BIOLOGY OF REPRODUCTION 85, 452–456 (2011)
Published online before print 18 May 2011.
DOI 10.1095/biolreprod.111.091876
Dose-Response of Female Llamas to Ovulation-Inducing Factor from Seminal Plasma1
Valeria M. Tanco, Marcelo H. Ratto, Maura Lazzarotto, and Gregg P. Adams2
Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
The present study was designed to determine if the dose of
purified ovulation-inducing factor (OIF) from llama seminal
plasma required to provoke an ovulatory response is physiologically relevant in terms of the proportion present in a normal
ejaculate and to test the hypothesis that corpus luteum (CL) form
and function are affected by OIF in a dose-dependent manner.
Female llamas were assigned randomly to five groups (n ¼ 10 per
group) and given a single i.m. dose of 500, 250, 125, or 60 lg of
purified OIF (representative of the amount present in 1/25th to
1/200th of a normal ejaculate) or 1 ml of PBS (control).
Ovulation and CL development were monitored by transrectal
ultrasonography. Blood samples were taken to measure plasma
progesterone concentrations and to determine changes in
plasma concentrations of luteinizing hormone (LH). The high
dose of OIF (500 lg) was associated with the highest incidence
of ovulation (P , 0.05), the greatest maximum CL diameter (P ,
0.05), and the largest day-to-day profiles of CL diameter (P ,
0.05) and plasma progesterone concentrations (P , 0.01). A rise
in plasma LH concentration was apparent in all llamas that
ovulated and was most rapid and highest in the high-dose group
(P , 0.01). The low dose of OIF (60 lg) was minimally effective
for induction of ovulation and the least luteotrophic, as
evidenced by the smallest maximum CL diameter and the
smallest day-to-day profiles for CL diameter and plasma
concentrations of progesterone and LH. Responses were
intermediate for the middle-dose groups (125 and 250 lg). We
conclude that OIF from llama seminal plasma has a dosedependent effect on ovulation rate and CL form and function in
llamas and that the biological effect of OIF is evident at
physiologically relevant doses (i.e., as little as 1/100th of that
present in an ejaculate).
corpus luteum, gonadotropins, ovary, ovulation, seminal plasma
INTRODUCTION
Ovulation is a process preceded by an elevation in
circulating plasma luteinizing hormone (LH) concentrations
[1] that culminates with the rupture and evacuation of the
contents of the dominant follicle [2]. Mammalian species have
been classified as either induced ovulators or spontaneous
ovulators [3]. In spontaneous ovulators, the preovulatory LH
surge is triggered by a positive-feedback effect of rising
circulating concentrations of estradiol, secreted mainly by the
dominant follicle, in the absence of progesterone [1, 4]. In
1
Supported by grants from the Natural Sciences and Engineering
Research Council of Canada, the Alpaca Research Foundation, and
donations from members of the Canadian Llama and Alpaca
Association.
2
Correspondence: FAX: 306 966 7405; e-mail: [email protected]
MATERIALS AND METHODS
Seminal Plasma Collection
Semen was collected from four mature male llamas (age, 5–7 yr) two or
three times a week over a period of 2 mo using an artificial vagina inserted into
a wooden phantom [18]. A total of 72 ejaculates were obtained (n ¼ 18 per
male). Semen samples were processed as previously described [9]. Briefly,
ejaculates were diluted 1:1 (v/v) with PBS (pH 7.4; Invitrogen), drawn backand-forth through an 18-guage needle attached to a 10-ml syringe, and
centrifuged at 1500 3 g for 30 min to separate plasma from spermatozoa. A
drop of the supernatant was then evaluated by microscopy to confirm the
Received: 19 February 2011.
First decision: 28 March 2011.
Accepted: 2 May 2011.
Ó 2011 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
452
Downloaded from www.biolreprod.org.
contrast, the trigger for eliciting the preovulatory LH surge in
induced ovulators, such as camelids, has been attributed
primarily to a neuroendocrine response to the physical
stimulation of the genitalia during copulation or other physical
stimulation, such as olfactory, auditory, or visual factors [5–7].
