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132
Braz. J. vet. Res. anim. Sci., São Paulo, v. 43, n. 1, p. 132-138, 2006
Effect of insemination-to-induced ovulation interval
on fertilization rate, embryo viability and number of
accessory sperms in sows
1 - Faculdade de Medicina Veterinária da Fundação de Ensino Otávio
Bastos, São João da Boa Vista – SP
2- Departamento de Reprodução Animal da Faculdade de Medicina
Veterinária e Zootecnia da Universidade de São Paulo - SP
Abstract
The ideal interval between AI and ovulation (OV) is not well
determined yet, varying from 12 to 28 h before up to 4 h after
ovulation. Utilization of gonadotrophins to synchronize ovulation
would allow the pre-determination of the groups’ size, according to
the AI-OV intervals, and would contribute to determine a secure
interval between AI-OV. 120 sows received 7.5 mg IM of Luprostiol,
between days 12 and 17 of the estrous cycle, 600 IU of eCG IM 24 h
after prostaglandin and 5.0 mg of LH IM 72 h after eCG injection.
The moment of ovulation was diagnosed by transrectal
ultrasonography at intervals of 6 h. There were 5 treatments according
to IA-OV interval: T1- 48 to 36 h before OV; T2- 36 to 24 h before
OV; T3- 24 to 12 h before OV; T4- 12 to 0 h before OV and T5- 0 to
12 h after OV. Sows were slaughtered 96.7±11.37 h after OV. Recovery
rate (RR), number of corpora lutea (NC), total number of structures
(ST), fertilization rate (FR), embryo viability (EV) and number of
accessory sperm (AS) were analyzed. The synchronization protocol
showed an homogenous distribution of the animals among
treatments (LH-OV interval 39.22±7.6h), and it didn’t influenced
the results. FR and EV results suggest that 36 h is the time of sperm
viability in sow genital tract. There was a strong decline of AS between
T3 and T4.
Carlos Henrique Cabral
VIANA1
Pedro Henrique CANDINI2
Rogério Dantas GAMA2
Adriana CARBONE2
Renato Campanarut
BARNABE2
Correspondência para:
CARLOS HENRIQUE CABRAL VIANA
Faculdade de Medicina Veterinária da
Fundação de Ensino Octávio Bastos
Av. Dr. Octávio Bastos, s/n - Jardim Nova
São João
13874-149 - São João João da Boa Vista
chcabral@usp.br
Key-Words:
Swine.
Synchronization.
Insemination.
Ovulation.
Introduction
The ideal moment to perform
artificial insemination (AI) in swine has
attracted the researchers’ attention since the
60’s. Until the end of the 80’s, the
determination of this moment based on data
obtained by the estrus observation1, by
induction of ovulation and fixed time
insemination2, and by determination of
hormone levels (LH and progesterone)
related to ovulation3. With the coming of
the ultrasound, as an accurate and not invasive
technique, for the diagnosis of ovulation4,
more consistent results surged in relation to
insemination-to-ovulation interval. Waberski
et al.5 didn’t observe significant differences
in fertilization rate of gilts inseminated in
intervals of 0 to 12, 12 to 24 and over 24
hours before ovulation, when the semen was
stored for a period of up to 48 hours.
However, the number of accessory sperms
was quite larger, but not significant, when
the inseminations were performed between
0 and 12 hours in relation to the other
intervals.
The evaluation of the percentage of
normal embryos, in sows inseminated from
48 hours before to 16 hours after ovulation,
showed significantly higher results when the
insemination happened between 24 and 0
hours before ovulation, in comparison to
intervals from 48 to 24 hours before and 0
to 16 hours after ovulation6.
Recebido para publicação: 13/04/2004
Aprovado para publicação: 01/06/2005
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Braz. J. vet. Res. anim. Sci., São Paulo, v. 43, n. 1, p. 132-138, 2006
According to the results of Nissen et
al.7, a safe insemination-to-ovulation interval
would be from 28 hours before up to 4
hours after ovulation, according to the results
of farrowing rate and litter size, and from
24 hours before up to the moment of
ovulation, according to embryo recovery
rate. Evaluation of the interval between
inseminations showed that there were no
significant differences between intervals of
12 and 24 hours, in relation to the results of
conception rate, farrowing rate and litter size.
However, there was a decrease of 1.3
inseminations per sow for the group of 24
hours compared to the group of 12 hours
interval7.
