Animal Reproduction Science 182 (2017) 28–34

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Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Effects of leptin administration on development, vascularization MARK

and function of Corpus luteum in alpacas submitted to pre-ovulatory
fasting
⁎
María Cecilia Norambuenaa,b, , Francisca Hernándeza, Jonathan Maureiraa,
Carolina Rubilara, Jorge Alfaroa, Gonzalo Silvaa, Mauricio Silvaa,b, César Ulloa-Lealc
a
School of Veterinary Medicine, Faculty of Natural Resources, Universidad Católica de Temuco, Manuel Montt 056, Temuco, PC 4780000, Chile
b
Núcleo de Investigación en Producción Alimentaria (NIPA), Universidad Católica de Temuco, Manuel Montt 056, Temuco, PC 4780000, Chile
c
Universidad de las Fuerzas Armadas ESPE, IASA II, Sangolqui, PC170501, Ecuador

AR TI CLE I NF O AB S T R A CT

Keywords: The objective of this study was to determine the effect of leptin administration on the
Leptin development, vascularization and function of Corpus luteum (CL) in alpacas submitted to pre-
Corpus luteum ovulatory fasting. Fourteen alpacas were kept in fasting conditions for 72 h and received five
Vascularization doses of o-leptin (2 μg/kg e.v.; Leptin group) or saline (Control group) every 12 h. Ovulation was
Camelids
induced with a GnRH dose (Day 0). The ovaries were examined every other day by trans-rectal
Fasting
ultrasonography (7.5 MHz; mode B and power Doppler) from Day 0 to 13 to determine the pre-
ovulatory follicle diameter and ovulation, and then to monitor CL diameter and vascularization
until the regression phase. Serial blood samples were taken after GnRH treatment to determine
plasma LH concentration; and every other day from Days 1 to 13 to determine plasma
progesterone and leptin concentrations. The pre-ovulatory follicle and CL diameter, LH,
progesterone and leptin plasma concentrations were not affected by treatment (P > 0.05).
The vascularization area of the CL was, nevertheless, affected by the treatment (P < 0.01) with
significant differences between groups at Days 3, 7 and 9 (P < 0.05). The Leptin group had a
larger maximum vascularization area (0.67 ± 0.1 compared with 0.35 ± 0.1 cm2; P < 0.05).
In addition, there was a positive correlation between CL vascularization, CL diameter and plasma
progesterone. The exogenous administration of leptin during pre-ovulatory fasting increased the
vascularization of the CL in alpacas in vivo.

1. Introduction

Leptin is a protein hormone (16 kDa) produced mainly by adipocytes, although also by the digestive and reproductive systems,
among others (Chehab, 2014; Friedman, 2014). Its role as a molecule linking nutrition and reproduction has been studied since the
discovery that hypoleptinemic rodents were obese and infertile and that both conditions were reversed following administration of
leptin (Barash et al., 1996; Farooqi et al., 1999).
In ruminants it has been documented that the relative abundance of adipose tissue leptin mRNA and leptin plasma concentration
is markedly decreased by nutritional restriction, and increases after feeding is resumed (Chilliard et al., 2005; Delavaud et al., 2007).

⁎
Corresponding author at: School of Veterinary Medicine, Faculty of Natural Resources, Universidad Católica de Temuco, Manuel Montt 056, Temuco, PC 4780000,
Chile.
E-mail address: mcnorambuena@uct.cl (M.C. Norambuena).

http://dx.doi.org/10.1016/j.anireprosci.2017.04.006
Received 29 October 2016; Received in revised form 20 February 2017
Available online 03 May 2017
0378-4320/ © 2017 Elsevier B.V. All rights reserved.
M.C. Norambuena et al. Animal Reproduction Science 182 (2017) 28–34

