Administration of testosterone from day 13 of the estrous cycle to estrus increased the number of corpora lutea and conceptus survival in gilts.

Abstract

The effects of exogenous androgens on the number of corporea lutea (CL) and conceptus survival were examined in crossbred gilts. In Exp. 1, gilts received 1 mg of testosterone per day from d 13 (d = 0 first day of estrus, n = 21) or d 16 until estrus (n = 23). Gilts in the vehicle group received corn oil (n = 20). Gilts were mated and on d 11.5 their concepti and CL were evaluated. In Exp. 2, conceptus survival was examined at the 4- to 8-cell, early blastocyst or hatching blastocyst stages for gilts given vehicle or 1 mg testosterone from d 13 (24 gilts per group). In Exp. 3, gilts received 1 mg of androstenedione (n = 20) or vehicle (n = 18) per day from 13 d to estrus and then were mated and evaluated on d 11.5. Results from Exp. 1 indicated that the number of CL was greater (P < .04) in gilts treated with testosterone from d 13 to estrus than in gilts receiving vehicle (16.4 vs 14.8, respectively). Similarly, the number (P < .01) and recovery rate (P < .04) of blastocysts were greater in gilts treated with testosterone from d 13 to estrus than in gilts treated with testosterone from d 16 to estrus in gilts receiving vehicle (number, 15.3 vs 12.8 or 12.8; recovery rate, 95 vs 87 or 86%, respectively). Gilts treated testosterone or vehicle did not exhibit differences (P > .05) in number of normal concepti at the 4- to 8-cell and hatching stages. However, prior treatment with testosterone delayed conceptus death; gilts treated with testosterone had more (P < .01) normal concepti at the intermediate stage (early blastocyst) than those treated with vehicle (treatment x embryo stage interaction, P < .05). In Exp. 3, androstenedione treatment did not influence (P > .10) the number of CL or the number and recovery rates of d-11.5 blastocysts. Treating gilts with testosterone from d 13 of the estrous cycle to the following estrus increased the number of CL and blastocyst survival, perhaps by improving some, as yet unknown, aspect(s) of oocyte quality.

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H. Cardenas and W. F. Pope
number of corpora lutea and conceptus survival in gilts
Administration of testosterone from day 13 of the estrous cycle to estrus increased the
1997, 75:202-207.
J ANIM SCI
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202
1Salaries and research support were provided by Hatch Project
814 and state and federal funds appropriate to The Ohio State
University, Columbus, OH 43210. Manuscript no 146−94.
2Present address: Departamento de Produccio Ân Animal, Univer-
sidad Nacional Agraria. Apartado 456, Lima, Peru Â.
3To whom correspondence should be addressed: 2029 Fyffe Road.
Received April 29, 1996.
Accepted September 4, 1996.
Administration of Testosterone from Day 13 of the Estrous Cycle
to Estrus Increased the Number of Corpora Lutea
and Conceptus Survival in Gilts1
Horacio Ca Ârdenas2and William F . Pope3
Department of Animal Sciences, The Ohio State University, Columbus 43210
ABST R ACT :
the number of corpora lutea (C L ) and conceptus
survival were examined in crossbred gilts. In Exp. 1,
gilts received 1 mg of testosterone per day from d 13
(d 0 = first day of estrus, n = 21) or d 16 until estrus
(n = 23). Gilts in the vehicle group received corn oil
(n = 20). Gilts were mated and on d 11.5 their
concepti and CL were evaluated. In Exp. 2, conceptus
survival was examined at the 4- to 8-cell, early
blastocyst or hatching blastocyst stages for gilts given
vehicle or 1 mg testosterone from d 13 (24 gilts per
group). In Exp. 3, gilts received 1 mg of androstenedi-
one (n = 20) or vehicle (n = 18) per day from d 13 to
estrus and then were mated and evaluated on d 11.5.
Results from Exp. 1 indicated that the number of CL
was greater ( P < .04) in gilts treated with testoster-
one from d 13 to estrus than in gilts receiving vehicle
(16.4 vs 14.8, respectively). Similarly, the number ( P
< .01) and recovery rate ( P < .04) of blastocysts were
The effects of exogenous androgens on
greater in gilts treated with testosterone from d 13 to
estrus than in gilts treated with testosterone from d
16 to estrus or in gilts receiving vehicle (number, 15.3
vs 12.8 or 12.8; recovery rate, 95 vs 87 or 86%,
respectively). Gilts treated with testosterone or vehi-
cle did not exhibit differences ( P > .05) in number of
normal concepti at the 4- to 8-cell and hatching stages.