The classic protocol of synchro-
nization of ovulation extols the eCG
administration 24 hours after weaning, and
the hCG4 or porcine LH8 administration 72
hours after eCG, with the occurrence of
ovulation between 32 and 44 hours after the
last injection. The use of gonadotropins to
synchronize ovulation, making it possible to
pre-determine the groups’ size, according to
the insemination-to-ovulation interval,
associated to the ultrasonography for the
diagnosis of ovulation, would complement
the information about the determination of
a safe insemination-to-ovulation interval.
The objectives of this experiment are:
To determine a sufficiently long interval
between inseminations to facilitate management
and sufficiently safe to guarantee appropriate
farrowing rate and litter size, by the evaluation
of fertilization rate and of the number of
accessory sperms;
To evaluate the use of a protocol, which
uses Luprostiol, eCG and LH, as a technique
of synchronization of the ovulation, to
elaborate fixed time AI protocols and make
the equalization of the groups size possible, in
experiments that evaluate the effect of
insemination-to-ovulation interval on
reproductive parameters.
Material and Method
The work was performed in an
Agroceres-PIC farm, Minas Gerais State. 120
cyclic sows were used after the 3rd
parturition. During lactation, sows received
a lactation diet and water ad libitum, staying
there for an average period of 5.35±1.13
days. Because of the early weaning
management in this farm, the second post-
weaning estrus was used to avoid possible
influences in ovulation rate, fertilization rate
and recovery of embryos. The 120 sows
were divided into 12 weekly groups, of
approximately 10 animals each, to make the
execution of the work possible. For the
diagnosis of ovulation, the technique of
trans-rectal ultrasound was used with the
Scanner 200 machine (Pie Medical©),
equipped with a sector transducer of 7.5
MHz. The examinations started 24 hours
after LH application with intervals of 6
hours, approximately at 08:00, 14:00, 20:00
and 02:00, continuing until the ovulation
occurred. The diagnosis of ovulation was
considered when no follicle was found or
when the number of follicles was smaller
than in the previous examination. The
moment of ovulation (MO) was defined as
the mean time between the last examination
where preovulatory follicles were detected
and the first examination where no follicles
could be detected on the ovary.
Of the 120 examined sows, 16 were
excluded from the experiment for having
ovulated within 24 hours after LH
application. In the first estrus (first cycle after
weaning), the day of ovulation was
determined and served as a reference for
the beginning of the hormonal applications.
Sows were injected with 7.5mg of Luprostiol
(Prosolvin®, an PGF2α analog), between 12
and 17 days after the first ovulation (D1);
600UI of eCG (Novormon®) 24 hours after
the Luprostiol injection (D2); and 5.0mg of
porcine LH (Lutropin®) 72 hours after the
eCG injection (D5). The prostaglandin
application was performed at 10:00, on
Fridays and on Saturdays (D1) (two weekly
groups of hormonal treatment), trying to
achieve a larger number of sows, which were
among 12 to 17 days after the first ovulation.
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The eCG injection was applied appro-
ximately at 10:00, on Saturdays and Sundays
(D2) and porcine LH at 10:00 on following
Tuesdays and Wednesdays (D5) (Figure 1).
The sows were submitted to 5
treatments. Only one insemination was
performed by sow. In treatment 1, sows
were inseminated between 48 and 36 hours
before the ovulation; in treatment 2, between
36 and 24 hours before; in treatment 3,
between 24 and 12 hours before; in
treatment 4, between 12 and 0 hours before
and in treatment 5, between 0 and 12 hours
after the ovulation. Heterospermic semen
from 3 boars was used in the concentration
of 3.5 billion sperm cells per dose. The sows
were slaughtered 96.7±11.37 hours after the
occurrence of ovulation and the number of
corpora lutea (NCL) was counted in both
ovaries. Each oviduct was flushed with 10
ml of solution of phosphate buffered saline
(PBS). Later, the oviducts were separate from
the uterus and each uterine horn was flushed
twice, and in the first flushing, it was used
20ml and in the second 30ml of PBS, in two
different plates to collect the embryos and
oocytes. The recovery rate (TR) was
determined by the total number of embryos
and collected oocytes (ET) in relation to the
number of corpora luteaa9. The evaluation
of the embryos was performed using stereo-
microscopy, in a magnitude of 40x and
based, firstly, in the presence and uniformity
of the perivitelline space with the cells evenly
distributed, in the development stages
according to the time between ovulation and
collection10, and in the presence of accessory
sperms in the zone pellucida (EA), which
were counted using optical microscopy in a
magnitude of 400X. The embryos were
deposited in a glass slide, where the zone
pellucida was disrupted with 0.5% pronase
solution. Later, it was covered with a slide
to make possible the count of the sperms.
