR), follicle-stimulating hormone receptor (FSHR), aquaporin-8 (AQP8), ubiquitin carboxyl-terminal esterase L1 (UCHL1), anti-Müllerian hormone (AMH), claudin 11 (CLDN11) and luteinising hormone receptor (LHR). In the current study, four reference genes were tested across all samples with RT-PCR, including ribosomal protein S9 (RPS9), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ) and ubiquitin (UBQ). Data were analysed using GeNorm (GenEx 5.2.1.3; MultiD Analyses, Gothenburg, Sweden). GeNorm is a software package that measures the total stability of the tested reference genes by calculating the intra- and intergroup CV and combining both CVs to give a stability value (M value) (Keogh et al. 2015). A lower M value indicates a greater stability in gene expression across all samples. In the current study, the M value scores in the testicular tissue for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ), ubiquitin (UBQ) and ribosomal protein S9 (RSP9) were 2.44, 1.23, 0.99 and 0.99 respectively. In anterior pituitary tissue, M value scores for YWHAZ, GAPDH, UBQ and RSP9 were 0.95, 0.65, 0.50 and 0.50 respectively. The M value scores for the arcuate region for GAPDH, YWHAZ, UBQ and RSP9 were 1.39, 0.50, 0.43 and 0.43 respectively. On this basis, UBQ and RSP9 were selected as suitably stable housekeeping genes for each of the tissues, and results are presented relative to an average of both genes.
Gene expression
The RT-qPCR assays were performed on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems) as per the protocol reported by Keogh et al. (2015). The formula E = 10(−1/slope)−1 was used where slope refers to the slope of the linear curve of CT values plotted against log dilution. Only primers with PCR efficiencies between 90 and 110% were used. Gene expression was analysed using GenEx software (www.multid.se/genex.html), which allowed for compensation of PCR efficiencies, before averaging for RT-PCR replicates. Each gene of interest was normalised using the geometric mean of the four reference genes.
Immunohistochemical analysis
Immunohistochemistry was used to assess cell proliferation in the arcuate nucleus by assessing KI67 staining and to validate the specificity of the tissue using proteins KISS1 (Kisspeptin) and NPY (Neuropeptide Y) that are highly specific to the arcuate nucleus; while Sertoli cell number and developmental stage was assessed in the testes using transcription factor GATA-binding protein 4 (GATA4) and anti-Müllerian hormone (AMH). Antigen retrieval of the deparaffinised tissue sections was performed using a PT-Link module (Dako) at 95–99°C for 20 min in a citric acid buffer (0.01 M, pH 6.0). Slide staining was performed using a Dako autostainer Link 48 (Dako) according to the manufacturer’s instructions. The primary antibodies used were anti-KI67 as a marker of cellular marker for proliferation (Ready-to-use, Dako), anti-KISS1 (1:1000, ABIN672726, Antibodies-online) and anti-NPY (1:150, ABIN724475, Antibodies-online) on the arcuate sections and anti-GATA4 (1:1000, LS-C355535, Source Bioscience) and anti-AMH (1:75, ab84952, Abcam) on the testicular sections. The secondary antibody was a rabbit-anti-goat antibody and slides were counterstained with haematoxylin, dehydrated, cleared and mounted. Bovine kidney, human breast cancer and bovine cerebrum sections were used as positive controls for KI67, KISS1 and NPY staining of the arcuate tissue respectively. Bovine heart and ovary sections were used as positive controls for GATA4 and AMH staining of the testes respectively.
All stained slides were analysed using the software Aperio ImageScope (Leica Biosystem, Dublin, Ireland). The cytoplasmic V2 algorithm was selected to calculate the number of cells containing stain within the nucleus and the cytoplasm per area (mm2) and percentage stained vs unstained for GATA4 (Fig. 1). The colour deconvolution algorithm was selected to analyse AMH staining as it separated a stained tissue image into two colour channels, corresponding to the actual colours of the stains used and facilitated measurement of intensity (weak, moderate and strong intensity) and provision of the number of cells containing stain per area (mm2; Fig. 1). The cytoplasmic V2 algorithm was selected to analyse DAB staining intensity (weak, moderate and strong intensity) and provide the number of cells containing stain within the nucleus and the cytoplasm per area (mm2) for KI67, NPY and KISS1 (Fig. 2).
AMH (anti-Müllerian hormone; ×400) and GATA4 (GATA-binding protein 4; ×100) staining in the testes. The images illustrate different staining intensities; (A) negative control, (B) AMH weak staining, (C) AMH moderate staining, (D) AMH strong staining, (E) GATA4-negative staining and (F) GATA4-stained cells. Tissues for analysis were collected at slaughter (18 weeks of age).
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
AMH (anti-Müllerian hormone; ×400) and GATA4 (GATA-binding protein 4; ×100) staining in the testes. The images illustrate different staining intensities; (A) negative control, (B) AMH weak staining, (C) AMH moderate staining, (D) AMH strong staining, (E) GATA4-negative staining and (F) GATA4-stained cells. Tissues for analysis were collected at slaughter (18 weeks of age).
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
AMH (anti-Müllerian hormone; ×400) and GATA4 (GATA-binding protein 4; ×100) staining in the testes. The images illustrate different staining intensities; (A) negative control, (B) AMH weak staining, (C) AMH moderate staining, (D) AMH strong staining, (E) GATA4-negative staining and (F) GATA4-stained cells. Tissues for analysis were collected at slaughter (18 weeks of age).