These authors suggested that the reduction of leptin may function as an acute signal to stimulate feeding behavior, reduce energy
expenditure and inhibit reproduction.
Several studies have determined that leptin administration reverses the negative effect caused by fasting on the secretion of LH in
cows and rodents (Nagatani et al., 2000; Zieba et al., 2003). It has also been shown that the hypothalamus, hypophysis, ovarian
follicles and Corpus luteum (CL) have leptin receptors in different species (Di Yorio et al., 2008; Sarkar et al., 2010). Interestingly, it
has been determined that the greatest leptin plasma concentration or relative abundances of leptin and leptin receptor mRNA are
observed in the middle and late luteal stages and decrease coincident with luteal regression in llamas (Norambuena et al., 2013),
cattle (Sarkar et al., 2010) and buffalo (Kumar et al., 2012). There is also a positive correlation between the amount of steroidogenic
acute regulatory (StAR) protein, side-chain cleavage cytochrome P450 (P450scc) and 3beta-hydroxysteroid dehydrogenase/
isomerase (3beta-HSD) factors and ObR receptor activity, suggesting a role of leptin in progesterone and other steroid production
(Kumar et al., 2012).
Only a few in vivo studies have been conducted to understand the role of leptin in luteal function. Kendall et al. (2004) reported
that ovarian leptin infusion during the early follicular phase resulted in an increase in progesterone production during the subsequent
luteal phase in sheep. Roman et al. (2005), however, observed that chronic leptin administration reversed the negative effect on
progesterone plasma concentration induced by severe food restriction in rodents.
Wiles et al. (2014) provided in vitro evidence that leptin may be involved in the luteal angiogenic process because it stimulated
Ang1, FGF2 and VEGF gene expression in luteal cell cultures. The aim of the present study was to determine the effect of leptin
administration on the development, vascularization and function of the CL in alpacas submitted to pre-ovulatory fasting.

2. Material and methods

2.1. Animal management

Eighteen adult non-lactating alpacas (Vicugna pacos), property of the Universidad Católica de Temuco, Chile (38° 46′ S latitude,
72° 38′ longitude, and 200 m above sea level) were used in this study. All procedures were reviewed and approved by the Universidad
Católica de Temuco Bioethics Committee and were performed in accordance with the animal care protocols established by the
University. Alpacas were kept indoors at night and during the afternoon (from 12:00 to 17:00) the animals grazed in natural pastures
of Ballica sp. The animals were also fed daily Ballica sp. hay (6.2% crude protein [CP], 4.4 Mcal/kg, metabolic energy [ME], 4.7%
total ash [TA)] – dry matter basis), water and mineral salts (Usablock®, Sweetlix, Mankato, Minnesota, USA) ad libitum; plus 200 g of a
commercial concentrate (16.0% CP, 5.4% TA, and 3.0 Mcal/kg ME – dry matter basis; Cosetán®, Iansagro S.A., Osorno, Chile).

