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Laparoscopic Ovum Pick-Up (LOPU) is the most reliable and efficient technique for collecting high quality oocytes from live animals in certain species or age groups, allowing its use for In Vitro Embryo Production (IVEP) and Somatic Cell Nuclear Transfer (SCNT). In order to maximize the number and quality of oocytes collected by donor, it is necessary to synchronize estrus and stimulate follicular growth using hormonal protocols that vary according to species. There are 2 big categories of applications for the LOPU-IVEP technology in production animals, those in which it acts as an alternative to MOET (competitive applications) and those in which it doesn’t compete with MOET as it cannot be done in those categories (non competitive applications). In wild animals, LOPU can play an important role in conservation programs for endangered species when associated with effective IVEP and has been done in several species. It has commercial application in sheep, goats, cattle and buffaloes calves. Repeating LOPU procedures in the same female does not cause sequels with impact on the female's reproductive life, even when performed on prepubertal or wild animals.
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Spermova 2018; 8(1): 61 - 67
Artículo de revisión - Review article
DOI. 10.18548/aspe/0006.07
LAPAROSCOPIC OVUM PICK-UP (LOPU): FROM ANIMAL PRODUCTION TO
CONSERVATION
Aspiración folicular por Laparoscopia (LOPU): de la producción a la conservación
Pedro Nacib Jorge Neto1*, Letícia Alecho Requena2, Cristiane Schilbach Pizzutto1, Hernan Baldassarre3
1 Faculdade de Medicina
Veterinária e Zootecnia,
Universidade de São Paulo,
São Paulo-SP, Brazil
2 Genética Bacurizinho,
Potirendaba-SP, Brazil
3 McGill University, Montreal,
Canada
* Corresponding author
Pedro Nacib Jorge Neto, R.
Vitoriano dos Anjos, 1081 - Vila
São Jorge, Campinas - SP,
13041-317.
E-mail: pepovet@usp.br
DOI. 10.18548/aspe/0006.07 Jorge Neto PN, Alecho Requena L, Schilbach Pizzutto C, Baldassarre H. SPERMOVA. 2018; 8(1): 61-67
62
INTRODUCTION
Reproductive biotechnologies are in constant development, and
In Vitro Embryo Production (IVEP) has become an important tool
for improving the genetic merit of cattle, buffaloes, sheep and
goats, as well as a technology with enormous potential for the
conservation of wild animals.
Laparoscopic Ovum Pick-Up (LOPU) is the most reliable and
efficient technique for collecting high quality oocytes from live
animals in certain species or age groups, allowing its use in IVEP
and Somatic Cell Nuclear Transfer (SCNT).
The technique is minimally invasive and has repeatability and
reliability, allowing the observation of the ovaries through the
laparoscope and the puncture of the follicles, besides a fast
recovery of the female after the procedure and in general,
without surgical complications. In contrast to Multiple Ovulation
and Embryo Transfer (MOET), LOPU brings the advantages of
being less invasive and causing less postoperative stress, and
can be repeated in a shorter interval (minimum of 60 days for
MOET against a minimum of 14 days for LOPU), besides
causing no adhesions or fibrosis and adhesions commonly
occurring after multiple MOETs.
LOPU also allows the year-round production of embryos, even
outside the breeding season.
OVARIAN SUPERSTIMULATION
In order to maximize the number and quality of oocytes
collected by donor, it is necessary to synchronize estrus and
stimulate follicular growth prior to LOPU, through protocols
based on gonadotropins, which exert a significant influence on
the results.
Synchronization is performed using intravaginal devices
containing progesterone or progestogen (eg, CIDR, Primer,
sponges) which are applied with a luteolytic dose of
prostaglandin or analog (e.g., cloprostenol, dinoprost) when
treatment with gonadotropins initiates.