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
KI67, NPY (Neuropeptide Y) and KISS1 (Kisspeptin) nuclear staining in the arcuate tissue (×400). The images illustrate different staining intensities; (A) KI67-negative staining, (B) KI67 weak staining, (C) KI67 moderate staining and (D) KI67 strong staining, (E) NPY unstained cells, (F) NPY-stained cells, (G) KISS1 negative staining, (H) KISS1 weak staining, (I) KISS1 moderate staining and (J) KISS1 strong staining. Tissues for analysis were collected at slaughter (18 weeks of age).
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
KI67, NPY (Neuropeptide Y) and KISS1 (Kisspeptin) nuclear staining in the arcuate tissue (×400). The images illustrate different staining intensities; (A) KI67-negative staining, (B) KI67 weak staining, (C) KI67 moderate staining and (D) KI67 strong staining, (E) NPY unstained cells, (F) NPY-stained cells, (G) KISS1 negative staining, (H) KISS1 weak staining, (I) KISS1 moderate staining and (J) KISS1 strong staining. Tissues for analysis were collected at slaughter (18 weeks of age).
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
KI67, NPY (Neuropeptide Y) and KISS1 (Kisspeptin) nuclear staining in the arcuate tissue (×400). The images illustrate different staining intensities; (A) KI67-negative staining, (B) KI67 weak staining, (C) KI67 moderate staining and (D) KI67 strong staining, (E) NPY unstained cells, (F) NPY-stained cells, (G) KISS1 negative staining, (H) KISS1 weak staining, (I) KISS1 moderate staining and (J) KISS1 strong staining. Tissues for analysis were collected at slaughter (18 weeks of age).
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
Each section stained with GATA4 (two per calf; n = 10) was examined at 400× magnification and ten round seminiferous tubules were randomly selected and assessed (Harstine et al. 2017). The number of stained Sertoli cell nuclei present in a circular monolayer at the wall of a round seminiferous tubule was recorded (Harstine et al. 2017).
Statistical analysis
All PCR data analysed using GenEx were log transformed to obtain normal distribution prior to subsequent statistical analysis. Area under the curve (AUC) for FSH, LH and testosterone was determined using Sigma Plot, version 11 (Systat Software, San Jose, CA, USA). All bodyweight, testicular measurements, histology, immunohistochemistry, PCR and monthly blood analysis data were analysed using the procedures of Statistical Analysis Software (SAS version 9.3). All data were tested for normality (UNIVARIATE procedure) and where appropriate, transformed to the power of lambda (TRANSREG procedure). Data were analysed using ANOVA (MIXED procedure). The covariance matrix (simple or compound symmetry) was determined for each variable by examining the Bayesian Information Criteria (BIC; smaller is better) value. Fixed effects included plane of nutrition, week of age and their interactions, where appropriate. Animal was the experimental unit. Sampling time was included in the statistical models as a repeated measure for weights and blood analysis. Differences were deemed statistically significant if P < 0.05, while P values >0.05 and <0.10 were deemed as indicating a tendency towards statistical significance. All results are presented as mean ± s.e.m.
Results
Animal performance
Pre-weaning average daily gain (ADG) was greater for calves on HIGH in comparison with LOW (P < 0.001; Table 3). The post-weaning ADG was also greater for HIGH calves (P < 0.001) compared to LOW, which resulted in pre-slaughter bodyweights being greater for HIGH calves compared with LOW (P < 0.001). Paired testicular weight at slaughter was also greater for the calves on HIGH compared with those on LOW (P < 0.001; Table 4). The MATURE bulls grew at 1.4 kg/day prior to slaughter.
Effect of a HIGH compared to a LOW plane of nutrition (mean ± s.e.m.) on the pre-weaning average daily gain, post-weaning average daily gain, total average daily gain and live weight at the time of slaughter (18 weeks of age) of Holstein–Friesian bull calves.
| HIGH | LOW | Significance | |
|---|---|---|---|
| Pre-weaning ADG (kg) | 0.73 ± 0.02 | 0.43 ± 0.02 | *** |
| Post-weaning ADG (kg) | 1.46 ± 0.03 | 0.57 ± 0.04 | *** |
| Overall ADG (kg) | 1.08 ± 0.03 | 0.50 ± 0.03 | *** |
| Bodyweight at slaughter (kg) | 160.9 ± 3.98 | 107.1 ± 3.19 | *** |
Pre-weaning: 2–12 weeks of age; post-weaning: 12–18 weeks of age.
***P < 0.001.
ADG, average daily gain.
Effect of a HIGH compared to a LOW plane of nutrition (mean ± s.e.m.) on the paired testes weight and on morphological properties including seminiferous tubule diameter and stage of spermatogenesis.
| HIGH | LOW | Significance | |
|---|---|---|---|
| Paired testes weight (g) | 55.4 ± 3.24 | 31.4 ± 2.57 | *** |
| Seminiferous tubule diameter (µm) | 85.4 ± 2.09 | 72.5 ± 1.43 | *** |
| % Gonocyte and Prespermatogonia | 31.5 ± 2.11 | 57 ± 1.20 | *** |
| % Spermatogonia | 68.5 ± 2.11 | 43 ± 1.20 | *** |
| No. of Sertoli cells | 28 ± 0.7 | 24 ± 0.6 | * |
| Volume density of Sertoli cells | 9.4 ± 0.41 | 8.4 ± 0.27 | *** |
Pre-weaning: 2–12 weeks of age; post-weaning: 12–18 weeks of age. Tissue samples collected at slaughter at 18 weeks of age.
***P < 0.001; *P < 0.05.
s.e.m., standard error of the mean.