2.2. Experimental design

Stage of follicle wave development was synchronized using intra-vaginal progesterone devices for 7 days (CIDR®, 0.3 g, Pfizer
Chile S.A., Chile) as described in previous studies (Chavez et al., 2002; Cavilla et al., 2016). After removal of the devices, the alpacas
were examined by trans-rectal ultrasonography (7.5 MHz linear-array transducer; Sonovet3, Samsung Medison, South Korea) every
other day for 10 days to evaluate follicle diameter and retrospectively, the emergence of the new wave of follicular development.
Four alpacas which had follicles > 5 mm after the removal of the progesterone devices were not used for the study. Eight days after
the removal of the devices, the remaining alpacas were fasted for 72 h with water ad libitum and randomly assigned to the Leptin or
Control group (n = 7 alpacas/group). Alpacas in the Leptin group received five intravenous doses (at 12, 24, 36, 48 and 60 h) of o-
Leptin (2 μg/kg; 0.1 mg/mL; e.v.), while those in the Control group received a similar number of saline administrations (1 mL; e.v.).
Six hours before the conclusion of the fasting period, ovulation was induced with an intramuscular dose of GnRH (Day 0 = GnRH
treatment; 50 μg; gonadorelin acetate; Gonasyn GDR, Syntex, Buenos Aires, Argentina). The ovaries were subsequently examined by
trans-rectal ultrasonography to determine the pre-ovulatory follicle diameter at Day 0 and to detect ovulation at Day 3. Ovulation
was defined as the disappearance of a large follicle (≥7 mm) that had been detected in the previous examination. The ovaries were
examined every other day thereafter until Day 13 to monitor CL diameter and vascularization. During each examination, power
Doppler mode was activated after initial B-mode evaluation to examine CL vascularization. Fixed, preinstalled Doppler system
controls were used, to exclude variations in recording. Cineloops (10 s in length) of the CL were recorded during power Doppler
imaging and downloaded onto a VLC media player (www.videolan.org, Version 2.0, Boston, MA, USA). The cineloops were examined
frame by frame to select three images that represented the maximum vascular signal near the maximum cross-sectional area of the CL.
Images were saved in JPG format with minimal compression, and analyzed by ImageJ software (National Institute of Health,
Bethesda, MD, USA). The degree of vascularization was estimated by measuring the area (cm2) of the vascular flow signals (power
Doppler mode) overlaying the B-mode image of the CL. The average of the three images was taken as the value for a given animal on a
given day. The maximum CL diameter, the maximum vascularization area and the CL lifespan were determined. The lifespan of the
CL was calculated using the interval in days between the 5 mm stage at the growing phase to the 5 mm stage during the regression
phase. The body live weight (BLW) and body condition score (BCS; 1–5 scale according to Van Saun, 2009) were determined before
and after fasting, and every 4 days from Day 1 to 13 (Day 0 = day of ovulation induction). Blood samples for measuring the plasma
concentration of triglycerides (GPO-PAD enzymatic method), cholesterol (CHOD-PAD enzymatic method) were collected from the
jugular vein in heparinized tubes before and after fasting. Metabolic variables were analyzed at the Universidad Católica de Temuco
using Metrolab 2300® plus Random Access Clinical Analyzer.

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2.3. Hormone assays

Blood samples (4 mL, heparinized tubes, jugular vein, 0800) were taken immediately before and after fasting to determine the
plasma concentration of leptin. Serial blood samples to evaluate plasma LH concentration were collected in heparinized tubes every
30 min for 6 h after ovulation induction treatment, for which a jugular catheter (inner and outer diameters of 1.0 and 1.5 mm,
respectively) was fixed in place 1 day before to minimize the effects of handling stress on hormone profiles. In addition, blood
samples were taken from the jugular vein in heparininized tubes (0800), every other day from Day 1 to 13 (Day 0 = Day of ovulation
induction treatment) to measure plasma concentrations of progesterone and leptin. All blood samples were centrifuged for 15 min at
1500 × g; plasma was stored at −20 °C and hormone assays were performed in the Laboratory of Veterinary Physiology and
Endocrinology of the University of Concepción, Chillán, Chile. Plasma LH concentration was determined using a double-antibody
radioimmunoassay as previously described (Recabarren et al., 1996). The LH assay was validated previously for LH determination in
llamas (Silva et al., 2011). Intra-assay coefficients of variation were 0.6% to 2.2%, and inter-assay were 1.2% to 3.1%. The minimum
detectable LH concentration, defined as 90% of buffer control, was 0.11 ng/mL. Plasma leptin concentration was determined using
the Sheep Leptin ELISA Kit® (MyBiosource, USA) which was validated previously. In brief, serially diluted plasma from alpacas
containing high concentrations of leptin produced a displacement curve parallel to the standard curve. Intra- and inter-assay
coefficients of variation were 1.9% to 3.7% and 0.9% to 3.1% respectively. The sensitivity of the assay was 0.1 ng/mL. Plasma
progesterone concentration was determined using a commercial solid-phase radioimmunoassay kit (Coat-A Count 17Alpha OH
Progesterone, Siemens, USA). The intra-assay coefficient of variation varied from 0.8% to 1.6%; the limit of sensitivity was 0.48 ng/
mL.

