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Livestock Science 12

Hypothalamic deafferentation in prepuberal beef heifers: Effects ofgonadotropin-releasing hormone and estradiol benzoate on

luteinizing hormone secretion

Jose R. Molina a, Allison M. Benoit a, Sender Lkhagvadorj a,c, Lloyd L. Anderson a,b,c,⁎

a Department of Animal Science, College of Agriculture and Life Sciences, 2356 Kildee Hall, Ames, Iowa, 50011-3150, USAb Department of Biomedical Sciences, College of Veterinary Medicine, USA

c Neuroscience Interdepartmental Program, Iowa State University, Department of Animal Science, 2356 Kildee Hall, Ames, Iowa, 50011-3150, USA

Received 9 October 2007; received in revised form 14 February 2008; accepted 18 April 2008

Abstract

Hypothalamic control of luteinizing hormone (LH) secretion was investigated in crossbred beef heifer calves by comparinganterior (AHD), posterior (PHD), and complete (CHD) hypothalamic deafferentation with sham operated controls (SOC). Heifers(n=16) were fitted with an indwelling jugular catheter for 6 days before cranial surgery, and assigned randomly to treatments. Bloodfor radioimmunoassay of LH was collected sequentially at 15-min intervals during an 8-h period on days −1 before and day 6 afterhypothalamic deafferentation or sham operation. On the day of surgery, blood samples were collected sequentially at 15-min intervals2 h before induction of anesthesia and throughout surgery and early recovery. Seven months after hypothalamic deafferentation, allexperimental and sham operated heifers were ovariectomized and treated with vegetable oil (i.m.) plus saline (i.v.), vegetable oil plusgonadotropin releasing hormone (GnRH), estradiol benzoate (EB, 1 mg) in vegetable oil. After ovariectomy basal plasmaconcentrations of LH increased (Pb0.01) compared with the low circulating hormone levels before ovariectomy. The amplitude ofLH response to GnRH was greater (Pb0.01) in CHD and PHDwhen compared with SOC and AHD heifers. Injection of EB failed toinduce a LH surge in CHD and PHD 900–1100 min later when compared with the robust response seen in SOC and AHD heifers.Injection of EB plus GnRH elicited LH release in all deafferentated and sham operated heifers. These results indicate a transientchange in LH secretion after AHD or CHD in prepuberal heifers with intact ovaries. After OVX, the integrity of the neural connectionof the posterior hypothalamus is required for EB-induced LH release in beef heifers.© 2008 Elsevier B.V. All rights reserved.

Keywords: Hypothalamic deafferentation; Beef calves; Ovariectomy; Gonadotropin-releasing hormone; Luteinizing hormone secretion;Estradiol benzoate

⁎ Corresponding author. Department of Animal Science, College ofAgriculture and Life Sciences, 2356 Kildee Hall, Ames, Iowa, 50011-3150, USA. Tel.: +1 515 294 5540; fax: +1 515 294 4471.

E-mail address: llanders@iastate.edu (L.L. Anderson).

1871-1413/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.livsci.2008.04.019

1. Introduction

In cattle luteinizing hormone (LH) is released in apulsatile pattern (Williams et al., 1987; Roberson et al.,1989; Kojima et al., 2003) that depends on age,reproductive stage of the estrous cycle, pregnancy andpost-partum (Roberson et al., 1989; Nakada et al.,

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2002) and effects of other factors, i.e., leptin, naloxone(an opiate antagonist), neuropeptide-Y (Zieba et al.,2003; Kadokawa et al., 2006; Stahringer et al., 1990;Thomas et al., 1999; Ahmadzadeh et al., 1998).Hypophysial stalk transection of prepuberal beef heifercalves results in complete inhibition of episodic LHsecretion compared with that seen in sham-operatedcontrols (Anderson et al., 1981). This indicatesepisodic LH release is regulated by hypothalamicgonadotropin-releasing hormone (GnRH). Pulsatileinfusion of GnRH in stalk-transected calves inducesLH release and results in ovarian follicular develop-ment (Awotwi et al., 1984). Intracerebroventricularcannulation of the third-ventricle of cattle allowedmeasure of GnRH secretion in cerebrospinal fluid(Gazal et al., 1998). Peak release of GnRH incerebrospinal fluid elicited a coincident LH peakrelease in the peripheral circulation in free-standingcows, clearly showing the neuroendocrine regulationof the hypothalamic-pituitary axis on GnRH and LHsecretion (Gazal et al., 1998). Tonic and surge releaseof LH and its tonic inhibition by suckling andneuropeptide Y was clearly demonstrated in theseanimals. Estradiol treatment induces a preovulatory LHsurge in cattle presumably by increasing pituitarysensitivity of hypothalamic GnRH and subsequentGnRH release. Unknown in cattle is whether neuro-surgical isolation of the hypothalamus near the medianeminence differentially affects GnRH neurons andfiber tracts to release GnRH and LH secretion.