Metabolic hormones and metabolites
Metabolite and metabolic hormone data are presented in Figs 3 and 4, respectively. There was a plane of nutrition × week interaction for albumin (P < 0.001); HIGH calves had greater albumin concentrations at 14 and 18 weeks of age. Total protein was greater in LOW compared to HIGH calves (P < 0.01) and total protein increased in both planes of nutrition between 2 and 18 weeks of age (P < 0.01). There was no plane of nutrition × week interaction or no effect of either plane of nutrition or week on triglyceride concentrations (P > 0.05). There was no effect of plane of nutrition on NEFA (P > 0.05); however, there was an effect of week (P < 0.01). There was a plane of nutrition × week interaction for glucose concentration (P < 0.01); glucose concentrations were greater in HIGH compared to LOW calves at 6, 14 and 18 weeks of age (P < 0.05) but not at other timepoints. There was a plane of nutrition × week interaction for globulin concentrations (P = 0.06); as LOW calves had greater globulin concentrations from 10 to 18 weeks of age (P < 0.05). There was a plane of nutrition × week interaction for creatinine (P < 0.01). Creatinine was not different between the planes of nutrition from week 2 to 10 (P > 0.05). At week 14, there was a tendency for creatinine to be greater on LOW compared to HIGH (P = 0.09) with concentrations greater on LOW at week 18 (P < 0.05). There was a plane of nutrition × week interaction for BHB (P < 0.001). At weaning, BHB was lower on LOW (P < 0.001). However, at week 14, LOW had a greater BHB concentration compared to HIGH calves (P < 0.001).
Effect of a HIGH compared to a LOW plane of nutrition on the plasma concentrations of metabolites of Holstein–Friesian bull calves. Black full line: low plane of nutrition, Black dotted line: high plane of nutrition; T, plane of nutrition; W, week; T*W, plane of nutrition by week interaction; NS, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001, error bars represent standard error of the mean. P = 0.07 shows a tendency for significance between the high and low planes of nutrition at week 6 and 18 for total protein.
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
Effect of a HIGH compared to a LOW plane of nutrition on the plasma concentrations of metabolites of Holstein–Friesian bull calves. Black full line: low plane of nutrition, Black dotted line: high plane of nutrition; T, plane of nutrition; W, week; T*W, plane of nutrition by week interaction; NS, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001, error bars represent standard error of the mean. P = 0.07 shows a tendency for significance between the high and low planes of nutrition at week 6 and 18 for total protein.
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
Effect of a HIGH compared to a LOW plane of nutrition on the plasma concentrations of metabolites of Holstein–Friesian bull calves. Black full line: low plane of nutrition, Black dotted line: high plane of nutrition; T, plane of nutrition; W, week; T*W, plane of nutrition by week interaction; NS, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001, error bars represent standard error of the mean. P = 0.07 shows a tendency for significance between the high and low planes of nutrition at week 6 and 18 for total protein.
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
Effect of a HIGH compared to a LOW plane of nutrition on the plasma concentrations of metabolic hormones of Holstein–Friesian bull calves. Black full line: low plane of nutrition, Black dotted line: high plane of nutrition; T, plane of nutrition; W, week; T*W, plane of nutrition by week interaction; NS, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001, error bars represent standard error of the mean.
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
Effect of a HIGH compared to a LOW plane of nutrition on the plasma concentrations of metabolic hormones of Holstein–Friesian bull calves. Black full line: low plane of nutrition, Black dotted line: high plane of nutrition; T, plane of nutrition; W, week; T*W, plane of nutrition by week interaction; NS, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001, error bars represent standard error of the mean.
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
Effect of a HIGH compared to a LOW plane of nutrition on the plasma concentrations of metabolic hormones of Holstein–Friesian bull calves. Black full line: low plane of nutrition, Black dotted line: high plane of nutrition; T, plane of nutrition; W, week; T*W, plane of nutrition by week interaction; NS, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001, error bars represent standard error of the mean.
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
There was no effect of plane of nutrition on adiponectin concentrations (P > 0.05). Adiponectin increased for both planes of nutrition between 2 and 18 weeks of age (P < 0.001). There was no effect of plane of nutrition on leptin (P > 0.05), but leptin increased linearly between 2 and 18 weeks of age (P < 0.01). There was a plane of nutrition × week interaction for IGF-1 (P < 0.001). There was no difference between planes of nutrition in IGF-1 concentration at either weeks 2 or 6 (P > 0.05). At all other timepoints HIGH had a greater concentration compared to LOW calves (week 10, 2.5-fold change in IGF-1 concentration; week 14, 5-fold change; week 18, 2.9-fold change). The calves on the low plane of nutrition maintained a constant IGF-1 concentration throughout the trial period. Insulin concentrations tended to be greater in HIGH compared to LOW calves (P = 0.06). Insulin concentrations showed little change between 2 and 10 weeks of age; however, concentrations increased between 14 and 18 weeks of age for both planes of nutrition (P < 0.01).
Reproductive hormones
There was no effect of plane of nutrition on mean concentrations of FSH or LH (P > 0.05; Fig. 5) but there was an effect of week on both hormones (P < 0.001). There was a tendency towards a plane of nutrition × week of age interaction for mean concentration of testosterone (P = 0.09) as testosterone was greater in HIGH compared to LOW calves at 14 and 18 weeks of age.
Effect of a HIGH compared to a LOW plane of nutrition on the serum concentrations of reproductive hormones. Black full line: low plane of nutrition, Black dotted line: high plane of nutrition; T, plane of nutrition; W, week; T*W, plane of nutrition by week interaction; NS, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001, error bars represent standard error of the mean; P = 0.08 shows a tendency for significance between the high and low planes of nutrition at week 10 for FSH.