However, the authors of an early study on Bactrian camels,
also considered induced ovulators, suggested the presence of
an ovulation-inducing factor (OIF) in the semen of this species
[8]. More recently, the presence of an OIF in the seminal
plasma of llamas and alpacas was convincingly documented by
the observation that ovulation occurred in more than 90% of
females treated intramuscularly with seminal plasma [9, 10].
Ovulation in seminal plasma-treated animals was consistently
preceded by a rise in plasma LH concentrations. The
magnitude of the LH response was greater in seminal
plasma-treated animals than in gonadotropin-releasing hormone (GnRH)-treated animals [9] and was associated with an
increase in corpus luteum (CL) diameter and plasma
progesterone concentration, suggesting a powerful luteotrophic
effect. Results of a study comparing routes of administration
were consistent with a systemic rather than a local mode of
action for OIF [10]. More females ovulated after i.m. treatment
than after intrauterine deposition of seminal plasma (93% vs.
41%), and the ovulation rate was intermediate (67%) for
animals treated by intrauterine deposition followed by
endometrial curettage [10]. This finding is also consistent with
the results of a study in which alpaca seminal plasma
stimulated secretion of LH from rat pituitary cells in vitro [11].
The biologically active fraction of llama and alpaca seminal
plasma was identified recently as a robust protein that is
resistant to enzymatic digestion with proteinase K and
incubation at 388C for 12 h or 658C for 1 h. Only enzymatic
digestion with pronase E abolished the ovulation-inducing
activity of llama seminal plasma [12]. Using a two-step
combination of hydroxylapatite and gel filtration chromatography, OIF was isolated from llama seminal plasma as a 14kDa protein molecule [13]. Previous studies of OIF [8–11, 14–
17] have involved treatment with whole seminal plasma;
however, purified OIF was used in the present study.
The present study aimed to determine if the minimum dose
of purified OIF required to provoke an ovulatory response is
physiologically relevant in terms of the proportion present in a
normal ejaculate. The present study also tested the hypothesis
that CL form and function are affected by OIF in a dosedependent manner.
ABSTRACT
453
DOSE-DEPENDENT EFFECT OF OVULATION-INDUCING FACTOR
absence of cells. If spermatozoa were detected, the sample was centrifuged and
evaluated again as described above until no spermatozoa were detected. The
average ejaculatory volume was 2 ml; therefore, the diluted volume per sample
was, on average, 4 ml. Diluted, sperm-free samples were stored at 708C until
further purification.
Protein Purification
Animals and Treatments
Mature, nonlactating female llamas (n ¼ 10 llamas/group; age, 4 yr;
weight, 90–120 kg) were selected from a herd of 53 llamas during July and
August at the University of Saskatchewan, Canada (528N, 1068W; 500 m above
sea level). Llamas were examined daily by transrectal ultrasonography using a
7.5-MHz linear-array transducer (Aloka SSD900) [20], and when a growing
follicle (diameter increase for three consecutive days) of 7 mm or greater was
detected [21], they were assigned randomly to one of four treatment groups (n ¼
10 llamas/group). Llamas were given a single i.m. injection (semimembranosus
or semitendinosus muscle) of 1 ml PBS (negative control) or 500, 250, or 125
lg of purified OIF in 1 ml of PBS. Doses were based on the results of previous
studies in which a relatively conservative i.m. dose of 1–2 ml of diluted (1:1, v/
v), unpurified seminal plasma (i.e., one quarter to one half of an ejaculate based
on an average volume of 2 ml/ejaculate [18]) resulted in 90%–100% ovulation
in female llamas [9, 10]. The mean total protein in llama seminal plasma was
12.4 mg/ml, and OIF constituted approximately 50% of total protein [13].
Hence, the doses used in previous studies are equivalent to 3–6 mg of OIF. The
doses in the present study, therefore, represent 1/100th (125 lg), 1/50th (250
lg), and 1/25th (500 lg) of the amount of OIF normally present in a single
ejaculate.