Embryos, which didn’t fit to the first two
evaluation criteria, were classified as
degenerate. The fertilization rate (TF) it was
calculated dividing the number of embryos
(degenerate and viable) by the total number
of embryos more oocytes and the embryo
viability rate (TEV), dividing the number of
embryos with normal development by the
total number of embryos more oocytes.
The data were analyzed using SAS
(1999) and were presented as the average ±
standard deviation and variation (minimum–
maximum). All variables were submitted to
the test of normality of the residues, to verify
if they followed the normal distribution. The
variables: NCL, TR and ET obeyed the
normal distribution and were analyzed by
ONEWAY procedure, according to the
factor insemination-to-ovulation interval,
divided in 5 classes (T1, T2, T3, T4 and T5).
The variables TF, TEV and EA didn’t
follow the normal distribution and they were
analyzed by the non parametric procedure
NPAR1WAY, according to the factor
insemination-to-ovulation interval, divided in
5 classes (T1, T2, T3, T4 and T5).
Results
Of the 120 used sows, 16 were
discarded of the experiment, in reason of
precocious ovulation (before the expected
interval, 9 sows) or metritis (7 sows). The
protocol of synchronization of ovulation
was satisfactory (LH application-to-ovulation
interval of 39.22±7.6 hours) in the
homogeneous distribution of the number
of animals for each treatment of
insemination-to-ovulation interval and it
didn’t influence the results, since there were
no significant differences among the averages
of the characteristics NCL, TR and ET,
according to the treatments (Table 1). A
significant difference was verified in the
characteristics TF and TEV, and, in both, the
insemination-to-ovulation interval from 48
to 36 hours before ovulation showed a
smaller average than all of the other intervals,
whose averages were not different between
each other. The number of accessory sperms
was significantly different, according to
insemination-to-ovulation interval, so that the
averages of the intervals 48 to 36, 36 to 24
and 24 to 12 hours before ovulation didn’t
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Braz. J. vet. Res. anim. Sci., São Paulo, v. 43, n. 1, p. 132-138, 2006
Table 1 - Average values, standard deviation and range NC, ET, EA, TR, TF and TEV, according to the insemination-to-ovulation interval, São Paulo, 2004
Means followed by similar superscripts, within the same line don’t differ statistically among each other (P<0,05)
Figure 1 - Schedule of synchronization of ovulation and treatments
Discussion
The LH application-to-ovulation
interval of 39.22±7.6 hours is in agreement
with the protocols of synchronization
ovulation developed before, which used
LH8,11 or hCG and GnRH2,12,13,14, and it made
possible the formation of groups of homo-
geneous size according to insemination-to-
ovulation interval.
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Weaning works very well as starting
point for protocols of induction of
ovulation in swine. However, in some
situations, as certain experiments13 or in of
embryo15 transfer programs, it is necessary
to use cyclic sows. In the present experiment
cyclic sows were used to avoid possible
effects of the short lactation period on
reproductive results16,17, showing satisfactory
results.
The average TR, TF, TVE and the
number of embryos of all treatments together
were satisfactory according to data obtained
previously, so much in experiments that used
the induction of the ovulation18 as in
experiments with spontaneous ovulation6,9.
NCL and TR were similar among the classes
of interval insemination-ovulation (Table 1),
and therefore, they didn’t influence the results
of the fertilization rates and of embryonic
viability, confirming once again, the usefulness
of the synchronization of the ovulation for
the equalization of the size of the groups.
According to the results of fertilization
rate and embryonic viability, an interval of
up to 36 hours can be considered as the sperm
viability in the sow reproductive tract.
According to the literature, the longest
minimal sperm viability considered was of
24 hours6,7, and other authors considered this
time 1619 or 12 hours5. A possible cause for
the longer sperm viability observed in the
present experiment related to the results of
Soede et al.6 and Nissen et al.7 is that these
authors used a semen dose of 3 and 2 x 109
sperms against 3.5 x 109 in this experiment.
However, Steverink et al.9 didn’t see any
effect of the number of sperms cells per
dose on fertilization rate and nor on the
number of accessory sperms.