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
Effect of a HIGH compared to a LOW plane of nutrition on the serum concentrations of reproductive hormones. Black full line: low plane of nutrition, Black dotted line: high plane of nutrition; T, plane of nutrition; W, week; T*W, plane of nutrition by week interaction; NS, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001, error bars represent standard error of the mean; P = 0.08 shows a tendency for significance between the high and low planes of nutrition at week 10 for FSH.
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
Effect of a HIGH compared to a LOW plane of nutrition on the serum concentrations of reproductive hormones. Black full line: low plane of nutrition, Black dotted line: high plane of nutrition; T, plane of nutrition; W, week; T*W, plane of nutrition by week interaction; NS, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001, error bars represent standard error of the mean; P = 0.08 shows a tendency for significance between the high and low planes of nutrition at week 10 for FSH.
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
In contrast to mean concentrations, calves offered a HIGH a plane of nutrition had greater (P < 0.01) LH AUC compared to LOW calves.
Testicular histology
Seminiferous tubule diameter was greater for HIGH compared to LOW calves (P < 0.001; Table 4). There was a greater percentage of seminal chords with germ cells and prespermatogonia in the LOW compared to the HIGH calves (P < 0.001). Conversely, there were a greater percentage of seminal chords with spermatogonia in the HIGH calves compared to the LOW (P < 0.001).
The lumen scores for seminiferous tubules for both planes of nutrition ranged between 1 and 4 (Fig. 6). Number of tubules with a lumen score of 1 or 2 did not differ between planes of nutrition (P > 0.05). However, LOW calves had a greater number of seminiferous tubules with a score of 3 (P < 0.001), but less tubules with a score of 4 (P < 0.001) compared to HIGH calves.
Effect of a HIGH compared to a LOW plane of nutrition on the lumen development of the seminiferous tubules ***P < 0.001; Lumen Score 1: Tubes lack a lumen (solid spermatic chords), Lumen Score 2: Tubules contain a single vacuole, Lumen Score 3: Tubules contain several independent vacuoles, Lumen Score 4: Tubules contain several aggregating vacuoles. Tissues for analysis were collected at slaughter (18 weeks of age).
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
Effect of a HIGH compared to a LOW plane of nutrition on the lumen development of the seminiferous tubules ***P < 0.001; Lumen Score 1: Tubes lack a lumen (solid spermatic chords), Lumen Score 2: Tubules contain a single vacuole, Lumen Score 3: Tubules contain several independent vacuoles, Lumen Score 4: Tubules contain several aggregating vacuoles. Tissues for analysis were collected at slaughter (18 weeks of age).
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
Effect of a HIGH compared to a LOW plane of nutrition on the lumen development of the seminiferous tubules ***P < 0.001; Lumen Score 1: Tubes lack a lumen (solid spermatic chords), Lumen Score 2: Tubules contain a single vacuole, Lumen Score 3: Tubules contain several independent vacuoles, Lumen Score 4: Tubules contain several aggregating vacuoles. Tissues for analysis were collected at slaughter (18 weeks of age).
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
Calves assigned to a LOW plane of nutrition had a lower number of Sertoli cells in the testes (P < 0.001; Fig. 7 and Table 4) and also a lower volume density of Sertoli cells compared to HIGH calves (P < 0.05; Table 4) when testes weight was included as a covariate (P < 0.001).
Effect of a HIGH compared to a LOW plane of nutrition on GATA4 stained Sertoli cell number (×400); (A) high plane of nutrition and (B) low plane of nutrition. The number of stained Sertoli cell nuclei present in a circular monolayer at the wall of a round seminiferous tubule was recorded (Harstine et al. 2017). There were a greater number of Sertoli cells in the testes of HIGH calves in comparison with their contemporaries on LOW (P < 0.001). Tissues for analysis were collected at slaughter (18 weeks of age).
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
Effect of a HIGH compared to a LOW plane of nutrition on GATA4 stained Sertoli cell number (×400); (A) high plane of nutrition and (B) low plane of nutrition. The number of stained Sertoli cell nuclei present in a circular monolayer at the wall of a round seminiferous tubule was recorded (Harstine et al. 2017). There were a greater number of Sertoli cells in the testes of HIGH calves in comparison with their contemporaries on LOW (P < 0.001). Tissues for analysis were collected at slaughter (18 weeks of age).
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
Effect of a HIGH compared to a LOW plane of nutrition on GATA4 stained Sertoli cell number (×400); (A) high plane of nutrition and (B) low plane of nutrition. The number of stained Sertoli cell nuclei present in a circular monolayer at the wall of a round seminiferous tubule was recorded (Harstine et al. 2017). There were a greater number of Sertoli cells in the testes of HIGH calves in comparison with their contemporaries on LOW (P < 0.001). Tissues for analysis were collected at slaughter (18 weeks of age).
Citation: Reproduction 156, 4; 10.1530/REP-17-0671
Effect of a HIGH compared to a LOW plane of nutrition (mean ± s.e.m.) on the expression of genes in the anterior pituitary.
| Gene | Plane of nutrition | Significance | |
|---|---|---|---|
| HIGH | LOW | ||
| FSHβ | 10.0 ± 2.89 | 9.2 ± 1.93 | NS |
| LHβ | 2.1 ± 0.42 | 1.8 ± 0.20 | NS |
| GHSR | 2.4 ± 0.31 | 3.6 ± 0.35 | P < 0.05 |
| GH1 | 1.9 ± 0.27 | 2.6 ± 0.42 | NS |
| GnRHR | 2.3 ± 0.33 | 2.7 ± 0.36 | NS |
| IGF-1R | 1.8 ± 0.21 | 2.0 ± 0.19 | NS |
| IGF-1 | 1.9 ± 0.37 | 2.3 ± 0.37 | NS |
The results are relative to the average of the reference genes UBQ and RSP9. Pre-weaning: 2–12 weeks of age; post-weaning: 12–18 weeks of age. Tissue samples collected at slaughter at 18 weeks of age.