The experiment was conducted in two replicates (first replicate, n ¼ 6 per
group; second replicate, n ¼ 4 per group). The second replicate involved
frequent blood sampling to determine the endogenous LH response to
treatment. To facilitate data collection in the second replicate, follicular wave
emergence was synchronized among animals by giving a single i.m. dose
of porcine luteinizing hormone (pLH; 5 mg Armour standard; Lutropin-V;
Bioniche Animal Health) [14] to all animals on the same day. Llamas were reexamined 12 days later—enough time to allow CL regression in those that
ovulated in response to pLH treatment and for all to have developed a new
mature dominant follicle. Llamas with a follicle 7 mm or larger and no CL were
assigned randomly to the respective treatment groups. Based on the
unexpectedly high ovulatory response to all doses of OIF in the first replicate,
an additional group was included in the second replicate (n ¼ 10) and treated
Statistical Analyses
Single-point measurements (i.e., follicle size at the time of treatment, day of
appearance of the CL, maximum CL diameter, and CL diameter on Days 8 and
14) were compared by two-way ANOVA to determine the effects of treatment
and replicate. Serial data (i.e., plasma progesterone and LH concentrations and
CL diameter profile) were compared among groups by two-way ANOVA for
repeated measures to determine the effects of treatment, replicate, and their
interaction with time (SAS software; SAS Institute, Inc.). In the absence of a
replicate effect, data from the two replicates were combined. Tukey multiple
comparison was used as a post-hoc test when a main effect of treatment or a
treatment interaction was detected. Ovulation rates were compared among
groups by chi-square analysis. Significance was assumed when the probability
of values differing by chance alone was 0.05 or less. Data were centralized to
the day of treatment (Day 0) for preparation of figures.
RESULTS
The diameter of the preovulatory follicle at the time of
treatment did not differ among groups (P ¼ 0.3) (Table 1). The
proportion of llamas that ovulated was lower (P , 0.05) in the
control and low-dose (60-lg) groups compared to the higherdose groups (Table 1).
TABLE 1. Effect of dose of OIF on ovulation in llamas (mean 6 SEM).
Group
Follicle diameter on day
of treatment (mm)*
Proportion that ovulated
Saline
60 lg
125 lg
250 lg
500 lg
10.6 6 0.6
10.6 6 0.3
9.0 6 0.2
9.1 6 0.2
10.5 6 0.1
0/10a (0%)
3/10a (30%)
7/10b (70%)
9/10b (90%)
9/10b (90%)
* No significant difference among groups (P ¼ 0.3).
a,b
Proportions with different superscripts are different (P , 0.05).
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Upon thawing, seminal plasma samples were pooled and purified in 12
elution replicates, each with approximately 12 ml of seminal plasma, as
previously described [13]. In brief, after sonication and centrifugation, the
seminal plasma was loaded onto a type 1 macroprep ceramic hydroxylapatite
column (20 lm; Bio-Rad Laboratories) that was previously equilibrated with
10 mM sodium phosphate (pH 6.8). Approximately 24–30 mg of total protein
were loaded onto the column (1 3 10 cm) for each replicate, and elution was
done at room temperature with a flow rate of 0.5 ml/min using a linear gradient
with 350 mM sodium phosphate (pH 6.8). Fractions were collected, and their
absorbance was measured at 280 nm. Fraction C [13] was concentrated and
buffer exchanged in PBS (pH 7.4) using a 20-ml ultrafilter device with a
membrane cutoff of 5 kDa (Vivaspin; Vivascience/Sartorius). Further
purification was done by loading fraction C (;6–8 mg/replicate) onto a 320ml size-exclusion column (Hi Prep 26/60 Sepahacryl S-100; GE Health
Sciences) attached to an AKTA fast protein liquid chromatography system
(Amersham Biosciences, Inc.). Samples were eluted isocratically using PBS
(pH 7.4) at a flow rate of 0.5 ml/min, and elution volumes corresponding to
fraction C2 (purified OIF [13]) were concentrated as described above. Samples
of fraction C2 were stored at 708C and, upon thawing, were diluted with PBS
to prepare doses of 500, 250, 125, and 60 lg/ml of total protein (purified OIF).