2.4. Statistical analysis

The Student's t test for independent samples was used to compare non-serial data (i.e., pre-ovulatory follicle diameter, maximum
CL diameter and maximum CL vascularization area, CL lifespan) and a Student's test for repeated measures was used to compare
leptin, BLW, BCS and plasma metabolite concentrations, before and after fasting. A mixed ANOVA with time points as repeated
measurements was used to compare serial data (BLW, LH, leptin and progesterone plasma concentration, CL diameter and
vascularization area). If significant main effects or interactions were detected (P < 0.05), ANOVA pairwise comparisons with
Bonferroni adjustment were used. Pearson's correlation was used to determine relationships between CL diameter, vascularization
area and plasma progesterone concentration. Statistical analyses were done using SPSS Program (V 15.0). Data are reported as
mean ± SEM.

3. Results

The BLW, BCS, plasma triglycerides, cholesterol and leptin concentrations before and after fasting did not differ between groups
(P > 0.05). Fasting for 72 h reduced BLW to about 6% (P < 0.05), and affected the Leptin and Control groups similarly (weight loss
of 3.2 ± 0.9 kg and 2.6 ± 0.5 kg respectively; P = 0.9). Fasting also increased the triglyceride plasma concentrations but did not
affect cholesterol, leptin and BCS in both groups (P > 0.05, Table 1). During the luteal phase, BLW did not differ between groups
(P = 0.5) or days (P = 0.2), and there was no interaction between these factors (P = 0.4).
Pre-ovulatory follicle diameter was similar in the Leptin and Control groups (10.5 ± 0.8 compared with 10.1 ± 0.6 mm
respectively; P = 0.7). All alpacas had ovulations after GnRH treatment. The CL diameter was different among days but not between
groups and there was no interaction between these factors (Fig. 1). Maximum CL diameter was also similar between Leptin and

Table 1
Body live weight (BLW), body condition score (BCS) and metabolic profile (mean ± SEM) before and after 72 h of fasting in alpacas of the Leptin
and Control groups.

Variable Fasting

Before After P

Leptin group
BLW (kg) 55.9 ± 2.5 52.7 ± 2.5 *
BCS 3.2 ± 0.2 3.1 ± 0.1
Triglycerides (mmol/L) 0.09 ± 0.01 0.16 ± 0.02 **
Cholesterol (mmol/L) 0.42 ± 0.07 0.59 ± 0.13
Leptin (ng/mL) 12.3 ± 3.5 12.3 ± 4.0

Control group
BLW (kg) 55.5 ± 2.7 52.9 ± 2.7 *
BCS 3.0 ± 0.1 3.0 ± 0.1
Triglycerides (mmol/L) 0.13 ± 0.02 0.22 ± 0.03 **
Cholesterol (mmol/L) 0.54 ± 0.08 0.7 ± 0.17
Leptin (ng/mL) 12.2 ± 3.0 10.8 ± 4.9

Values with this designation differed *P < 0.05; **P < 0.01.

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M.C. Norambuena et al. Animal Reproduction Science 182 (2017) 28–34

Fig. 1. Diameter of the Corpus luteum (mean ± SEM) of alpacas submitted to 72 h of pre-ovulatory fasting. Day 0 = GnRH treatment. The Leptin group (n = 7; —)
received five doses of leptin, while the control group (n = 7; – – –) received five doses of saline during fasting.