Several treatments have been proposed for the stimulation of
follicular growth, being the two most popular the protocol with
multiple FSH injections and the Oneshot protocol. The first one
consists of 3 FSH injections of 1.5, 1.5 and 1 mL, totaling 80 mg
FSH (Folltropin-V, Vetoquinol N.-A inc, Canada) administered
at 12-hour intervals and starting 36 hours before LOPU. The
second consists on the application of 4 mL of FSH together with
300 IU of eCG simultaneously (in different syringes), 36 hours
before LOPU. Work performed by Baldassarre et al. (2012)
did not show significant differences between both protocols,
making Oneshot more attractive due to simplicity. Recently,
Baldassarre et al (2012) reported an alternative protocol to
the Oneshot-FSH without co-injecting eCG, by using 0.5%
hyaluronic acid solution for the reconstitution of the lyophilized
FSH. Hyaluronate acts as a slow release factor of FSH. The
results demonstrated the efficiency of this protocol, with no
differences between the control and the new treatment in the
mean number of aspirated follicles (goats: 17.8 vs. 17.9; sheep
12.6 vs. 12.4) and recovered oocytes (goats: 13.7 vs. 14.0;
sheep: 10.9 vs. 10.8).
In buffalo calves (2-6 months), Baldassarre et al. (2017a)
tested two different superovulation protocols, both starting with
the implantation of a progesterone device (Eazi-breed CIDR,
Zoetis, 330 mg P4) five days before LOPU. The first, FSH-only
starting 72 hours before the LOPU and consists of 6 injections
with interval of 12h, totaling 140 mg of FSH. The second, with
4 injections of FSH (100mg) along with an injection of eCG (400
IU) simultaneously the last injection of FSH, 36h before the
LOPU. The result of this protocol had no statistical differences
in follicle number (18.0 vs. 20.3) and oocytes recovered (15.0
vs. 17.5). In bovine calves, Galli et al (2001) proposed the use
of 5mg of estradiol valerate and progesterone sponge (80mg
of fluorogestone) on day 0, 400 IU of eCG on days 3 and 11,
1000 IU of eCG on day 16 and LOPU in D18, showing a
difference in the number of oocytes recovered from the
untreated group (27.2 vs. 16, respectively). Recently,
Baldassarre et al (2018) compared three treatments, all
starting with the insertion of progesterone device for small
ruminants (Eazi-breed CIDR, Zoetis) five days before LOPU, as
well as gonadotrophin stimulation was initiated 72 h prior to
the procedure. In the FSH-12 group the animals were treated
intramuscularly (IM) with a total of 140 mg FSH (Folltropin-V)
in 6 injections at 12 h intervals (7:00 a.m. and 7:00 p.m.). To
assess the impact of more frequent stimulation of
gonadotrophin, two additional protocols were tested. In the
FSH-8h group, FSH was administered IM every 8 h (at 7:00
a.m., 3:00 p.m. and 11:00 p.m.) for a total dose of 180 mg (9
injections). In the FSH8h-eGG group, the animals received the
same injections of FSH as FSH-8h until injection number 5, when
they received 400 IU. of eCG (Folligon, Intervet, The
Netherlands) and no further injections until LOPU (total of 120
mg FSH in 5 injections). No statistical differences were observed
between the 3 treatments in terms of average follicles
available for aspiration (35.7 ± 16 vs. 38.5 ± 25 vs. 31.1 ±
22), mean oocytes recovered 21.6 ± 10 vs. 19.4 ± 14) and
cleavage rate (66.0 ± 14 vs. 61.1 ± 11 vs. 72.2 ± 8) for
FSH12h, FSH8h and FSH8h-eCG, respectively. However,
FSH8h-eCG resulted in a significantly higher rate of
transferable embryos (17.5 ± 8%) compared to FSH12h (8.9
± 5%, P <0.05).