However, prior treatment with testosterone delayed
conceptus death; gilts treated with testosterone had
more ( P < .01) normal concepti at the intermediate
stage (early blastocyst) than those treated with
vehicle (treatment × embryo stage interaction, P <
.05). In Exp. 3, androstenedione treatment did not
influence ( P > .10) the number of CL or the number
and recovery rates of d-11.5 blastocysts. Treating gilts
with testosterone from d 13 of the estrous cycle to the
following estrus increased the number of CL and
blastocyst survival, perhaps by improving some, as yet
unknown, aspect(s) of oocyte quality.
Key Words: Pigs, Testosterone, Ovulation Rate, Blastocyst
J . Anim. Sci. 1997. 75:202±207
Introduction
Administration of testosterone to gilts on d 17 and
18 of the estrous cycle increased ovulation rate and
the number of d-11 concepti (Ca Ârdenas and Pope,
1994) and could, theoretically, increase litter size.
Exogenous testosterone also increased plasma concen-
trations of estradiol in gilts (Ca Ârdenas and Pope,
1994) and cows (K otwica and Williams, 1982). These
observations suggested that the effects of testosterone
on increasing ovulation rate in gilts might be medi-
ated through the actions of estradiol, because the
health and development of follicles are usually as-
sociated with amounts of estradiol within the antral
fluid (Peters and McNatty, 1980; Hsueh et al., 1984).
If the effects of testosterone are mediated by estradiol,
then perhaps androstenedione, the other substrate
utilized by pig follicles for the synthesis of estradiol
(Anderson et al., 1979; Schomberg, 1979; Evans et al.,
1981), might produce similar effects to those observed
with testosterone.
Stimulation of follicular
ogenous androgens might, in turn, augment oocyte
maturation and improve the proportion of concepti
surviving during early gestation. Various investiga-
tors have hypothesized that the degree of follicular
maturation and oocyte development is related to
conceptus survival (Pope et al., 1988; Hunter and
Wiesak, 1990). For example, Xie et al. (1990)
observed that the diversity of follicular development,
in turn, influenced the diversity among littermate
concepti.
developmentwith ex-
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EXOGENOUS ANDROGENS AND SWINE REPRODUCTION
203
Some early relationships between the dose of
testosterone and number of corpora lutea ( CL ) or
conceptus survival have been described for swine
(Ca Ârdenas and Pope, 1994). In the present experi-
ments the effects of different durations of testosterone
treatment and the effects of androstenedione on
number of CL, conceptus survival, and diversity
among littermate concepti were examined in multies-
trous gilts.
Materials and Methods
Crossbred gilts ( Ø Duroc × Ô Landrace × Ô
Yorkshire, 6 to 8 mo of age, 110 to 130 kg of body
weight initially) were observed at approximately
12-h intervals for estrous behavior in the presence of
intact boars. Gilts that exhibited at least one period of
estrus were used in the following experiments.
Experiment 1. Gilts were allocated to receive daily
intramuscularinjections of one of the following
treatments: 1) vehicle (corn oil) from d 13 of the
estrous cycle (d 0 = first day of estrus) until the first
day of the next estrus (n
testosterone (Sigma Chemical, St. Louis, MO) dis-
solved in corn oil from d 13 to estrus, as above (n =
21), or 3) vehicle on d 13, 14, and 15 and then 1 mg of
testosterone from d 16 of the estrous cycle to estrus (n
= 23). Gilts that displayed estrus 12 h after the last
injection of vehicle or testosterone did not receive
additional injections.
Gilts within each treatment group were assigned to
receive testosterone dissolved in 1 or 7 mL of corn oil.
The 7-mL volume of vehicle was used in previous
experiments to facilitate dissolving of larger amounts
of testosterone (Ca Ârdenas and Pope, 1994). Charac-
teristics evaluated were not altered by volume of
vehicle; therefore, data were pooled for the final
statistical analyses. Furthermore, in a small trial
performed simultaneously using ovariectomized gilts,
plasma concentrations of testosterone at 0, 1, 2, 4, 6, 8,
12, 16, and 24 h after administration of 1 mg of
testosterone were not altered ( P = .30) by volume (1
or 7 mL, n = 5 gilts/group) of vehicle or the interaction
of volume of vehicle with time ( P = .77).