We hypothesize that surgical isolation of medialbasal hypothalamus in beef heifer calves differentiallyaffects estrogen-induced LH secretion. The objectiveswere to: 1) determine the effects of anterior, posterior,complete hypothalamic deafferentation (AHD, PHD andCHD, respectively) as compared with sham-operatedcontrols (SOC) on LH secretion; 2) determine the effectsof GnRH injection on LH secretion in deafferentatedprepuberal beef heifers as compared with SOC beforeand after ovariectomy (OVX); and 3) determine theeffect of estrogen administration on the preovulatory-like LH surge in these deafferentated and SOC heifercalves.

2. Materials and methods

2.1. Experimental animals

Sixteen crossbred beef heifer calves (Hereford×AberdeenAngus) averaging 210±18 days (±SEM) of age and 182±6 kgbodyweight were used in this study. Calves were fed alfalfahay supplemented with 3–4 kg of corn and soybean mealration (14.1% protein) twice daily. Average daily gain of all

experimental and sham operated control heifers was 1.1±0.03 kg. All animals had free access to mineralized salt andwater. Six days before cranial surgery each heifer was fittedwith an indwelling jugular catheter (Tygon microbore tubing1.27 mm id and 2.29 mm od, Fisher Scientific, Pittsburgh, PA,USA); the catheter was secured around the neck with elastictape (Elasticon, Johnson & Johnson, Inc., New Brunswick, NJ,USA). Animals were randomly assigned to one of fourexperimental groups: SOC (n=4), AHD (n=4), PHD (n=4),and CHD (n=4). Animal care and experimental protocols werein accordance with the guidelines of the Iowa State UniversityCommittee on Animal Care.

2.2. Surgical procedures for hypothalamic deafferentationand post operative care

Each calf was isolated and fasted for 36 h and water wasrestricted 12 h before surgery. Anesthesia was induced by i.v.injection of thiamylal sodium (1.5–2.0 g; Surital, Parke Davis,Inc., Morris Plains, NJ, USA) for endotracheal intubation. Thetube (15 mm id and 22 mm od) was inserted and securedwithin the mouth of the animal by a bite block. Anesthesia wasmaintained by a closed-circuit system of halothane (3–8%;Ayerst Laboratories, Inc., New York, NY, USA) and O2 (0.5–1.5 l/min).

The calf was placed in ventral recumbency with the headimmobilized in a specially designed large-animal stereotaxicinstrument (David Kopf Instruments Inc., Tujunga, CA, USA).The calvarium was exposed, and the point on the surface of theskull at the junction of the coronal and sagittal sutures(bregma) was located. A surgical bone saw (OrthopedicEquipment Co., Bourbon, IN, USA) was used to cut the frontalbone. The calvarium was carefully removed with the aid of adouble action Rongeur (Weck Inc., Research Triangle Park,NC, USA) to expose the dorsal aspect of the dura mater. Theexcised frontal bone (3×4 cm) was placed in sterilephysiological saline containing penicillin-G (100,000 IU; EliLilly Co., Indianapolis, IN, USA) and dihydrostreptomycin(100 mg; Sigma-Aldrich Co., St. Louis, MO, USA). Afterremoval of the sharp edges of the frontal bone and calvariumthe smooth surfaces were covered with bone wax (Ethicon,Inc., Somerville, NJ, USA) to control bone bleeding. Asolution of 20% mannitol (250–300 ml; Abbott Laboratories,Inc., North Chicago, IL, USA) was given i.v. to reduce thevolume of cerebrospinal fluid and cerebral pressure.

The dura mater was incised along the midline lateral tothe falx cerebri. The dorsal sagittal sinus was gently deflectedand with the aid of stereotaxic instrument a knife designedfor cattle was placed in between the cerebral hemispheres. Alateral radiogram confirmed proper alignment of the head inthe stereotaxic instrument. The relative position of the tip ofthe blade and the tuberculum sellae were located on the film.After corrections were made the knife was moved anteriorly/rostrally, or posteriorly/caudally to the desired location withrespect to the tuberculum sellae and the optic chiasm. Theknife was lowered between the cerebral hemispheres into