In wild animals the protocols have great variations due to the
particular physiology of each species. In previous studies of our
team with cougars (Puma concolor) and jaguars (Panthera
onca), in order to prepare the females for LOPU, 750 IU of
eCG were used to stimulate multiple follicular development 5
days before the procedure. In some donors, 500 IU of hCG
were also used to try to mature the oocytes in vivo before
LOPU, but the results obtained were very variable (Baldassarre
et al, 2017b and unpublished results). In gazelle dama
(Gazella dama mhorr), Berlinguer et al (2008) synchronized
estrus with intravaginal progesterone device (CIDR) for 15
days. CIDRs were replaced on day 10 and removed during
LOPU on day 15. Follicular growth was stimulated by
administration of a total of 5.28 mg ovine FSH (Ovagen, ICPbio
Ltd, New Zealand) applied in four equal doses given at 8:00
p.m. and 6:00 p.m. on days 13 and 14.
In red deer (Cervus elaphus), Bainbridge et al. (1999) used the
same CIDRs for 10 days, applying 1,000 IU of eCG 48 h prior
to removal and performed LOPU 24 h after. Locatelli et al.
(2006) performed weekly LOPUs on sika deer (Cervus nippon
nippon), synchronizing estrus intravaginal sponges (45 mg FGA,
Intervet, France) during the breeding season and stimulated the
ovaries with three injections of FSH of 0.2, 0.2 and 0.1 IU, at
12 hour intervals, starting 48 h before LOPU.
Description of the LOPU Technique
The equipment used for LOPU consists of a laparoscope
(diameter of 5 or 7mm, angle of or 30º and length of ~
30cm) connected to a cable and light source. One pump of
insufflation, three trocars (two of 5mm being one with valve for
insufflation and one of 3,5mm) and an atraumatic endoscopic
DOI. 10.18548/aspe/0006.07 Jorge Neto PN, Alecho Requena L, Schilbach Pizzutto C, Baldassarre H. SPERMOVA. 2018; 8(1): 61-67
63
forceps. The suction system is composed of a suction pump
connected to the OPU stopper connected to a 50 ml Falcon-
type tube and connected to the LOPU pipette. The pressure of
the vacuum pump should be measured in drops per minute
(velocity of the aspiration medium through the aspiration
pipette and falling into the collection tube) and adjusted to 50-
70 drops min (Baldassarre et al., 1994), the system must be
washed with aspiration medium before and after LOPU.
The LOPU technique was described by Baldassarre et al.
(1994). Animals should be fasted for 12 (water) to 24 hours
(solids) before LOPU. The anesthetic protocol varies according
to the species, consisting of a sedative to position the animal on
a cradle typically used for laparoscopi insemination and then
is then connected to the anesthesia machine ane maintained
under general anesthesia with isoflurane. A tricotomy is
performed in the abdominal region followed by disinfection of
the area cranial of the mammary gland. Once the female
reaches an anesthetic plane, the cradle is tilted to
Trendelenburg position (variation of the dorsal decubitus
position where the upper back is lowered and the legs are
elevated, keeping the intestinal loops in the upper abdominal
cavity) at 45 degrees. Three small incisions are made with the
aid of a scalpel blade for the easy entrance of the trocars, and
the location of the incisions will vary according to the species.
The first trocar (with insufflation valve) must be introduced with
counter-traction for abdominal elevation, avoiding sudden
movements or excessive force (Campos & Roll, 2003). Then,
filtered air is blown into the abdominal cavity to separate
organs and facilitate visulization. In sequence, the other two
trocars are inserted, under direct visualization by the
laparoscope. The ovary is then exposed for visualization and
puncture of the follicles, moving the fimbria in different
directions with the endoscopic forceps. The aspiration needle
should be entered by the side of the follicle, avoiding
vascularized areas, parallel to the base of the follicle. If this is
not possible, the puncture occurs perpendicular to the follicle
wall (Baldassarre et al., 1994). At the end of follicular
aspiration, both ovaries are washed with 0.9% sodium chloride
(physiological solution) to eliminate any clots formed by the
follicular puncture, avoiding adhesions. The trocars are then
removed and the incisions are glued with instant adhesive or
sutured, depending on the species. They are given antibiotic
and anti-inflammatory and the female is withdrawn from the
anesthetic plane, recovering in a calm and safe place. The
collection tube is passedon to the lab for finding and grading
of the oocytes under the streomicroscope
LOPU in Farm Animals
LOPU presents exceptional performance in specific
applications in production animals, allowing to increase the
production of offspring fromfemales of high genetic merit and
also the production of progeny from categories that are not
eligible for MOET (Baldassarre et al., 2004).