NS, non-significant; s.e.m., standard error of the mean.
Gene expression
Arcuate RT-PCR data highlight a tendency for an effect of plane of nutrition on GHSR mRNA expression (P = 0.09), which was greater for LOW compared to HIGH calves. There was no effect of plane of nutrition on relative transcript abundance KISS1, GPR54, GnRH, AGRP, NPY, POMC, MC4R, IGF-1, IGF-1R and OBR expression in the accurate tissue (P > 0.05; Supplementary table 1).
Effect of a HIGH and a LOW plane of nutrition (mean ± s.e.m.) compared to mature bulls on the expression of genes in the parenchyma of the testes.
| Gene | MATURE | HIGH | LOW | Significance | ||
|---|---|---|---|---|---|---|
| M–H | H–L | M–L | ||||
| AMH | 2.6 ± 0.12 | 5.2 ± 0.59 | 6.7 ± 0.31 | *** | * | *** |
| PCNA | 5.6 ± 0.29 | 1.4 ± 0.10 | 1.3 ± 0.07 | *** | NS | *** |
| GATA4 | 14.6 ± 0.56 | 31.7 ± 1.96 | 27.1 ± 1.89 | *** | NS | *** |
| ZO1 | 3.8 ± 0.09 | 3.7 ± 0.47 | 4.0 ± 0.15 | * | NS | NS |
| CLAUDIN11 | 14.7 ± 0.11 | 15.3 ± 0.76 | 12.9 ± 0.48 | NS | * | ** |
| THY1 | 4.8 ± 0.23 | 5.1 ± 0.92 | 5.5 ± 0.41 | NS | NS | NS |
| AR | 0.6 ± 0.01 | 0.4 ± 0.06 | 0.5 ± 0.03 | * | P = 0.06 | NS |
| FSHR | 9.4 ± 0.12 | 11.5 ± 0.37 | 10.2 ± 1.18 | ** | NS | P = 0.06 |
| AQP8 | 12.4 ± 0.21 | 14.5 ± 1.70 | 12.2 ± 1.41 | NS | NS | NS |
| UCHL1 | 4.8 ± 0.41 | 5.8 ± 0.57 | 5.6 ± 0.76 | NS | NS | NS |
| LHR | 0.7 ± 0.17 | 5.4 ± 1.55 | 2.6 ± 0.62 | *** | NS | ** |
The results are relative to the average of the reference genes UBQ and RSP9. Pre-weaning: 2–12 weeks of age; post-weaning: 12–18 weeks of age. Mature bulls offered a commercial ration and slaughtered at 19 months of age. Tissue samples collected at slaughter at 18 weeks of age.
*P < 0.05; **P < 0.01; ***P < 0.001.
H, high; L, low; M, mature; NS, non-significant; s.e.m., standard error of the mean.
Anterior pituitary RT-PCR data are presented in Table 5. There was an effect of plane of nutrition on GHSR mRNA expression (P < 0.05), which was greater for LOW compared with HIGH calves. There was no effect of plane of nutrition on FSHβ, LHβ, IGF-1, GH1, GnRHR or IGF-1R (P > 0.05; Table 5).
For testicular tissue, we compared relative transcript abundance (i) within the calves on the two planes of nutrition and (ii) between each of the two planes of nutrition and the mature bulls. In the testes, PCNA mRNA expression was greater in both prepubertal groups of calves when compared with the mature bulls (P < 0.001; Table 6). AR mRNA expression was upregulated in the mature bulls compared to both the HIGH (P < 0.01) and the LOW (P = 0.06) calves. FSHR mRNA expression was upregulated in the HIGH calves compared to the mature bulls (P < 0.01). GATA4 mRNA expression was upregulated in both HIGH and LOW calves (P < 0.001) compared with the mature bulls. AMH in the LOW was upregulated compared with both the HIGH calves (P < 0.05) and the mature bulls (P < 0.001). CLDN11 mRNA expression was upregulated in the HIGH calves (P < 0.05) and the mature bulls (P < 0.001) compared with the LOW. LHR mRNA expression was upregulated in both HIGH calves and LOW (P < 0.01) in comparison with the mature bulls. There was no effect of plane of nutrition on the relative expression of APQ8, THY1, UCHL1 or ZO1 (P > 0.05; Table 6).
Immunohistochemistry
The arcuate nucleus was stained using KI67, KISS1 and NPY. The staining for NPY was located cytoplasmically, and there was no difference in the number of protein-positive cells per unit of area between planes of nutrition (P > 0.05). There were no differences in KI67 and KISS1 protein intensity or in the number of protein-positive cells per area between planes of nutrition (P > 0.05).
The testicular parenchyma was stained for the presence of AMH and GATA4. There was a greater percentage of weak AMH staining in HIGH calves in comparison with LOW (P < 0.05). There were no differences found between overall GATA4 expression within the testes (P > 0.05).