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position and a final radiogram confirmed its location. Fordeafferentation of the medial basal hypothalamus (MBH) theknife formed an arc 11.0 mm height and a radius of 5.0 mm.For AHD, the knife was inserted between the cerebralhemispheres. With the arc pointed rostrally, it was loweredslowly to the desired location based on the first radiogram,the position was confirmed and slowly rotated 90°. The knifethen was rotated 180° in the reverse direction. The motionwas again reversed and the knife again rotated 180°. Finally,it was returned to midline and removed. For PHD, the knifewas inserted between the cerebral hemispheres. With the arcpointed caudally, it was lowered slowly to the desiredposition and rotated 90°. The knife then was rotated 180° inthe reverse direction. The motion was again reversed and theknife rotated 180°, returned to midline and removed. ForCHD, the knife was inserted between the cerebral hemi-spheres with the arc pointed rostrally. It was lowered aspreviously described, and rotated 360° clockwise. Then theknife was rotated 360° in reverse direction and removed. ForPHD and CHD, the knife was rotated slowly and carefully toensure deafferentation especially when approaching theridges formed by the trigeminal nerves at the base of thesphenoid bone and to avoid bouncing of the knife whichcould result in incompleteness of the cuts. For SOC, heifercalves were subjected to the same surgical proceduresdescribed for hypothalamic deafferentation with the excep-tion that the knife was not rotated and therefore no cut wasperformed in the hypothalamus. As in the hypothalamicdeafferentated animals the location of the knife and thealignment of the head in the stereotaxic instrument wereconfirmed by a lateral radiogram. After the knife waswithdrawn, the edges of the dura mater were sutured with 4-0silk ligatures (Ethicon, Inc.). The incision on the dura materwas covered with gel-foam (Pfizer Animal Health, Kalama-zoo, MI, USA), soaked in bovine thrombin (100 U/ml; ParkeDavis, Inc.), and the exposed surface was powdered withpenicillin-G (500,000 IU, Eli Lilly Co.), and dihydrostrepto-mycin (500 mg, Sigma-Aldrich Co). The excised frontal bonewas replaced and the skin sutured with silk ligatures.

Immediately after surgery, an i.m. injection of 100 mg ofcortisone acetate (25 mg/ml; Cortone, Merck & Co., Inc., WestPoint, PA, USA) was given to all animals in the study tocontrol hyperemia. Rectal temperature was monitored hourlyfor 5 h after surgery and twice daily for at least 1 week afterhypothalamic deafferentation or sham operation. All animalswere checked twice daily at feeding time to determine changesin behavior or eating patterns.

2.3. Surgical procedure for OVX

Seven months after hypothalamic deafferentation or shamoperation the 16 heifer calves were bilaterally OVX by leftparalumbar laparotomy under local anesthesia (Xylocain HCl;15–20 cm3 i.m. injection, Astra Pharmaceutical Products, Inc.,Westborough, MA, USA). Ovaries underwent écrasement andwere excised and each ovary was fixed in 10% buffered neutralformalin for histological examination.

2.4. Blood collection and hormone treatments

Five days before cranial surgery (day −5) at 0800 h theheifers were penned individually with access to water only.Blood samples (8 ml) were drawn sequentially from theindwelling jugular catheter at 15-min intervals for an 8 h-period.Blood samples were placed in disposable borosilicate culturetubes (16×100 mm) containing 20 IU heparin sodium(100 IU/ml; ICN Pharmaceuticals, Inc., Cleveland, OH,USA) and immediately centrifuged (1500 ×g) upon collec-tion. Plasma was decanted and stored in disposable culturetubes (12×75 mm) at −20 °C for LH radioimmunoassay. Oneday before cranial surgery (day −1) the same procedure forday −5 was followed. At the end of the 8 h bleeding period theanimal remained isolated and the water source was removed.On the day of cranial surgery (day 0) blood samples werecollected sequentially every 15-min for 2 h before inductionof anesthesia and continued throughout anesthesia andsurgery. Six days after cranial surgery (day 6) the animalwas again isolated and blood samples collected sequentially at15-min intervals for an 8-h period.

On day 7 after hypothalamic deafferentation or shamoperation calves were treated with a 50 μg i.v. injection ofGnRH (Cystorelin #8100 50 μg/ml Abbott Laboratories, Inc.)via the indwelling catheter. The bleeding protocol was asfollows: blood samples were collected sequentially at 15-minintervals for 2 h before GnRH injection and at 5, 10, 15-minand then every 15-min for 2 h after GnRH injection.

Twenty-eight days after OVX all deafferentated and shamoperated heifers were assigned randomly to four treatmentgroups: vegetable oil i.m. plus saline i.v., vegetable oil plus50 μg GnRH, EB in vegetable oil (i.m.) plus PBS, and EB invegetable oil plus 50 μg GnRH. Calves were stanchioned andthe experiment performed during a 2-week period. Bloodsamples were collected sequentially hourly 2 h before and 10 hafter injection of vegetable oil (2 ml, i.m.) or 1 mg of EB in2 ml of vegetable oil. Immediately after the last blood samplewas collected, an i.v. injection of PBS (1 ml) or 50 μg GnRHwas given and blood samples were collected sequentially at 5,10, 15-min and every 30-min for a 20-h period.