According to Tervit (1996), the technology has been applied to
species or age groups where it is not possible or easy to
manipulate the reproductive tract through the rectum during
oocyte retrieval.
There are 2 big categories of applications for the LOPU-IVEP
technology in production animals, those in which it acts as an
alternative to MOET (competitive applications) and those in
which it doesn’t compete with MOET as MOET cannot be done
in those categories (non competitive applications).
COMPETITIVE APPLICATIONS
This application refers to the use of non-gravid, non-
lactating/weaned, adult females as donor of oocytes for
LOPU-IVEP. Used in this category, LOPU-IVEP is for the exact
same objective as MOET, i.e. production of more progeny from
outstanding adult females. In a 1:1 comparison, MOET should
win this competition because in normal conditions MOET should
result in an average of 8-10 transferable embryos/flush while
LOPU-IVEP will produce an average of 4-5 blastocysts for
transfer, and the viability of in vivo produced embryo tends to
be better than the in vitro counterparts. So, in this specific
application, the advantage of using LOPU-IVEP applies only to
customers willing to produce many more progenies from their
top females, that what can be achieved by 1-2 MOET flushes
per year. In those cases, LOPU-IVEP offers the option of oocyte
collection every 2 weeks almost unlimitedly (30 times in the
same animals in 4 years, Baldassarre et al. personal
communication). For example, 3 MOET in one year can result in
24-30 transferable embryos, but in the same period one can
easy conduct 1 LOPU evry 2 weeks and end with 48-60
transferable embryos for transfer. And, 3 surgical embryo
collections has a high chance of developing adhesions, while 12
LOPU will produce no sequels in the donors.
NON-COMPETITIVE APPLICATIONS:
This refers to the categories where MOET doesn’t work so it is
not a reproductive technology that can be applied for
exponential multiplication of valuable females. Some of these
categories are age-related, specifically the use of LOPU-IVEP
for early reproduction of elite females before reaching
puberty a sexual maturity; as well as older animals that have
become unfertile with age or uncapable of carrying a
pregnancy themselves but are genetically superior. Other non-
competitive categories refer to animal “conditions”, most of
them temporary conditions that prevent the animals from
producing embryos by MOET for a period, such as early
pregnant animals and early post partum animals. Finally,
another category of non-competitive application refers to the
practice of LOPU-IVEP in MOET failures, specifically animals
with history of repeated failure. These are animals that have
been subjected to MOET programs repeatedly and they hav
always failed to produce transferable embryos for reason of
a) repeated luteal regression; b) failure to superovulate (non-
responders), c) failure to fertilize (only unfertile eggs are
recovered). Finally, LOPU-IVEP offers opportunities for
reproductive rescue of females that have been “crippled” by
previous surgeries ending in a uterus with so many adhesions
that they can’t be flushed any more, maybe even not get
pregnant any more. As long as their ovaries are clean, oocytes
can be collected and embryos produced in vitro to continue
propagating their outstanding genetics.