In order to decrease variability due to duration of
the estrous cycle, 7 out of 64 gilts (3 and 4 gilts that
received testosterone beginning on d 13 or d 16,
respectively) were excluded from the experiment
because their estrous cycles did not fall within the
range of 18.0 to 22.0 d. For gilts that remained in the
experiment, duration of the estrous cycle of gilts did
not differ ( P = .32). Estrous cycle durations in vehicle
gilts and gilts treated with testosterone beginning on
d 13 or d 16 were 19.4 ± .2, 19.3 ± .2, and 19.0 ± .2 d,
respectively.
Gilts were mated to boars at 12, 24, and 36 h after
onset of estrus and were subsequently ovariohysterec-
tomized on d 11.5. Corpora lutea were counted,
= 20), 2) 1 mg of
dissected from the ovaries, and weighed. Blastocysts
were recovered by flushing the uterus with physiologi-
cal saline (.9% NaCl) within 10 to 15 min after
surgery. Before flushing, the mesometrium was sepa-
rated from the uterus and the tip of each uterine horn
was cut approximately .5 cm from the uterine-tubal
junction. The uterus was flushed twice by infusing 50
mL (total of 100 mL ) of saline into the tip of one horn
and recovering the flushing through the tip of the
opposite uterine horn. Concepti were examined using
a stereomicroscope and then were measured to the
nearest millimeter at their largest diameter. Number
of filamentous blastocysts entangled during flushing
were estimated based on the number of trophoblastic
ends.Morphologicaldiversity
blastocysts was estimated by calculating the standard
deviation of blastocyst diameter.
Experiment 2. This experiment examined conceptus
survival at earlier stages of development than those in
Exp. 1. Gilts (n = 24 per group) were administered 1
mg of testosterone or vehicle from d 13 of the estrous
cycle until estrus and then mated as described in Exp.
1. This treatment was selected because it increased
the number of CL and recovery rates of blastocysts in
Exp. 1. Corpora lutea and concepti were evaluated
after ovariohysterectomies on d 4.0, 5.5, or 7.0.
Concepti were recovered by flushing each oviduct and
uterine horn three times with 25 mL of modified
Tyrode's medium buffered with Hepes (Hagen et al.,
1991). This medium was used instead of saline
because it took longer to search and recover the
concepti at these earlier stages of development than at
the elongating stage (d 11.5).
Morphological development of concepti was deter-
mined by microscopic examination at magnifications
up to 100×, and then concepti were classified as
normal or degenerating. Concepti, which were de-
veloping normally, were at the 4- to 8-cell, early
blastocyst and hatching blastocyst stages on d 4.0, 5.5,
and 7.0, respectively. The early blastocyst stage
included the period of blastocoele formation, whereas
the hatching stage comprised expanding and hatched
blastocysts.
Experiment 3. Gilts were administered 1 mg of
androstenedione (Sigma Chemical, n = 20) or vehicle
(n = 18) per day from d 13 of the estrous cycle to the
first day of their next estrus. Gilts were mated and
concepti were recovered on d 11.5 as described in Exp.
1.
Statistical Analyses. Data from Exp. 1 and Exp. 3
were analyzed by one-way ANOVA. Seven gilts, with
some or all filamentous blastocysts, that were un-
measurable were not included in the analysis of
conceptus diameter in Exp. 1. Recovery rate of
concepti was calculated by dividing the number of
concepti by the number of CL and multiplying by 100.
Because of the possibility that gilts from which no
concepti were recovered had total fertilization failure,
they (one gilt per group in Exp. 1 and 3) were not
among littermate
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CARDENAS AND POPE
204
Table 1. Number and weight of corpora lutea (CL) in
vehicle- and testosterone-treated giltsa
aMean ± SE.
bCorpora lutea were collected in Exp. 1. Corpora lutea from two gilts were accidentally lost.
c,dMeans within a column lacking a common superscript differ (P < .05).
Mean no. of CL Mean CL
weight (mg)
per gilt (n)b
Treatment Exp. 1 (n)Exp. 2 (n)
Vehicle
Day 13
Day 16
14.8 ± .5c
16.2 ± .5d
15.0 ± .5c,d
(20)
(18)
(19)
13.3 ± .4c
14.9 ± .4d
(24)
(24)
501.8 ± 25.0 (20)
471.6 ± 27.1 (17)
452.2 ± 26.3 (18)
Table 2. Characteristics of day 11.5 concepti from
vehicle- and testosterone-treated giltsa
aMean ± SE.
bEffect of treatment group ( P = .10).
cGilts having unmeasurable filamentous blastocysts were not included (2, 1, and 4 for vehicle, d 13,
and d 16, respectively). In each group one additional gilt had no concepti.
d,eMeans within a column lacking a common superscript differ (P < .05).