2.5. Radioimmunoassay (RIA) of LH

Concentrations of LH were quantified in duplicate inaliquots of 100 or 200 μl plasma by a double antibody RIA.Purified bovine LH (LER-1716-2 and USDA-bLH-I-1) wasused for labeling with 125I (chloramine-T method) and forassay standards (0.25, 0.5, 1.0, 2.5, 5, 10, 20, and 40 ng/ml).Radioiodination was done by adding 5 μl of a 1 μg/μl bLH,50 μl 0.5 м sodium phosphate buffer pH 7.5, 1 mCi 125I(100 mCi/ml, IMS-30, Amersham Corp., Arlington Heights,IL, USA) and 15 μl of 2 mg/ml chloramine-T (Sigma-AldrichCo.) solution into a 1 ml Wheaton vial (Wheaton ScientificInc., Millville, NJ, USA). The reaction mixture was agitatedfor 2 min before adding 30 μl of 2 mg/ml sodium metabisulfite(Sigma-Aldrich Co.) followed by 100 μl 0.05 м PBS-16%sucrose as transfer solution. A sephadex G-25-150 (Sigma-

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Aldrich Co.) column (18×0.9 cm) was used to separatethe labeled antigen and the peak fraction (1.0 ml) containing125I-bLH was diluted to 30,000 to 35,000 cpm in 0.01 м PBS0.1% gel buffer and used as assay tracer. After dilution ofovine LH antiserum (GDN #15, 1:40,000) with 0.05 м EDTA-PBS 1:400 normal rabbit serum, 200 μl were added to 100 or200 μl of plasma unknown plus 0.01 м PBS 1% gelatin,pH=7.0 to a final volume of 500 μl. Samples were incubatedfor 24 h at 4 °C and 100 μl of 125I-bLH (30,000 to 35,000 cpm)was added and incubation continued for additional 24 h at4 °C. Then 200 μl of 1:45 dilution of goat anti-rabbit-γ-globulin (#20321, Cappel Laboratories, Inc., Malvern, PA,USA) were added and incubated 72 h at 4 °C. Three milliliters0.01 м PBS pH 7.0 were added to each assay tube and boundand free fractions were separated by centrifugation at 2200 ×gfor 30 min, the supernatant was decanted and the radioactivityof the pellet determined in a γ-well spectrometer. The antibodyat the dilution used, bound 28–32% of the 125I bLH. Assaysensitivity was defined as the amount of LH standard whichyielded 95% of the radioactive cpm in plasma control tubes.Average sensitivity of assay (n=6) was 0.25 ng/ml. Intraassayvariability of LH was determined from replicates of twoplasma pools (n=10) a low one from a hypophysial stalktransected heifer (0.67+0.13 ng/ml) and a high one from anovariectomized cow (13.7+0.61 ng/ml). Interassay variationwas determined by assaying samples (n=10) of the sameserum pools in each assay. The intraassay and interassaycoefficients of variation were 8.17 and 11.21%, respectively.

Precision and accuracy of the RIA were evaluated byadding 0.25, 0.5, 1.0, 2.5, 5, 10, 20, and 40 ng LH on an ng/ml

Fig. 1. Peripheral LH plasma concentrations in prepuberal beef heifers randobefore surgery, on day 0 surgery, and day 6 after surgery with four calves in

basis (n=6) to plasma from a long-term hypophysial stalk-transected heifer, and to plasma from a long-term ovariecto-mized cow. After subtraction of the plasma blank anddemonstration of parallelism between the plasma pools, theLH concentrations (+ SEM) obtained were 0.26+0.03 and10.7+0.36 ng/ml, respectively.

2.6. Postmortem, brain tissue collection and histology

Calves were harvested at the university abattoir and thehead immediately removed at the occipital bone. Each brainwas perfused with buffered neutral formalin (pH 7.2) via thecommon carotid arteries. The brain was removed, and a blockof tissue containing the hypothalamus and preoptic regionwas excised and placed in buffered neutral formalin for 24 h.The block was dehydrated in a sequence of ethanol solutions(25–100%), imbedded in paraplast, and coronal sectionscut at 7 μm. Tissue sections were stained with solvent blue 38(S-3382, Sigma-Aldrich Co.) and cresyl violet acetate (C-1893,Sigma-Aldrich Co.), whereas others were stained with hematox-ylin and eosin. The knife cuts were reconstructed by examiningevery 10th coronal section with a Stereomicroscope IVb, (CarlZeiss, Inc., New York, New York, USA). Brain tissue wascollected from SOC calves to establish the typographicallocation of nuclei within the hypothalamus by staining of thecells and fiber tracts (Klüver and Barrera, 1953). Criteria fordelineating nuclei were a) density of cells in relation to adjacenttissue and b) location of the dense mass with respect to the opticchiasm, rostral supraoptic nucleus, fornix, anterior commissure,middle forebrain bundle, third ventricle, paraventricular nucleus,

mly assigned to SOC, AHD, PHD, and CHD on day −5 and day −1each group. Values are mean+SEM.