Protein concentration was determined using the microassay method (Protein
Assay; Bio-Rad) based on the method first described by Bradford [19].
with a lower dose (60 lg) of purified OIF representative of approximately 1/
200th of the total amount of OIF present in a single ejaculate. Llamas were
examined daily by transrectal ultrasonography until Day 4 (Day 0 ¼ day of
treatment) to detect ovulation and every other day thereafter until Day 16 to
monitor CL form and function [22]. Ovulation was defined as the
disappearance of a large follicle (8 mm) from one examination to the next
and was confirmed by the detection of a CL in subsequent examinations [20].
The onset of CL regression was defined as the first day the CL began a
progressive decrease in diameter [9].
For measurement of plasma progesterone concentration in both replicates,
blood samples were collected from the jugular vein into heparinized tubes
(Vacutainer Systems; Becton Dickinson) every other day starting from the day
of treatment. Plasma progesterone concentration was measured using a
commercially available double-antibody radioimmunoassay kit (Coat-A-Count
Total Progesterone; Diagnostic Products Corporation) [22]. Samples were
analyzed in two consecutive assays with intra-assay coefficients of variation of
4.5%, 3.2%, and 8.8% for reference concentrations of 1.9, 3.6, and 16.6 ng/ml,
respectively, for the first assay and of 6.8%, 2.0%, and 1.7% for reference
concentrations of 1.6, 3.2, and 16.0 ng/ml, respectively, for the second assay.
Interassay coefficients of variations were 9.2%, 6.5%, and 6.9% for reference
concentrations of 1.78, 3.49, and 16.36 ng/ml, respectively.
More frequent blood samples were collected from four animals per group in
the second replicate for measurement of plasma LH concentration [9]. Samples
were taken immediately before treatment (Hour 0), every 15 min until Hour 2,
and again at Hours 3, 4, 5, 6, 7, 8, and 12. A catheter was placed in the jugular
vein on the day before treatment to minimize the effect of stress at the time of
sampling. All samples were centrifuged within 2 h of collection at 1700 3 g for
20 min, and plasma was stored at 208C. Plasma LH concentrations were
measured using a double-antibody radioimmunoassay [9] and are expressed in
terms of NIAMDDK-oLH-24. The minimum detectable limit of the assay was
0.1 ng. The range of the standard curve was 0.06 ng (80% ligand-labeled LH)
to 8.0 ng (20% ligand-labeled LH). The intra- and interassay coefficients of
variation for the low reference plasma LH concentration (0.13 ng/ml) were
10.3% and 8.3%, respectively. The intra- and interassay coefficients of
variation for the high reference plasma LH concentration (0.99 ng/ml) were
11.1% and 10.0%, respectively.
The experimental protocol was approved by the University of Saskatchewan Committee on Animal Care and Supply in accordance with the guidelines
of the Canadian Council on Animal Care.
454
TANCO ET AL.
TABLE 2. Effect of dose of OIF on CL development in llamas that ovulated in response to treatment (mean 6 SEM).
End point*
Day of 1st detection of CL
Maximum CL diameter (mm)
CL diameter on Day 8 (mm)
CL diameter on Day 14 (mm) 60 lg (n ¼ 3)
3.3
10.9
8.5
7.8
6
6
6
6
0.3a
1.0a
2.0a
0.9
125 lg (n ¼ 7)
2.3
11.6
11.3
7.5
6
6
6
6
0.2b
0.7ab
0.8ab
1.2
250 lg (n ¼ 9)
2.5
10.8
10.4
7.2
6
6
6
6
0.2b
0.7a
0.7ab
0.3
500 lg (n ¼ 9)
2.1
13.5
12.8
8.3
6
6
6
6
0.1b
0.5b
0.6b
0.6
* Day 0 = day of treatment.
No significant difference among groups.
a,b
Values with different superscripts are different (P , 0.05).