The prepubertal females of the ovine, caprine, buffalo and
bovine species between two and seven months of life have a
greater response to gonadotrophins in relation to adult
animals, allowing to produce embryos of females with only two
months of age, with successive collections every two weeks to
seven months of age. Lohuis (1995) reports that prepubertals
are ideal from the perspective of genetics companies because
they reduce the intergenerational gap and thus accelerate the
process of genetic improvement. They have high production of
oocytes and, consequently, of embryos. This category is not
eligible for MOET, with studies showing that bovine calves in
this category, even when superovulated and inseminated with
fresh semen by laparoscopy, have many follicles, few
ovulations, and all structures recovered were non fertilized
(Tervit, 1996). Baldassarre & Karatzas (2004) point out that
DOI. 10.18548/aspe/0006.07 Jorge Neto PN, Alecho Requena L, Schilbach Pizzutto C, Baldassarre H. SPERMOVA. 2018; 8(1): 61-67
64
the application of this technology in prepubertal goats results
in the birth of their progeny at about the same time that donors
reach the age and weight for first breeding, and highlight the
reduction in the interval of generation that can be achieved.
Genetic merit senile females who generally present a high risk
of MOET failure due to reasons of low response to
superovulation protocol, regression of corpus luteum or infertile
embryos, are generally successful in the production of offspring
with the use of LOPU-IVEP, making it the tool of choice for
obtaining viable embryos of this category (Baldassarre et al.,
2007).
LOPU also allows oocytes to be obtained from pregnant
females throughout early pregnancy. In postpartum females, it
allows the recovery of oocytes before uterine involution, which
occurs between the third and fourth week postpartum and
consequently, are in this period unfit for MOET.
Table 1. Total recovered oocytes (T-COC). Mean recovered
oocytes (M-COC) and maximum number of recovered oocytes
from a single donor (MAX) in adult and elderly donors in
commercial LOPU services between 2016 and 2017 (Alecho
Requena et al., Unpublished).
Breed / Specie
No.
LOPU
T-
COC
M-
COC
MAX
Alpino / Goat
11
191
17,36
36
Anglo / Goat
6
145
24,16
73
Saanen / Goat
38
570
15,00
37
Dorper / Sheep
58
641
11,05
24
White Dorper / Sheep
42
496
11,80
27
Table 2. Results of LOPU at farm animals. Total of LOPU procedures (N-LOPU), Total Aspirated Follicles (TAF), Total Recovered
Oocytes (COC). Mean Recovered Oocytes (A-COC) and Average of Oocytes Recovered per LOPU (M-LOPU).
Species
Category
N-LOPU
TAF
COC
M-LOPU
Author
Goats
Adults
21
399
334
15,9
Koeman et al., 2003
Adults
18
221
146
8,1
Mendes et al., 2018
2-3 mo.
20
1.186
994
49,7
Baldassarre et al., 2002
3-5 mo.
36
1.238
987
27,4
Baldassarre et al., 2002
3-5 mo.
23
897
653
28,4
Koeman et al., 2003
Senil
43
772
676
15,7
Baldassarre et al., 2007
Various
1.580
~26.523
21.219
13,4
Baldassarre et al., 2004
Sheep
Adults
142
1.941
1.522
10,7
Baldassarre et al., 1996
Cattle
2-6 mo.
63
~1.953
1.351
21,4
Baldassarre et al., 2018
2-6 mo.
24
N/A
111
4,6
Armstrong et al., 1992
2-4 mo.
20
1.025
853
42,6
Taneja et al., 2000
Buffalo
2-6 mo.
47
903
774
16,5
Baldassarre et al., 2017a
LOPU in the Conservation of Wild Animals
Assisted Reproduction Technologies (ART) can be applied to
species conservation. Among the ARTs, LOPU has an important
role in endangered species, allowing the efficient recovery of
oocytes for use in IVEP and SCNT, tools to reconstruct the
balance in the number of animals for these endangered
species. Such technologies are useful in rare and endangered
animals, as well as species that have difficulty breeding in
captivity. They also allow the exchange of genetic material
between captive and free-living animals, increasing genetic
variability in captivity and restoring free populations with a
high degree of inbreeding.
Tervit (1996) points out that the technique may play an
important role in endangered species when associated with
effective PIVE and it is anticipated that the application of LOPU
will increase as biological differences between species are
understood.