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median eminence, medial basal hypothalamus and arcuatenucleus.

2.7. Statistical analysis

In this experiment heifer calves were assigned randomlyto one of four treatments in a split-plot design. Plasmaconcentrations of LH were averaged for each heifer duringthe sampling period. Values greater than two standarddeviations above the mean during the sampling period ofeach calf were defined as a secretory spike. Data were subjectedto analysis of variance (ANOVA) using the general linearmodel of SAS (SAS User's Guide, 2002). Basal and meanplasma concentrations of LH and amplitude of pulses of LHwere subjected to repeated measures analysis. Data are shownas the mean+SEM. Differences were considered statisticallysignificant at Pb0.05.

Fig. 2. Peripheral LH plasma concentrations in SOC, AHD, PHD andCHD prepuberal beef heifers on day 7 after surgery. Calves were bloodsampled 120 min before administering an i.v. injection of 50μg GnRH attime 0, and continuing blood sampling for 120 min. Values are mean+SEM.

3. Results

3.1. Hypothalamic deafferentation on LH secretion

LH concentrations in peripheral plasma were similar(PN0.05) in all treatment groups before hypothalamicdeafferentation or sham operation (day 0). The pattern ofLH secretion was episodic in all 16 heifers and thefrequency of the spikes was 1 to 2 during an 8-h periodwith a maximum amplitude of 13.7 ng/ml. Basal levels ofthe hormone were maintained between 0.2 to 0.5 ng/ml,between episodes. Mean LH values 5 days before surgery(day −5) were 0.9+0.1, 0.6+0.1, 0.7+0.1, and 0.5+0.1 ng/ml for animals randomly assigned to SOC, AHD,CHD and PHD, respectively (Fig. 1).

On the day before surgery (day −1) LH concentra-tions were similar (PN0.05) to those found on day −5and the pattern of hormone secretion also was similar inall experimental and sham operated heifers. Mean LHvalues at day −1 were 0.7+0.1, 0.6+0.1, 0.8+0.1, and0.4+0.1 ng/ml in SOC, AHD, CHD and PHD heifers,respectively (Fig. 1). During the day of surgery (day 0)LH levels remained similar in all treated and shamoperated heifers; anesthesia did not affect either basallevels or in the pattern of the hormone secretion. MeanLH values on day 0 were 0.6+0.1, 0.4+0.1, 0.7+0.1and 0.8+0.1 ng/ml in SOC, AHD, CHD and PHDheifers, respectively (Fig. 1). Six days after hypotha-lamic deafferentation or sham operation, LH concentra-tions were similar (PN0.05) in all treated and shamoperated calves. However, the amplitude of the LHspikes was reduced (Pb0.01) in AHD when comparedSOC, (6+0.8 vs. 2+0.4 ng/ml). Likewise the amplitudeof the LH spikes was reduced (Pb0.05) in CHD whencompared with SOC (6+0.8 vs. 4+1.1 ng/ml). No

changes (PN0.05) in amplitude of the LH spikes wereevident when SOC and PHD groups were compared(6.2+ 0.8 vs. 8.8+3.1 ng/ml). Overall mean LHconcentrations on day 6 were 0.8+0.2, 0.6+0.2, 0.7+0.2 and 1.3+0.2 ng/ml in SOC, AHD, CHD and PHD,respectively (Fig. 1).

3.2. Effect of GnRH after hypothalamic deafferentationon plasma LH levels

Administration of 50 μg of GnRH i.v. on day 7after hypothalamic deafferentation of prepuberalcalves resulted in an abrupt increase (Pb0.0001) inperipheral plasma LH concentrations from baseline(0.5+0.08 ng/ml) to peak levels of 17+0.8, 13+0.8,16+0.8, and 13+0.6, ng/ml in SOC, AHD, CHD andPHD, respectively, within 30 min after GnRHinjection (Fig. 2; Table 1). The amplitude of the LHspike in response to GnRH was similar (PN0.05) inall deafferentated and sham operated heifers. Overallmean LH concentrations during the 2-h bleeding periodbefore LHRH treatment were 6+0.3, 4+0.3, 6+0.3and 5+0.3 ng/ml in SOC, AHD, CHD and PHD heifers,respectively (Fig. 2). Plasma circulating LH levelsdecreased rapidly from peak values but remained abovebaseline by 2 h after GnRH injection.