In those llamas that ovulated, the CL was detected earliest
(P , 0.05) and reached the greatest diameter (P , 0.05) in the
high-dose (500-lg) group (Table 2). Conversely, CL detection
was latest (P , 0.05) and diameter was smallest (P , 0.05) in
the low-dose (60-lg) group. By Day 14 after treatment, the
diameter of the regressing CL did not differ among groups
DISCUSSION
FIG. 1. CL diameter (a) and plasma progesterone concentrations (b and
c) in llamas given a single i.m. dose of OIF (60 lg, *; 125 lg, &; 250 lg, m;
500 lg, &) or PBS (*; n ¼ 11 per group). In c, data from llamas that did not
ovulate were excluded (i.e., n ¼ 3, 7, 9, and 9 in groups treated with 60,
125, 250, or 500 lg, respectively, of OIF). Data are presented as the mean
6 SEM.
Ovulation-inducing factor is a proteinase K-resistant,
pronase-sensitive protein constituent of the seminal plasma of
llamas and alpacas [12] responsible for eliciting ovulation in
these species [9]. Using a combination of hydroxylapatite and
gel-filtration chromatography, purified OIF was isolated as a
protein with a molecular mass of 14 kDa [13] but with
bioactive forms at both 14 kDa and approximately 30 kDa [13].
The purified protein elicited a preovulatory LH surge followed
by ovulation and CL formation in llamas after i.m. administration [13]. Compared to animals treated with GnRH, the OIFinduced preovulatory surge of LH lasted longer, and
subsequent CL development was larger and plasma progesterone concentrations twice as high [9].
Results of the present study support the hypothesis that OIF
exerts a dose-dependent effect on ovulation and CL form and
function. Four different doses were chosen based on the
estimate that an average ejaculate of 2 ml [18] contains
approximately 3–6 mg of OIF [13]. Thus, the maximum dose
in this experiment (500 lg) represents 1/25th of what is
normally present in an ejaculate, and because an intermediate
dose (125 lg) still induced ovulations in 70% of the females in
the first replicate of the present study, a low dose representative
of approximately 1/200th of the normal amount present in a
single ejaculate was added in the second replicate. The dose
effect was manifest by higher ovulation rates (90% in the highdose group, P , 0.05), earlier appearance of the CL, a larger
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(Table 2). The day-to-day CL diameter profile was greatest in
the 500-lg group, intermediate for the 250- and 125-lg groups,
and lowest for the 60-lg group (day effect, P , 0.01; group
effect, P , 0.05; day 3 group interaction, P , 0.01) (Fig. 1).
Plasma progesterone concentrations in those llamas that
ovulated were highest in the high-dose group (day effect, P ,
0.01; group effect, P ¼ 0.03; day 3 group interaction, P ¼ 0.15)
(Fig. 1). This effect was more evident when all animals,
regardless of whether they ovulated, were taken into consideration (day effect, P , 0.01; group effect, P , 0.01; day 3
group interaction, P , 0.01) (Fig. 1).
A rise in plasma LH concentration was apparent in all
llamas that ovulated and was absent in all that failed to ovulate.
Data from all animals were included in the analysis of the dayto-day LH profile (Fig. 2) except for one llama from each of the
60-, 250-, and 500-lg groups in which plasma concentrations
of LH, averaged over all days, was more than 14 SD above the
average for the group (outliers). Despite the high LH values,
ovulation was not detected in these individuals. Over the 12-h
sampling period, a time 3 group interaction in plasma LH
concentrations (P , 0.01) was the result of a more rapid
increase in progressively higher-dose groups and a greater and
more sustained elevation in the high-dose group than in all
other groups. The magnitude of the LH profile decreased in a
dose-related manner.
DOSE-DEPENDENT EFFECT OF OVULATION-INDUCING FACTOR
455
FIG. 2. Plasma LH concentrations (mean
6 SEM) in female llamas that ovulated
following treatment with OIF (500 lg, &;
250 lg, m; 125 lg, &; 60 lg, *; PBS, *).
a
Within group, the maximum concentration
(P , 0.05); bwithin group, the first increase
(P , 0.05); cwithin group, the first decrease
from maximum (P , 0.05); k within group,
the last value higher than pretreatment
levels (P , 0.05).
incidence of ovulation, 2) larger CL diameters, and 3) higher
plasma progesterone concentrations in animals treated with a
higher dose (500 lg) of purified OIF compared with lower
doses. Results document that OIF exerts its effects at
physiologically relevant doses; the minimum effective dose
used in the present study was 125 lg of purified OIF,
equivalent to that present in approximately 1/100th of a normal
ejaculate.
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