The accessory sperm count technique
has been used as parameter to evaluate
experiments related to artificial insemination,
by expressing the amount of sperms
capable of fertilization, at the place and
moment of occurrence of ovulation and for
being correlated positively to embryo
viability5,6,9,20,21. As in all the consulted
experiments, which used accessory sperm
count as a parameter, in the present
experiment this parameter was highly
variable, what is observed by the standard
deviations in table I. The abrupt decrease in
the average accessory sperms among the
classes of insemination-to-ovulation interval
from 0 to 12 and 12 to 24 hours also
happened in the experiment of Waberski et
al.5. However, this decrease didn’t reflect a
decrease in the fertilization rate and in the
embryonic viability (Table 1), in disagreement
to the positive correlation between the
number of accessory sperms and these
variables, described in other experiments21.
Steverink et al.9 reported that several sows
showing 100% of normal embryos had a
relatively low number of accessory sperms
and found negative correlation (R2=0,24; P
< 0,0001) between the number of accessory
sperms and the insemination-to-ovulation
interval. These authors suggest that the
relationship between accessory sperms and
fertilization rate and embryonic viability can
be indirect, once there is a clear effect of the
insemination-to-ovulation interval on these
variables.
Conclusions
It was not possible to show significant
differences in the results of fertilization rate
and of embryonic viability when the sows
were inseminated between 36 hours before
and 12 hours after ovulation. There was a
decrease in the number of accessory sperms
when the insemination-to-ovulation interval
was longer than 12 hours.
The use of the synchronization of
ovulation technique in cyclic sows
(Luprostiol, eCG and LH) and fixed time
insemination, resulted in the expected pattern
of synchronization, making possible the
equalization of the group’s size, without
influence on the reproductive parameters.
Acknowledgements
Agroceres PIC Melhoramento
Genético de Suínos.
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Resumo
O intervalo ideal entre a AI e ovulação (OV) não está bem determinado
ainda, variando entre 12 a 28 h antes até 4 h depois da ovulação. A
utilização de gonadotrofinas para sincronizar ovulação permitiria a
pré-determinação do tamanho dos grupos, de acordo com os
intervalos IA-OV, e possibilitaria determinar um intervalo seguro
entre IA-OV. 120 porcas receberam 7.5 mg IM de Luprostiol, entre os
dias 12 e 17 do ciclo estral, 600 IU de eCG IM 24 h após o Luprostiol
e 5.0 mg de LH IM 72 h após a injeção de eCG. O momento de
ovulação foi diagnosticado pela ultra-sonografia trans-retal a intervalos
de 6 h. Definiu-se 5 tratamentos de acordo com o intervalo IA-OV:
T1 - 48 a 36 h antes da OV; T2 - 36 a 24 h antes da OV; T3 - 24 a 12 h
antes da OV; T4 - 12 a 0 h antes da OV e T5 - 0 a 12 h após a OV. O
abate ocorreu 96.7±11.37 h após a OV. A taxa de recuperação (RR),
número de lutea de corpos (NC), número total de estruturas (ST),
taxa de fecundação (FR), viabilidade embrionária (EV) e número de
espermatozóides acessórios (AS) foram analisados. O protocolo de
sincronização mostrou uma distribuição homogênea dos animais
entre os tratamentos (intervalo LH-OV de 39.22±7.6h), e não
influenciou os resultados. A FR e os resultados de EV sugerem que
36 h seja o tempo de viabilidade do espermatozóide trato genital da
porca. Houve um forte declínio do AS entre T3 e T4.
Palavras-chave:
Suínos.
Sincronização.
Inseminação.
Ovulação.
References
1 WILLEMSE, A. H. Estimation of optimun time for
insemination of gilts and ewes by double mating at
certain times relative to ovulation. Journal of
Reproduction and Fertility, v. 22, n. 2, p. 277-282,
1970.
2 DZIUK, P. Estimation of optimun time for
insemination of gilts and ewes by double mating at
certain times relative to ovulation. Journal of
Reproduction and Fertility, v. 22, n. 2, p. 277-282,
1970.
3 HELMOND, F.; AARNINK, A.; OUDENAARDEN,
C. Periovulatory hormone profiles in relation to
embryonic development and mortality in pigs. In:
SREENAN, J. N.; DISKIN, M. G. Embryonic mortality
in farm animals. Dordrecht: Martinus Nijhoff , 1986.
p. 119-125.
4 WEITZE, K. F. et. al. Detection of ovulation in sow
using transcutaneous sonography. Zuchthygiene, v.
24, n. 1, p. 40-42, 1989.
5 WABERSKI, D. et al. Effect of time of insemination
relative to ovulation on fertility with liquid and frozen
boar semen. Theriogenology, v. 42, n. 5, p. 831-840,
1994.
6 SOEDE, N. M. et al. Effects of time of insemination
relative to ovulation, as determined by ultrasonography,
on fertilization rate and accessory sperm count in sows.