Table 1Luteinizing hormone (LH) secretion after estradiol benzoate and LH-releasing hormone treatment

aAUC bCmaxcTmax

Fig. 2 LHRH, 50 μgSOC 1287 17.5 30AHD 862 13.2 20PHD 1006 13.7 30CHD 1238 16.8 20

Fig. 4 Vegetable oil and LHRH, 50 μgSOC 7898 65.9 610AHD 5909 59.7 630PHD 9916 172.6 630CHD 14,302 180.4 630

Fig. 5 E2B, 1 mg and PBSSOC 65,159 191.0 1080AHD 46,155 148.4 990PHD 18,314 59.8 960CHD 15,073 47.2 1110

Fig. 6 E2B, 1 mg and LHRH, 50 μgSOC 34,122 194.3 690AHD 24,073 153.4 690PHD 27,896 185.9 750CHD 26,578 196.4 660

a Area under the curve (min×ng/ml).b Maximum LH concentration (ng/ml).c Time (min) to reach Cmax.

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3.3. Effect of OVX, estrogen replacement and GnRH onLH secretion

Removal of the endogenous estrogen source 7monthsafter hypothalamic deafferentation caused a significant

Fig. 3. Peripheral LH plasma concentrations in SOC, AHD, PHD and CHD ovat time 0 and an i.v. injection of PBS diluent at time 600 min. Values are m

increase (Pb0.01) in basal LH levels in all deaf-ferentated and sham operated heifers. After i.m. injectionof either vegetable oil or PBS 600min later no changes inthe overall mean LH plasma concentrations wereobserved, and an episodic pattern of LH release wasevident during the 30 h bleeding period (Fig. 3).

Six days after the injection of vegetable oil or PBS, alldeafferentated and sham operated heifers were injectedwith vegetable oil i.m. and 50 μg GnRH i.v. 600 min later,after which, a significant increase (Pb0.001) in peripheralplasma concentrations of LH was detected. Basal LHlevels were elevated from 10+0.9 ng/ml to peak levels of66+9, 60+6, 173+56, and 180+42, ng/ml in SOC,AHD, CHD and PHD calves within 30 min after GnRHinjection, respectively (Fig. 4; Table 1). The amplitude ofthe LH spike in response to GnRH was greater (Pb0.01)in CHD and PHD heifers when compared with SOC andAHD heifers. Overall mean plasma LH concentrationsduring the 30-h bleeding period were 14+3, 11+3, 26+3and 17+2 ng/ml for SOC, AHD, CHD and PHD heifers,respectively (Fig. 4; Table 1). Statistical analyses of thedata indicated a significant increase (Pb0.05) in LHconcentrations in CHD animals when compared withSOC.

One week after vegetable oil or PBS treatment, eachheifer was challenged with 1 mg EB in vegetable oil i.m.and i.v. injection of PBS 600 min later. Intramuscularinjection of 1 mg EB to ovariectomized SOC and AHDheifers produced a sharp elevation on circulating levelsof LH from baseline (6.1+0.7 ng/ml) to an average peak

ariectomized beef heifers given an i.v. injection of vegetable oil diluentean+SEM.

Fig. 4. Peripheral LH plasma concentrations in SOC, AHD, PHD and CHD ovariectomized beef heifers given an i.v. injection of vegetable oil diluentat time 0 and an i.v. injection of 50 μg GnRH at time 600 min. Values are mean+SEM.

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levels of 196+23 ng/ml within 17+0.3 h after EBadministration (Fig. 5; Table 1). A negative feedbackeffect of EB on LH secretion was evident within 2 h

Fig. 5. Peripheral LH plasma concentrations in SOC, AHD, PHD and CHD otime 0 and an i.v. injection of PBS diluent at time 600 min. Values are mean

after injection; LH levels were reduced and maintainedbelow average basal concentrations for at least 10 h afterEB. Thereafter, LH plasma concentrations increased

variectomized beef heifers given an i.v. injection of 1 mg EB in oil at+SEM.

Fig. 6. Peripheral LH plasma concentrations in SOC, AHD, PHD and CHD ovariectomized beef heifers given an i.v. injection of 1 mg EB in oil attime 0 and an i.v. injection of 50 μg GnRH at time 600 min. Values are mean+SEM.

Fig. 7. Specially designed knife for hypothalamic deafferentation incattle.