Discussion
Calves offered a high plane of nutrition were heavier at slaughter, had larger testes, larger seminiferous tubule diameter, more mature spermatogenic cells and more Sertoli cells. The weights of the calves at slaughter are comparable to that of Holstein–Friesian bull calves employed in other studies on similar nutritional regimes (Dance et al. 2015, Byrne et al. 2017b ). Larger testes would suggest greater spermatogenic potential and is thought to reflect a greater abundance of Sertoli cells, and therefore, the capacity to support more sperm throughout the bull’s lifetime (O’Donnell et al. 2000). These advancements in testicular morphology were validated by transcriptomic analysis. Additionally, systemic metabolites and metabolic and reproductive hormone profiles broadly reflected the plane of nutrition offered (Byrne et al. 2017b ). However, there were no clear differences between the two planes of nutrition with regard to targeted gene and protein expression in either the arcuate nucleus or anterior pituitary.
The systemic concentrations of the various metabolites measured in this study were within the normal range for calves and, in general, reflected the divergent metabolic status of the animals on the two contrasting planes of nutrition employed, as well as the switch-over from mainly liquid to solid feed post-weaning at 10 weeks. For example, BHB concentrations pre-weaning were low for both groups reflecting the relatively low quantity of concentrate consumed (Overvest et al. 2016). However, BHB concentrations increased as calves were transitioned to solid feed post weaning and were similar for both groups prior to slaughter, indicating normal rumen development (Khan et al. 2011). Glucose concentrations mapped, in general, the prevailing plane of nutrition, with concentrations markedly greater for calves on the high plane of nutrition post weaning, reflecting their much greater concentrate intake. Such conclusions have been drawn in previous studies for calves on a similar feeding regime (Byrne et al. 2017a ). Creatinine, decreased with age for both planes of nutrition, indicative of normal renal development and is consistent with other recent reports in the literature (Byrne et al. 2017a ). Plasma concentrations of urea were greater in the HIGH calves, reflecting their greater dietary protein consumption (Bartlett et al. 2006). Similar results for globulin and total protein concentrations have been observed where calves were offered restricted compared with an unlimited milk allowance (Hammon et al. 2002). Several studies including a recent report by Schäff et al. (2016) have shown that systemic concentrations of albumin increase steadily in the weeks after birth. In our study, we observed a steady increase in concentrations of albumin over time in HIGH calves, with greater concentrations compared to their contemporaries on LOW during the post-weaning period.
Systemic concentrations of leptin increase proportionally with increases in body fat (Landry et al. 2013) and the hormone plays an important role in energy homeostasis (Ahima et al. 2000). However, in our study, we did not find any difference between planes of nutrition with respect to leptin. This is in agreement with other studies using both dairy bull calves (Dance et al. 2015, Byrne et al. 2017b ) and may be due to the typical low abundance of subcutaneous fat in young growing calves. However, following RNASeq analysis of the subcutaneous adipose tissue collected at slaughter, calves on HIGH had >4.5 log fold greater LEP expression compared to their contemporaries on LOW (English et al. 2018). In contrast to leptin, the adipokine adiponectin is inversely related to body fat in cattle (Sauerwein & Häußler 2016) and thus we expected that calves on the high plane of nutrition would have lower concentrations. However, adiponectin concentrations in our study increased in all calves over time; age-associated increases in adiponectin concentrations have been previously reported (Heinz et al. 2015). It has been widely suggested that IGF-1 is a mediator of the effect of nutrition on the functionality of the HPT axis (Brito et al. 2007b ). Consistent with this, we observed 1.5–3.5 fold greater concentrations of IGF-1 in the systemic circulation of calves on the HIGH compared with the LOW, throughout the experimental period.
The GH secretagogue receptor also known as GHSR has been found to have the greatest expression in the anterior pituitary and hypothalamus relative to any other tissue in cattle (Komatsu et al. 2012). Thus, ghrelin via GHSR can stimulate the direct release of GH from the somatotropic cells in the anterior pituitary or indirectly via the GHSR in the arcuate nucleus in the hypothalamus (Reichenbach et al. 2012). Ghrelin concentrations in the systemic circulation are regulated by food intake (Stanley et al. 2005). Our finding of an upregulation of GHSR in both the arcuate nucleus and anterior pituitary in 18-week-old bull calves on a low plane of nutrition is consistent with the inhibitory effect of ghrelin on GnRH pulsatility in heifers (Chouzouris et al. 2016). Despite the well-documented relationship between GHSR and GH secretion (Sun et al. 2004) as well as the aforementioned positive effect of a high plane of nutrition on systemic concentrations of IGF-1, transcript abundance for GH in the anterior pituitary was not affected by diet in the current study. The disparity in these findings may be due to the transitory nature of gene transcripts in the brain (Bondy & Lee 1993).
Low systemic concentrations of leptin stimulate NPY/AGRP release and feed intake and inhibit POMC/CART/MC4R activity and energy expenditure (Schwartz et al. 2000). NPY/AGRP have opposing roles to that of POMC/α-MSH in the control of feeding and energy expenditure (Stanley et al. 2005). It has been reported that NPY and AGRP were downregulated and POMC and α-MSH were upregulated reported in heifer calves fed a high concentrate diet in order to achieve rapid bodyweight gain from three to seven months of age compared to contemporaries offered a high forage diet (Allen et al. 2012). As previously outlined, we failed to observe any effect of plane of nutrition on systemic concentrations of leptin, consistent with the lack of difference also observed for transcript abundance of NPY, AGRP, POMC or MC4R between the two planes of nutrition. Additionally, the lack of difference at the transcript level for NPY levels was validated using immunohistochemistry.