Baldassarre et al. (2017b) demonstrated that LOPU is a safe
procedure for oocyte collection in wild animals, reporting
fertility after LOPU is repeatedly conducted, validating the
procedure as safe for wild feline multiplication as part of
conservation strategies, specifically cougars and jaguars which
are considered vulnerable in Brazil. Some other studies were
carried out by the authors on wild animals in Brazil and by
Baldassarre in the United States and South Africa. Results of
oocytes were reported in table 3, to the best of our knowledge,
the record oocyte harvest from a live donor by LOPU in wild
animals, is 106 oocytes obtained by Requena et al. in a Puma
concolor. Also, LOPU was successfully performed on at least
seventeen other wild species (table 3).
All those consulted, with several species, demonstrated that
LOPU can be applied repeatedly to the same female with no
or few adverse effects. As a consequence, the application of
the technology to conservation projects only further
development or optimization of methodologies for the
production of in vitro embryos from the oocytes collected by
LOPU.
DOI. 10.18548/aspe/0006.07 Jorge Neto PN, Alecho Requena L, Schilbach Pizzutto C, Baldassarre H. SPERMOVA. 2018; 8(1): 61-67
65
Table 3. Total recovered oocytes (COC). Mean recovered oocytes (A-COC) and Maximum number of recovered oocytes from a
single donor (MAX).
Species
No. LOPU
COC
A-COC
MAX
LOPU Technichian
Acinonyx jubatus
12
277
23,1
35
Donoghue et al, 1992
Antidorcas marsupialis
10
73
7.3
15
Baldassarre Unpublished
Cervus elaphus
36
44
1,2
N/A
Bainbridge et al, 1999
Cervus nippon nippon
96
348
3,6
N/A
Locatelli et al, 2006
C. nippon pseudaxis
16
94
47
N/A
Locatelli et al, 2012
Connochaetes taurinus
118
75
7,5
N/A
Baldassarre Unpublished
Cuniculus pacas
30
155
5,2
N/A
Barros et al, 2016
Damaliscus pygargus phillipsi
12
83
6.9
16
Baldassarre Unpublished
Felis chaus
8
115
14,3
N/A
Pope et al, 1993
Felis nigripes
1
31
31
31
Pope et al, 1993
Felis silvestris ornata
9
134
14,9
N/A
Pope et al, 1993
Gazella dama mhorr
6
35
5,8
11
Berlinguer et al, 2008
Leptailurus serval
9
234
26
N/A
Pope et al, 2005
Odocoileus virginianus
43
851
19.8
85
Baldassarre Unpublished
Oryx gazela
15
49
3.3
6
Baldassarre Unpublished
Connochaetes taurinus
10
75
7.5
15
Baldassarre Unpublished
Panthera leo
3
26
8,7
12
Armstrong et al, 2004
Panthera onca
8
116
14,5
33
Baldassarre/Requena
Unpublished
Panthera tigris
11
384
34,9
62
Crichton et al, 2002
Panthera tigris
16
456
28,5
52
Donoghue et al, 1990
Prionailurus viverrinus
2
107
53,5
N/A
Pope et al, 1993
Puma concolor
12
421
35,1
106
Baldassarre/Requena
Unpublished
Puma concolor
7
140
20
52
Miller et al, 1990
CONCLUSIONS
The recovery of oocytes from live animals is of great
importance for the production of embryos by PIVE and also for
SCNT, and LOPU is the procedure of choice for the recovery of
oocytes from species and categories where transvaginal
ultrasound-guided aspiration is difficult or not possible, such as
small ruminants, prepubertal bovine and buffalos, and a range
of wild species. The procedure is safe and efficient, resulting in
high oocyte numbers and quality. LOPU can be repeated in the
same female multiple times as it doesn’t cause sequels with
negative impact on the female's reproductive life, even when
performed on prepubertal or wild animals. It has realistic
commercial application in sheep, goats, prepubertal cattle and
buffaloes. It can also be used for species conservation.
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Fotos anexas.