Percentage of gilts
having 100%
recovery rateb(n)
Mean conceptus
diameter per
litter, mmc(n)
No. ofRecovery rate
of concepti (n) Treatmentconcepti (n)
Vehicle
Day 13
Day 16
12.8 ± .5d(19)
15.3 ± .6e
12.8 ± .6d(18)
86.6 ± 2.7d
94.8 ± 2.8e
85.8 ± 2.7d
(19)
(17)
(18)
21.1 ± (19)
47.1 ± (17)
16.7 ± (18)
6.4 ± .7 (17)
7.2 ± .7 (16)
5.1 ± .8 (14)
(17)
included in the calculations of recovery rates. Propor-
tions of gilts from which concepti were recovered
(pregnancy rates) and proportions of gilts having
100% recovery rate of concepti were analyzed using
chi-square tests of independence. Experiment 2 was a
factorial (two doses of testosterone by three stages of
conceptus development), and data regarding CL and
concepti were analyzed accordingly by ANOVA proce-
dures. As expected, the number of CL in Exp. 2 was
not influenced by day (stage of conceptus develop-
ment) and was, therefore, pooled across days and then
reanalyzed byone-wayANOVA.
means in Exp. 1 and 2 were performed using LSD
tests. All analyses were performed using the statisti-
cal program SYSTAT (Wilkinson, 1990).
Comparisons of
Results
Experiment 1. Gilts treated with testosterone from d
13 to estrus had greater ( P < .04) numbers of CL than
gilts treated with vehicle and tended ( P = .07) to have
more CL than gilts treated with testosterone from d 16
to estrus. Number of CL
testosterone from d 16 to estrus and those treated
with vehicle were not different ( P = .77, Table 1).
The number of concepti ( P < .01) and percentage of
concepti recovered ( P < .04) were greater in gilts
treated with testosterone from d 13 to estrus than in
in gilts treated with
gilts treated with testosterone from d 16 to estrus or in
thosetreatedwith vehicle.
testosterone from d 16 to estrus and those receiving
vehicle were not different in number ( P = .93) or
recovery rate ( P = .84) of concepti. The proportion of
gilts having 100% of their concepti recovered on d 11.5
tended to be greater ( P = .10) in gilts treated with
testosterone from d 13 to estrus than in the other
groups (Table 2).
MeanCLweight(Table 1),
diameter (Table 2), and standard deviation of con-
cepti diameter (estimator of morphological diversity of
littermate concepti) were not influenced ( P > .10) by
treatment. The standard deviations of conceptus
diameter per litter were 1.6 ± .2, 1.9 ± .3, and 1.3 ± .3
mm (mean ± SE ) for gilts receiving vehicle and those
treated with testosterone from d 13 and d 16 to estrus,
respectively. The proportion of gilts from which
concepti were recovered on d 11.5 was not influenced
( P = .99) by treatment and was 19 out of 20 (95%),
17 out of 18 (94.4%), and 18 out of 19 (94.7%) for
gilts receiving vehicle or testosterone beginning on d
13 and d 16, respectively.
Experiment 2. Gilts treated with testosterone from d
13 to estrus had more CL ( P < .01) than those
administered vehicle (Table 1). Similarly, the mean
number of total concepti was influenced by the main
effect of testosterone treatment: number of total
concepti was greater ( P < .01) in gilts treated with
Gilts treatedwith
mean conceptus
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EXOGENOUS ANDROGENS AND SWINE REPRODUCTION
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Table 3. Number and recovery rate of total concepti at different stages
of development from vehicle- and testosterone-treated gilts (n)a
aMean ± SE.
b,cMeans within a row or column lacking a common superscript differ (P < .01).