It has been reported that hypothalamic expression of KISS1 and GPR54 transcripts are at their lowest prior to puberty with maximal expression occurring at puberty, for example, in both male and female rats (Navarro et al. 2005). This suggests that although GPR54 expression may increase from pre to post puberty; the number of GnRH neurons remains unchanged (Ezzat Ahmed et al. 2009). Short-term fasting of prepubertal female and male rats resulted in a decline in KISS1 mRNA expression and an increase in GPR54 mRNA expression in the hypothalamus (Castellano et al. 2005). These studies are contradictory of the results from our study. In addition, there were no differences observed in expression of GnRH either in the arcuate region of either plane of nutrition or in the expression of GnRHR, FSHβ and LHβ in the anterior pituitary gland in the current study. The lack of differences in KISS1 mRNA was further corroborated at the protein level by immunohistochemistry. The lack of a difference in systemic leptin may contribute to the similar KISS1 mRNA expression in the current study as kisspeptin is hypothesised to play a mediatory role between leptin- and GnRH-secreting neurons in the hypothalamus. This in turn may also explain the lack of mRNA expression difference in the anterior pituitary (Clarke et al. 2015).
In the seminiferous tubules, fluid filled vacuoles multiply and aggregate to form the lumen, which is not fully functional in bulls until after 20 weeks of age (Wrobel et al. 1986). While a previous study has been carried out on stallions (Rode et al. 2015), to the author’s knowledge, this is the first study to evaluate the effect of plane of nutrition on seminiferous tubule lumen development in bull calves. The HIGH calves had a more mature stage of lumen development and while no fully formed lumens were observed, nonetheless our data suggest that a more favourable metabolic status in early calfhood advances testicular functionality. Curtis and Amann (1981) reported outer seminiferous tubule diameter of 79 ± 2 μm and 110 ± 5 μm for Holstein–Friesian bull calves at 16 weeks and 20 weeks of age respectively and the diameters measured in the HIGH calves in our study were consistent with this range. Consistent with more mature seminiferous tubule development, there was a greater percentage of spermatogonia cells observed in the HIGH calves compared with the LOW in the current study. This indicates that both treatment groups were within the stage of development appropriate for their age. Along with the advanced germ cell development, our results also show that a greater plane of nutrition had a positive influence on the rise of testosterone, which may stimulate an early onset of puberty (Chandolia et al. 1997). While directly related to spermatogenic potential (Johnson et al. 2000), there has been no information to-date on the effect of early life plane of nutrition on Sertoli cell number in calves. Harstine et al. (2017) demonstrated that Angus calves treated with FSH from 1 to 3 months of age had a greater number of Sertoli cells. Sertoli cells harbour a fixed number of germ cells and Sertoli cell number can only be influenced before puberty (Johnson et al. 2000). Thus, our data suggest that calves offered a high plane of nutrition during the first 4–5 months of life may have the capacity to produce more sperm during their life time. Nevertheless, recent data from our research group indicate that while a bull which has been subjected to an enhanced nutritional regimen in early life may not necessarily produce more sperm at a particular ejaculate during the early post-pubertal period hastening of puberty does lead to earlier production of high-quality saleable semen and thus such bulls will typically yield a greater overall quantity of semen over the first 6–8 months post puberty (Byrne et al. 2018). Consistent with this, we observed a tendency towards greater systemic concentrations of FSH in the HIGH calves at 10 weeks of age, which agrees with the positive relationship between Sertoli cell number and systemic FSH (Bagu et al. 2004). Brito (2014) summarised work carried out on Angus and Angus × Charolais bulls receiving adequate nutrition and reported that FSH concentrations are at their highest between 10 and 14 weeks of age. This is similar to the pattern observed in the current study.
It has been reported that AMH controls the recession of Müllerian ducts in the male foetus (Rota et al. 2002) and is secreted from Sertoli cells from the time of prenatal sexual differentiation until puberty (Vigier et al. 1984). AMH secretion is at its maximum at birth, and it declines after 8 weeks of age, in bulls (Kitahara et al. 2016). This validates the results of the current study where transcript abundance for AMH in the LOW calves was upregulated compared with both HIGH calves and mature bulls and was greater, in turn, for HIGH calves compared to mature bulls. This again was corroborated by our immunohistochemical (IHC) work. Sertoli cell proliferation predominantly occurs in the early life of the bull (Rawlings et al. 2008) and GATA4 is used as a specific immunological marker for Sertoli cell nuclei in bulls (McCoard et al. 2001). This is consistent with the significantly greater GATA4 expression levels in both groups of calves in comparison with the mature bulls in the current study, which is possibly due to the crossover of presumptive Sertoli cells and mature Sertoli cells. As mentioned previously, there was a greater number of GATA4-stained Sertoli cells in HIGH compared with LOW calves. This is consistent with recent work carried out by Harstine et al. (2017) who found an increase in GATA4 stained Sertoli cells in FSH treated animals compared with untreated contemporaries. However, no differences in systemic FSH concentrations were observed in our study.
Testicular expression of FSHR decreases with testes development in bulls (Dias & Reeves 1982) with the number of receptors decreasing quickly from birth to 2 months of age and from then decreasing at a slower rate to 2–5 years of age (Dias & Reeves 1982). This is consistent with the greater expression of FSHR observed for both groups of calves compared to the adult bulls in the current study. Other studies have reported a decline in FSHR levels in rat and mice testes as they age (Ketelslegers et al. 1978). However, this result may be affected by an increase in germ cell population, causing an overall dilution of FSHR (Faucette et al. 2014). Given the absence of an effect of plane of nutrition on systemic FSH concentration or indeed FSHβ transcript abundance in the anterior pituitary, in our study, it is not entirely surprising that there was no effect on testicular FSHR expression.