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markedly as evident by 14 h after EB administration.Overall mean plasma LH concentrations during the 30 hbleeding period were 42+2.8 and 30+2.7 ng/ml in SOCand AHD heifers, respectively (Fig. 5). EB failed toinduce a preovulatory-like LH surge in 5 of 7 heifers in theCHD and PHD groups. Overall mean LH concentrationsduring the 30-h bleeding periodwere reduced (Pb0.01) inCHD and PHD when compared with SOC (12+3.2 and13+2.7 vs. 42+2.8 ng/ml, respectively) (Fig. 5).

Six days after EB injections all deafferentated andsham operated heifers were injected i.m. with 1 mg EBplus a 50-μg GnRH injection i.v. 10 h after EB injection.In every case, heifers released LH to peak levels within30 min after GnRH injection. Mean LH peak concen-trations were 209+32, 153+11, 196+8, and 185+9 inSOC, AHD, CHD and PHD animals, respectively(Fig. 6; Table 1). Administration of EB followed byGnRH reduced (Pb0.05) the overall mean LH con-centrations in SOC and AHD heifers when comparedwith mean LH levels in the same animals when GnRHinjection was not given (29+2 and 19+2 vs. 42+2 and30+2 ng/ml, respectively). CHD and PHD heifersreleased more LH after EB plus GnRH injection than EBalone during the 30-h bleeding period (23+2.5 and 20+2.7 vs. 12+3.2 and 13+2.8 ng/ml, respectively).

3.4. Histology of bovine hypothalamus

Histological reconstruction of the deafferentatedhypothalamus verified tissue isolation in each of the

14 prepuberal calves in the AHD, PHD and CHDgroups. In AHD and CHD calves, the rostral knife cutwas caudal to the anterior hypothalamic area and rostralto the median eminence (Figs. 7 and 8a,b,c). In the areanear the midline, the rostral knife cut likely affected the

Fig. 8. Photomicrographs a, b and c are representative of coronal sections cut at 7 μm of bovine hypothalamus after anterior hypothalamicdeafferentation (AHD) and d a coronal section from a control heifer for orientation. The arrows indicate the location and completeness of the bilaterallesion. The mammillothalamic tract (MTT), 3rd ventricle (III), paraventricular nucleus (PVN), dorsal medial nucleus (DMN), medial forebrain bundle(MFB), ventral medial hypothalamus (VMH), fornix (F), arcuate nucleus (AR) and median eminence (ME) are indicated. In none of the 14 heifercalves was there histological evidence of necrosis of brain tissue or accumulation of blood in the area of the hypothalamic deafferentation.Magnification ×2.8.

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arcuate nuclei. PHD lesions were located within oranteriorly to the mammillary bodies.

4. Discussion

This is the first report on the effects of hypothalamicdeafferentation of the MBH on CNS regulation of LHrelease in cattle. A major finding was the importance ofthe integrity of the posterior neural connections as wellas the anterior neural connections in the regulation ofLH release as emphasized by the magnitude of EB-induced release of this hormone. Amplitude of LHrelease was significantly reduced after AHD and CHDcompared with the robust response seen in SOCprepuberal beef heifers. The incidence of LH episodesup to 4 per 24 h in prepuberal beef heifers has beendocumented (McLeod et al., 1984). As frequency of

GnRH pulses decreases, the amplitude of LH responsesincreases in direct proportion to the size of thereleasable LH pool. A reduction in amplitude of LHrelease in calves in this study may relate to reducedavailability of GnRH to the pituitary caused bydeafferentation of neural pathways traversing theanterior hypothalamus.

In cattle and other species, GnRH containingneuronal cell bodies, axons and axon-terminals arescattered throughout the hypothalamus as well as themidbrain and limbic forebrain (Barry, 1973; Barry et al.,1973; Silverman and Zimmerman, 1978). Anterior,complete or posterior hypothalamic deafferentation hadlittle or no effect on episodic LH secretion in theseprepuberal heifers. Pulsatile infusion of GnRH intohypophysial stalk-transected beef heifers elicited anincrease in peripheral serum LH concentrations (Awotwi

22 J.R. Molina et al. / Livestock Science 120 (2009) 13–24

et al., 1984). A significant increase in peripheral plasmaLH concentrations was observed within 30 min after i.v.injection of 50 μg GnRH in all deafferentated and shamoperated heifers. A pharmacological dosage of GnRHstimulates LH release in intact postpuberal heifers andpostpartum dairy cows (Kittok et al., 1972; Fernandeset al., 1978). For example, 150 μg GnRH given at 4-hintervals during days 16 to 19 of the estrous cycleproduces mean LH peak blood levels of ~5 ng/ml whichwere higher than controls ~1 ng/ml (Kinder et al., 1975).These LH peaks occurred within 30 min after GnRH andreturn to basal levels within 4 h. In the present study, LHpeaks observed in all deafferentated and sham operatedOVX heifers were higher than those reported by otherinvestigators. Prepuberal heifers may be more respon-sive to GnRH than postpartum cows. During pregnancycows produce high levels of estradiol and estrone ofplacental origin that peak just before parturition(Anderson et al., 1999). It is also possible that theamount of releasable LH stored in the pituitary glandwas dampened immediately after 50 μg GnRH. Inprepuberal intact calves the daily administration ofGnRH causes an increase in peripheral serum levels ofLH which peak 2 h after injection and return to basallevels within 6 h (Mellin et al., 1975). An increase incirculating levels of estrogen similar to or less than thosethat occur during proestrus will induce preovulatory-likesurges of LH and FSH in OVX heifers (Kesner et al.,1982).