Control groups (13.7 ± 0.5 compared with 11.9 ± 0.6 mm respectively; P = 0.06). The CL vascularization was different among
days and between groups, and there was a significant interaction between these factors (Fig. 2). The Leptin group had greater CL
vascularization than the Control group on Days 3 (P = 0.01), 7 (P = 0.008) and 9 (P = 0.02; Fig. 2). The maximum vascularization
area of the CL was greater in the Leptin group than in the Control group (0.67 ± 0.09 compared with 0.35 ± 0.07 cm2 respectively;
P < 0.05). The CL lifespan was similar in both groups (12 ± 3.5 compared with 12 ± 1.6 d; P = 0.4). Plasma LH concentration
increased after GnRH treatment, but did not differ between groups and the interaction between factors was not significant (Fig. 3).
Plasma leptin concentration determined during the luteal phase did not differ among days or between groups and day by group
interaction was not significant (Fig. 4). Plasma progesterone concentration differed among days but not between groups and there
was no interaction (Fig. 4). The data for maximum and mean concentrations of the hormone profiles are given in Table 2.
Interestingly, a positive correlation was detected between CL vascularization area and CL diameter (r = 0.7, P < 0.001), as well as
between CL vascularization area and plasma progesterone concentration (r = 0.5, P < 0.001). Also, CL diameter was correlated
with plasma progesterone concentration (r = 0.4, P < 0.001).

4. Discussion

The administration of o-leptin during pre-ovulatory fasting did not affect the pre-ovulatory follicle and CL diameter, the pre-

Fig. 2. Vascularization of the Corpus luteum (mean ± SEM) of alpacas submitted to 72 h of pre-ovulatory fasting. Day 0 = GnRH treatment to induce ovulation. The
Leptin group (n = 7; —) received five doses of leptin, while the control group (n = 7; – – –) received five doses of saline during fasting. *P < 0.05.

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Fig. 3. Plasma LH concentration (mean ± SEM) after GnRH administration to induce ovulation. The Leptin group (n = 7; —) received five doses of leptin, while the
control group (n = 7; – – –) received five doses of saline during 72 h of fasting.

Fig. 4. Plasma concentrations of progesterone (A) and leptin (B) (mean ± SEM) after treatment with GnRH to induce ovulation. The Leptin group (n = 7; —)
received five doses of leptin, while the control group (n = 7; – – –) received five doses of saline during fasting.

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Table 2
Maximum and mean plasma concentrations (mean ± SEM) of LH and leptin immediately after GnRH treatment, and progesterone and leptin during Days 3
to 13 following GnRH treatment in alpacas.

Variable Maximum plasma concentration Mean plasma concentration

Leptin group
LH surge (ng/mL) 3.9 ± 0.5 2.1 ± 0.8
Progesterone (ng/mL) 3.9 ± 0.5 1.9 ± 0.4
Leptin luteal phase (ng/mL) 15.0 ± 2.5 12.3 ± 2.4

Control group
LH surge (ng/mL) 2.8 ± 1.1 1.8 ± 0.7
Progesterone (ng/mL) 3.5 ± 0.9 1.4 ± 0.2
Leptin luteal phase (ng/mL) 16.6 ± 4.4 14.2 ± 3.8