Figure 1. LOPU-Jaguar. Dr. Baldassarre conducting
LOPU in jaguars (Panthera onca)
Figure 2. Ovario-Puma. Superestimated Ovary of Puma
concolor, that result in 106 oocytes in a single collection.
DOI. 10.18548/aspe/0006.07 Jorge Neto PN, Alecho Requena L, Schilbach Pizzutto C, Baldassarre H. SPERMOVA. 2018; 8(1): 61-67
67
Figure 3. Oócitos-Puma.106 oocytes recovered from a
single donor from Puma concolor.
Figure 4. LOPU-Búfala. First LOPU performed on 3 month
old buffalo calf in Brazil , conducted by Drs. Baldassarre,
Requena and Jorge Neto (Dec, 2014).
Figure 5. LOPU-Puma. Dr. Requena conducting LOPU in
Puma Concolor that resulted in 106 oocytes in single
collection
Figure 6. LOPU-Saanen.Commercial LOPU conducted by
Dr. Requena in goat saanen.
... Data for goats has been reported to vary from 4.3 to 18 oocytes (Cognie, 1999, Graff et al., 1999, Samaké et al., 2000. Recent reports on LOPU for several species (Baldassarre, 2021, Neto et al., 2018 have . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
... ; https://doi.org/10. 1101/2022 indicated the advantages of collecting good quality oocytes for IVEP; oocyte crops vary from 10 to 15 oocytes (Baldassarre, 2021), whereas, the oocytes collected vary from 1.2 to 6 for deer and unpublished results for oocyte collection from WTD hinds of 19.8 oocytes (see review by Neto et al., 2018). Further, data for IVEP in these two latter reports was not provided. ...
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Assisted reproductive technologies (ARTs), such as artificial insemination, semen sorting and freezing, embryo production in vivo and in vitro, are all methods in which animal reproduction management of several species has become considerably more efficient. These ARTs have been applied both in domestic and wild ruminants. In this study, in vitro embryo production was attempted with oocytes collected surgically during the mating season, from live white-tailed deer (WTD) hinds, maintained under captivity, in Northeast Mexico. The study was conducted in two WTD farms nearby Cd. Victoria, Tamps., Mexico. The laboratory work was carried at the Centro de Desarrollo de la Capacidad Productiva y Mejoramiento Genetico de la Ganaderia, property of the Union Ganadera Regional de Tamaulipas (Centro-UGRT). The deer hinds are kept in captivity year-around, with feeding and health management provided accordingly to their requirements (50-60 kg females). Fresh green forage and clean fresh water were supplied daily. Oocytes were collected on each farm, while the hinds were maintained under general anesthesia and by means of a mid-ventral laparotomy, ovaries were exposed and follicles greater than 1-2 mm were aspirated with a 20 G needle, connected by a two-way plastic and latex hose to a vacum machine (WTA, Brazil); the oocytes were collected into a 50 ml. centrifuge tube containing wash media with heparine (Vitrogen, Brazil). Each oocyte collection lasted approximately 20 minutes, after which and while still on the farm, the oocytes were filtered with 50 microns mesh filters and rinsed several times with wash media, then placed on search Petri plates. Oocytes were then counted and classified (total, viable and non-viable oocytes) and placed into cryovials with maturation media (3 ml. MIV, Vitrogen, Brazil) and placed into a portable incubator with 5 % CO2 gas mix (LabiMix WTA, Brazil), after all hinds were done, oocytes were transported to the in vitro fertilization laboratory (IVF Lab) at the Centro-UGRT. Once at the IVF Laboratory, the oocytes were placed on a larger incubator (Eve, WTA, Brazil) and kept there for 18-20 hours, after which, the MIV media was changed for fertilization media (FIV, Vitrogen, Brazil) and fertilization was initiated by adding 10,000-12,000 live sperm per cryovial, and incubated for an additional period of 24 hours; after which, FIV media was replaced by the same media and at this point, cleavage rate was estimated by counting the oocytes that initiated cell division. At 72 hours after cell division started, fertilization rate was estimated; and 7 days after, the blastocysts were counted and classified. The whole process of oocyte maturation and embryo production in vitro, was conducted based on a beef cattle embryo production system and adapted to a deer embryo production system using media for small ruminants (Vitrogen, Brazil). Data collected per hind included total number of oocytes, viable and non-viable oocytes, cleavage rate (ratio of viable oocytes that initiated cell division over viable oocytes), fertilization rate (ratio of embryos that initiated cleavage over those that continued development to the blastocyst stage) and blastocyst rate (embryos reaching the blastocyst stage over cleaved embryos); averages were calculted for each parameter. The main results from this study on a per hind basis for total viable and non-viable oocytes were 9.8, 6.3 and 4.5, respectively; cleavage and blastocyst rates were 39.5 and 36.8, respectively and 2.3 blastocysts. In conclusion, oocyte collection from live WTD hinds and in vitro embryo production were succesfully done under farming conditions in Northeast Mexico. Key words: White-tailed deer, ARTs, oocytes, embryos, ovum pick-up and in vitro fertilization.