This study demonstrated that i.m. injection of EB toOVX SOC and AHD heifers produced a sharp elevationin circulating LH levels within 17.3 h after injection.Similar results occurred in OVX cows and in steersgiven GnRH 2 or 8 h after estradiol administration(Kesner et al., 1981). EB produced a negative feedbackeffect on LH secretion during the first 10 h afterinjection in deafferentated heifers. A decrease in the LHpulses as well as a reduction in basal levels of thehormone was evident.

In sheep, deafferentation of AHD regulates thepreovulatory LH surge (Jackson et al., 1978) andsuppresses estradiol benzoate-induced LH surge anddamping of the mean basal hormone level (Thiéry et al.,1978). Thus, an essential role of anterior and/or dorsalinputs in the regulation of estradiol-induced LH surge isindicated (Thiéry et al., 1978; Thiéry and Martin, 1991).The results suggest that antero-dorsal inputs, comingfrom preoptic-suprachiasmatic areas are required bothfor the positive feedback of estrogens on LH secretion,and for maintenance of the basal level of secretion. Inthe rat, surgical disconnection of hypothalamic areasdemonstrated that the MBH regulates tonic secretions

of gonadotropins whereas AHD is responsible forcyclic LH release which triggers ovulation (Halász andPupp, 1965). Frontal deafferentation in the rat onlydepressed the LHRH content in the MBH while totaldeafferentation diminished it completely (Sétáló et al.,1976). Thus, the mechanism responsible for thepulsatile pattern of LH secretion resides in MBH assuggested by Blake and Sawyer (1974). In the monkey,two main functions regulating gonadotropin secretion(i.e., tonic release and preovulatory surge) may belocalized in the MBH (Krey et al., 1975); however,Norman et al. (1976) with the same techniquessuggested a possible role for a more anterior part ofthe brain. Results from rat, sheep and monkeyemphasize the importance of anterior neural connec-tions in the regulation of LH secretion, whereas incattle integrity of the neural connection of the posteriorhypothalamus as well as the anterior hypothalamus isrequired for EB-induced LH release.

In five of seven heifers in CHD and PHD groups ofthe present study, EB failed to induce a preovulatory LHsurge comparable to that in SOC and AHD animals.These findings indicate that the posterior neural links ofhypothalamus play a major role in regulating thepreovulatory-like LH surge in beef heifers. Resultsfrom this study indicate that the posterior but not theanterior integrity of neural connections of the medialbasal hypothalamus is required for the EB-induced LHsurge in OVX beef heifers. LH peaks were similar(PN0.05) in all deafferentated and sham operatedheifers after EB plus GnRH and the overall mean LHconcentration in CHD and PHD heifers during the 30 hbleeding period was higher (Pb0.05) than after EBtreatment alone.

5. Conclusion

In conclusion, AHD and CHD reduced the ampli-tude of the LH peak secretion within 30 min inprepuberal beef heifers with intact ovaries. After OVX,a significant increase in LH basal levels was evidentbut no differences were observed in the overall LHsecretion in deafferentated and sham-operated calves.EB administered to OVX beef heifers bearing PHD orCHD was unable to release an LH surge at 900–1100 min compared with robust response seen in SOCcalves. Therefore, these results indicate that there is atransient change in LH secretion after AHD or CHD inprepuberal heifers with intact ovaries. Furthermore, theintegrity of the neural connection of the posteriorhypothalamus is required for EB-induced LH release inbeef heifers.

23J.R. Molina et al. / Livestock Science 120 (2009) 13–24

Acknowledgements

This is a journal series paper from the IowaAgriculturaland Home Economics Experiment Station, Ames; sup-ported by Hatch and State of Iowa funds together withMultistate Regional Research Grant NC-1006 Methods toIncrease Reproductive Efficiency in Cattle and withCSREES Special Research Grant 59-2191-1-2-033-0from the U.S. Department of Agriculture. We thankDr. D.F. Cox, Department of Statistics for advice onstatistical analyses; and M.E. Shell and C.R. Bohnker fortechnical assistance.

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