Journal of Reproduction and Fertility, v. 104, n. 1, p.
99-106, 1995.
7 NISSEN, A. K. et al. The influence of time of
insemination relative to time of ovulation on farrowing
frequency and litter size in sows, as investigated by
ultrasonography. Theriogenology, v. 47, n. 8, p. 1571-
1582, 1997.
8 CANDINI, P. H. et al. Utilização de gonadotrofinas
(eCG e LH) para synchronization da ovulação em fêmeas
suínas desmamadas. In: CONGRESSO BRASILEIRO DE
VETERINÁRIOS ESPECIALISTAS EM SUÍNOS, 9. Belo
Horizonte, 1999. Anais.. Belo Horizonte: Associação
Brasileira de Veterinários Especialistas em Suínos, 1999.
p. 375-376.
9 STEVERINK, D. W. B. et al. Influence of insemination
to ovulation interval and sperm cell dosage on
fertilization in sows. Journal of Reproduction and
Fertility, v. 111, p. 165-171, 1997.
10 HUNTER, R. H. F. Chronological and cytological
details of fertilization and early embryonic development
in the domestic pig, Sus scrofa. Anatomical Records, v.
178, p. 169-186, 1974.
Efeito do intervalo inseminação-ovulação induzida sobre a taxa de fecun-
dação, viabilidade embrionária e número de espermatozóides acessórios
em porcas
039_04.pmd 26/9/2006, 14:17137
138
Braz. J. vet. Res. anim. Sci., São Paulo, v. 43, n. 1, p. 132-138, 2006
11 CANDINI, P. H. et al. Utilization of synchronized
ovulation protocol, induced by porcine LH, to perform
insemination programs in fixed time, with one or two
inseminations per sow. In: INTERNATIONAL
CONFERENCE ON PIG REPRODUCTION, 6., 2001,
Columbia-Missouri, 2001. Anais.. Columbia: University
of Missouri, 2001. p.99.
12 HUNTER, R. H. F. Porcine ovulation after injection
of human chorionic gonadotropin. Veterinary Record,
v.81, n. 1, p. 21-23, 1967.
13 SOEDE, M. N.; NOORDHUIZEN, J. P. T. M.;
KEMP, B. The duration of ovulation in pigs, studied by
transrectal ultrasonography, is not related to early
embryonic diversity. Theriogenology, v. 38, n. 4, p.
653-666, 1992.
14 HÜHN, U.; JÖCHLE, W.; BRÜSSOW, K. P.
Techniques developed for the control of estrus, ovulation
and parturition in the east german pig industry: a review.
Theriogenology, v. 46, n. 6, p. 911-924, 1996.
15 HAZELEGER, W.; KEMP, B. Farrowing rate and
litter size after transcervical embryo transfer in sows.
Reproduction in Domestic Animals, v. 29, p. 481-
487, 1994.
16 TUBBS, R. C. Factors that influence the weaning-to-
estrus interval in sows. Compendium of Continuing
Education Practice Veterinary, v. 12, n. 1, p. 105-115,
1990.
17 Le COZLER, Y. Effect of weaning-to-conception
interval and lactation length on subsequent litter size in
sows. Livestock Production Science, v. 51, p. 1-11,
1997.
18 SOEDE, M. N.; KEMP, B. In synchronized pigs, the
duration of ovulation is not affected by insemination
and is not a determinant for early embryonic diversity.
Theriogenology, v. 39, n. 5, p. 1043-1053, 1993.
19 UEMOTO, D. A. et al. Sobrevivência embrionária e
número de embriões viáveis em leitoas submetidas à
inseminação artificial em diferentes intervalos pré-
ovulatórios. In: CONGRESSO BRASILEIRO DE
VETERINÁRIOS ESPECIALISTAS EM SUÍNOS, 9., 1999,
Belo Horizonte, Anais.. Belo Horizonte: Associação
Brasileira de Veterinários Especialistas em Suínos, 1999.
p. 359-360.
20 BERTANI, G. R. et al. Effect of the time of artificial
insemination with frozen-thawed or fresh semen on
embryo viability and early pregnancy rate in gilts.
Theriogenology, v. 48, p. 933-945, 1997.
21 DeJARNETTE, J. M. et al. Accessory Sperm: Their
importance to fertility and embryo quality, and attempts
to alter their numbers in artificially inseminated cattle.
Jornal of Animal Science, v. 70, p. 484-491, 1992.
039_04.pmd 26/9/2006, 14:17138