No. of total conceptiRecovery rate of total concepti, %
Stage of development VehicleTestosterone
Mean effect of
stage Vehicle Testosterone
Mean effect of
stage
4 to 8-cell
Early blastocyst
Hatching blastocyst
Main effect
13.6 ± .7
12.0 ± .7
10.5 ± .7
12.5 ± .4b(24) 13.6 ± .4c
(8) 13.9 ± .7
(8) 15.1 ± .7
(8) 11.9 ± .7
(8) 13.8 ± .5b(16)
(8) 13.6 ± .5b(16)
(8) 11.2 ± .5c
(24)
96.8 ± 3.2
94.3 ± 3.2
79.7 ± 3.2
90.3 ± 1.9 (24)
(8)
(8)
(8)
95.0 ± 3.2
97.7 ± 3.2
81.8 ± 3.2
91.5 ± 1.9 (24)
(8) 96.9 ± 2.3b
(8) 96.0 ± 2.3b
(8) 80.7 ± 2.3c
(16)
(16)
(16) (16)
Table 4. Number and recovery rate of normal concepti at different stages
of development from vehicle- and testosterone-treated gilts (n)a
aMean ± SE.
bNumber of normal concepti was influenced ( P < .05) by the interaction of treatment and stage.
c,d,eMeans for number of normal concepti lacking a common superscript differ (P < .01).
f,gMeans within stage lacking a common superscript differ (P < .001).
No. of normal conceptib
Recovery rate of concepti, %
Stage of development VehicleTestosterone Vehicle TestosteroneMean effect of stage
4 to 8-cell
Early blastocyst
Hatching blastocyst
Main effect
13.4 ± .8c,e
10.8 ± .8d
9.9 ± .8d
(8)
(8)
(8)
13.8 ± .8c
14.9 ± .8c
11.5 ± .8d,e
(8)
(8)
(8)
95.4 ± 3.5
84.7 ± 3.5
74.9 ± 3.5
85.0 ± 2.0 (24)
(8)
(8)
(8)
94.1 ± 3.5
95.5 ± 3.5
79.2 ± 3.5
89.6 ± 2.0 (24)
(8)
(8)
(8)
94.8 ± 2.5f
90.1 ± 2.5f
77.1 ± 2.5g
(16)
(16)
(16)
testosterone than in those treated with vehicle (Table
3).The mean number of total
4- to 8-cell and early blastocyst stages did not differ ( P
= .80); however, total concepti at these stages were
greater ( P < .01) than at the hatching stage (main
effect of stage of embryonic development, P < .01,
Table 3). The proportion of gilts from which concepti
were recovered was 100% in all groups.
The number of normal concepti was influenced (P <
.05) by the interaction of testosterone treatment and
stage of conceptus development (Table 4). The mean
number of early blastocysts, classified as normal, was
greater ( P < .001) in gilts treated with testosterone
than in those treated with vehicle. However, the mean
number of 4- to 8-cell embryos ( P > .73) and hatching
blastocysts ( P = .13) were not different between these
groups. The number of normal concepti in gilts treated
with testosterone did not change ( P = .30) from the
4- to 8-cell to the early blastocyst stage but decreased
( P < .01) by the hatching stage. In contrast, the
number of normal concepti in gilts treated with
vehicle decreased( P<
8-cell stage to the early blastocyst stage but did not
change ( P = .41) by the hatching stage (Table 4).
The recovery rate of total concepti was not in-
fluenced by the interaction ( P = .72) or the main
effect of treatment ( P = .65); however, it was highly
influenced ( P < .001) by stage of conceptus develop-
ment (Table 3). Similarly, recovery rates of normal
conceptiatthe
.02) fromthe4-to
concepti were not influenced ( P = .12) by testosterone
treatment but were influenced ( P < .001) by stage of
conceptus development (Table 4). Although recovery
rates of total ( P = .97) or normal ( P = .20) concepti at
the 4 to 8-cell and early blastocyst stages did not
differ, recovery rates at these stages were greater (P <
.01) than at the hatching blastocyst stage (Table 4).
Experiment 3. Administration of 1 mg of an-
drostenedione per day from d 13 of the estrous cycle to
the first day of the following estrus did not influence
the number of CL ( P = .28) or and the number ( P =
.63) or recovery rates ( P = .69) of concepti on d 11.5
(Table 5). The proportion of gilts from which concepti
were recovered were 94.4% and 95.0% for gilts treated
with vehicle or androstenedione, respectively.
Discussion
Administration of 1 mg of testosterone on d 17 and
18 of the estrous cycle was previously observed in our
laboratory to increase the number of CL and the
number of d-11 concepti. Recovery rates of concepti in
that experiment were not different between gilts
previously treated with testosterone and those receiv-
ing vehicle (89.9% and 84.7% respectively, Ca Ârdenas
and Pope, 1994). In the present experiments, a longer
treatment with testosterone, from d 13 to estrus,
consistently increased the number of CL (E xp. 1 and
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