ovulatory LH surge, or the progesterone and leptin plasma concentrations, but induced a greater vascularization area of the CL.
Three days of fasting resulted in increased concentrations of triglycerides but did not affect the cholesterol, or leptin plasma
concentrations in either group. Lipolysis increases the availability of free fatty acids in blood which are precursors for the synthesis of
triglycerides in the liver of true ruminant species (Mazuri et al., 2009). In other studies in camelids, with a greater energetic
challenge, the plasma cholesterol concentration also was increased (Norambuena et al., 2013). The loss of BLW may have primarily
resulted from loss of gastrointestinal content which has been calculated at approximately 6 kg in alpacas (Cristofanelli et al., 2005).
Consistent with other studies where there was no nutritional restriction of alpacas there were similar values in the present study as
those for the Leptin and Control groups for pre-ovulatory follicle and CL diameter (Fernández-Baca et al., 1970; Vaughan, 2011), pre-
ovulatory LH surge (Bravo et al., 1991); progesterone (Summar, 1996; Kershaw-Young et al., 2012) and leptin plasma concentrations
(Enciso et al., 2007). A decrease, however, in leptin gene expression and plasma leptin concentrations has been described in
ruminants submitted to 2–3 days of fasting (Amstalden et al., 2000, 2002). In addition, Maciel et al. (2004) found that there was an
increase in the GnRH-induced release of LH in pre-pubertal heifers treated with o-leptin (12 h intervals, s.c.) during 72 h of fasting.
Also, o-leptin administration increases circulating LH in a dose dependent manner in 60 h fasted mature cows (Zieba et al., 2003).
Interestingly, o-leptin administration positively affected LH secretion in 60 h fasted cows, but control-fed animals were insensitive to
leptin treatment (Amstalden et al., 2002). Camelids have metabolic adaptations, such as a lesser metabolic rate than true ruminant
species and have a greater capacity to reduce metabolic rate during fasting (Wensvoort et al., 2001; Nielsen et al., 2014) which could
explain the absence of any effect on the leptin and LH plasma concentrations in the present study.
Leptin administration resulted in a significant increase in the vascularization area of the CL in the present study. In previous
studies there has also been reported an increase of the CL vascularization area in llamas subjected to different doses of Beta Nerve
Growth Factor (β-NGF), a luteotropic factor, but the values were less than those of the Leptin group in the present study (Fernández
et al., 2014; Ulloa-Leal et al., 2014). The vascularization area of the CL in the Control group of the present study and that of Ulloa-Leal
et al. (2014) were similar.
Growing follicles have active angiogenesis after the formation of the theca layer that promotes the permeability of blood vessels,
follicle growth, ovulation and the formation of the developing CL (Acosta and Miyamoto, 2004; Bruno et al., 2009). It is possible that
o-leptin may stimulate the blood vessels of the ovary directly without affecting the hypothalamic–hypophyseal–gonadal axis.
Expression of the main angiogenic factor genes occurs in the granulosa, theca and endothelial cells of the ovary (Redmer et al., 2001;
Shimizu et al., 2002). The expression of leptin or its receptor genes in these same cells has also been reported (Archanco et al., 2003;
Barkan et al., 2005; Muñoz-Gutierrez et al., 2005; Sarkar et al., 2010) and this would support a direct (autocrine/paracrine) effect of
these molecules at the ovary. Treatment of human hepatic cells with leptin caused an increase of VEGF protein and messenger RNA
(Allefi et al., 2005). Wiles et al. (2014) determined that the addition of leptin stimulated the expression of the angiogenic factor genes
(Ang1, FGF2 and VEGF) in a culture of luteal cells from the early developmental stage CL of goats.
The leptin plasma concentration was similar in the Leptin and Control groups before and after fasting and during the luteal phase.
In the research of Maciel et al. (2004), the heifers which received o-leptin had as much as six times greater blood hormone
concentrations on treatment days than the group without treatment. The differences observed between the present and previous
studies may be due to the leptin dose of the present study being 10 times less (19.2 compared with 2 μg/kg), differences in the
techniques used in hormone measurement (RIA compared with ELISA), or to metabolic differences between the species.
The progesterone values were similar between groups in the present study. The positive correlations obtained between CL
vascularization, CL diameter, and progesterone plasma concentration, however, suggest that further studies with a larger number of
animals are needed to enhance the understanding of the role of leptin in luteal function in alpacas.
In summary, the administration of leptin during 72 h of pre-ovulatory fasting increased the vascularization area of the CL without
affecting the pre-ovulatory follicle and CL diameter, preovulatory LH surge release, or the progesterone and leptin plasma
concentrations.

Conflict of interest

The authors do not have any conflict of interest.

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Acknowledgements

This study was supported by: Fondecyt/Initiation grant No. 11130206 (C Norambuena), Government of Chile.

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