We observed a tendency for greater systemic concentrations of LH in HIGH calves. Other studies have shown that circulating LH is highest in bull calves between 12 and 20 weeks of age, after which concentrations begin to decline (Rawlings et al. 2008) and our study corroborates these findings. Similarly, it has been reported that the concentration of LH receptors in Hereford × Charolais bulls was high postnatally but decreased from 13 to 25 weeks of age, which may be due to the decline in foetal Leydig and undifferentiated Leydig progenitor cell numbers (Bagu et al. 2006). These authors also found that testicular LHR number increased from 25 to 56 weeks in the same bulls, when the study concluded (Bagu et al. 2006). It is postulated that an increase in LHR at this stage may be due to Leydig cell maturation or an increase in Leydig cell number (Amann 1983). In our study, we also observed greater LHR expression in the testicular tissue of calves when compared with adult bulls. Systemic concentrations of testosterone have been shown to increase in beef bred bull calves following the early LH rise, which typically occurs between 8 and 20 weeks (Amann 1983). Even though no significant differences were found in peripheral LH concentration between planes of nutrition, the tendency for greater concentrations in the high plane of nutrition may have a knock on positive influence on testosterone concentration. The greater AUC recorded in HIGH compared to LOW bull calves, for LH, is possibly as a result of greater LH pulsatility in HIGH. Such a finding was reported by Brito et al. (2007c) when Angus and Angus × Charolais bulls offered a high vs a low plane of nutrition had, on average, 3–4 more LH pulses during a 10-h period, between 14 and 18 weeks of age. The greater testosterone concentration in the HIGH calves is consistent with the aforementioned greater systemic concentrations of IGF-1 for these animals. Given the well-documented positive effects of IGF-1 on testosterone (Dance et al. 2015), it is possible that the lower concentrations of IGF-1 in the LOW group affected the peri-pubertal testosterone concentrations also, by influencing Leydig cells (Brito et al. 2007c ). This may be a contributory factor for the lower rise in testosterone concentration in the LOW calves.
The expression of AR increases during postnatal and prepubertal phases of development in the testes of primates (McKinnell et al. 2001) and humans (Chemes et al. 2008). In addition, it has been reported that AR expression fluctuates cyclically in the Sertoli cells depending on the stage of spermatogenesis in the seminiferous tubule (Smith & Walker 2014). In part agreement with the aforementioned studies, we observed greater in expression of AR in testicular tissue of adult bulls compared with the HIGH calves only.
A study by Wrobel (2000) divided postnatal Simmental bull calves into groups based on their seminiferous tubule diameter and found that animals with a seminiferous tubule of 50–80 µm (5–15 weeks old) have increased PCNA staining at the pre-Sertoli stage; however, this decreased if the pre-Sertoli cells were differentiating. Whereas, in the 80–120 µm (18–27 weeks old), the PCNA staining was restricted to all germ cells present as pre-Sertoli cells have fully transformed into adult cells and no longer have proliferative ability. In accordance with this work, the LOW calves should have had a downregulation of PCNA compared with the HIGH, due to having less mature seminiferous tubules. Despite this obvious difference in seminiferous tubule development; however, we failed to detect a difference in PCNA expression between the groups. This highlights the discrepancies that can exist between transcript abundance, or lack there-of, at a point in time and biological function.
In the current study, the mature bulls and the HIGH calves had a similar level of testicular expression of CLDN11 with both groups having greater transcript abundance compared to LOW calves and suggests a favourable developmental benefit to the HIGH calves. In agreement, CLDN11 mRNA and protein levels were lower in the immature testes of rabbits (postnatal Day 10) in comparison with those of adults (postnatal Day 180) (Park et al. 2011). Zonula occludens protein 1 (ZO1), also referred to as tight junction protein 1, is involved in blood–testis barrier function. Expression of ZO1 was found to be decreased in undernourished mature rams (Guan et al. 2014, 2015). However, in contrast, in our calves, all of which were in positive energy balance, we did not observe an effect of either stage of maturity or plane of nutrition (within calves) on transcript abundance for ZO1.
Conclusion
In summary, this is the first published study to investigate the effect of early life plane of nutrition on the molecular control and cross-talk of the HPT axis in Holstein–Friesian bull calves. While there was no clear difference between the two planes of nutrition with respect to targeted gene and protein expression in the arcuate nucleus region of the hypothalamus or in the anterior pituitary per se, it is clear from the analyses conducted on testicular tissue that an improved metabolic state during early calfhood advances testicular development.
Supplementary data
This is linked to the online version of the paper at https://doi.org/10.1530/REP-17-0671.
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 jointly funded by the Irish Department of Agriculture, Food and the Marine under the Research Stimulus Fund (Ref: 11/S/116) and the Irish Research Council (GOIPG/2013/1391).
Acknowledgements
The authors gratefully acknowledge the contribution of the staff at Grange laboratories especially J Larkin as well as that of the farm staff at Teagasc, Grange Beef Research Centre, for care and management of the animals. The authors acknowledge J Roser (Department of Animal Science, University of California, Davis, CA, USA) for providing LH antiserum, the NHPP of the NIH for provision of FSH antiserum (AFP-C5288113) and FSH for iodination (AFP-4177A) and D Bolt (USDA, Beltsville, MD, USA) for providing bovine FSH standard. They thank Dr E Matthews and S Ni Cheallaigh (UCD, Dublin, Ireland) for her technical assistance with hormone assays. They thank M Cirot (INRA, Tours, France) for the processing of the testicular histology blocks. They also thank Dr L J Spicer for assistance with the IGF-1 radioimmunoassay and Dr A F Parlow and the National Hormone & Peptide Program (NHPP) for supplying the anti-IGF-1, NHPP-NIDDK. They thank staff at the Research Pathology Facility, Conway Institute, University College Dublin, Ireland for their help with IHC analysis.
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