... However, in species that are too small for oocyte collection via OPU (e.g., sheep, goat, deer), a laparoscopic ovum pick-up (LOPU) procedure was developed in the early 90s [10]. Since then, it has been refined and adapted for use in a wide range of both domestic and wild species [10,11,12,13,14,15,16,17,18]. The LOPU approach has several advantages over OPU, including that the ovary is viewed directly with a depth of field, rather than on a two-dimensional sonogram, enabling superficial follicles to be aspirated accurately without risking injury to the ovarian stroma [19]. ...
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Laparoscopic ovum pick-up (LOPU) coupled with in vitro embryo production (IVEP) in prepubertal cattle and buffalo accelerates genetic gain. This article reviews LOPU-IVEP technology in prepubertal Holstein Cattle and Mediterranean Water Buffalo. The recent expansion of genomic-assisted selection has renewed interest and demand for prepubertal LOPU-IVEP schemes; however, low blastocyst development rates has constrained its widespread implementation. Here, we present an overview of the current state of the technology, limitations that persist and suggest possible solutions to improve its efficiency, with a focus on gonadotropin stimulations strategies to prime oocytes prior to follicular aspiration, and IVEP procedures promoting growth factor metabolism and limiting oxidative and endoplasmic reticulum stress.
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Big cats are apex predators with an essential role in global ecosystems. The One Conservation concept proposes artificial reproduction as one of the tools to reduce the vulnerability of these species. This manuscript aimed to assess what is new in big cat reproduction in the last decade. Knowledge of reproductive physiology and behavior is the first step towards developing reproductive technologies in wild animals. In big cats, copulatory behavior is of fundamental importance because they need ovulation induction mechanisms, which can be mechanical, sensory, or via the administration of the luteinizing hormone. The success in neonatal care represents the success of reproductive technology in females. In the last decade, successful artificial insemination was only reported in Siberian tigers and Anatolian leopards. Jaguar artificial insemination focuses on research at the Reprocon Institute, exchanging genetic material between in situ and ex situ environments thru artificial insemination. The technique of choice is laparoscopic ovum pick-up to obtain high-quality viable oocytes. The production of in vitro embryos faces challenges for the efficient maturation of oocytes and their efficient vitrification. Reproductive technologies need in-depth studies in big cats to achieve the repeatability necessary for efficient application in conservation.
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The potential of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production (IVEP) as a tool for accelerated genetic programs in ruminants is reviewed in this article. In sheep and goats, the LOPU-IVEP platform offers the possibility of producing more offspring from elite females, as the procedure is minimally invasive and can be repeated more times and more frequently in the same animals compared with conventional surgical embryo recovery. On average, ~10 and ~14 viable oocytes are recovered by LOPU from sheep and goats, respectively, which results in 3–5 transferable embryos and >50% pregnancy rate after transfer. LOPU-IVEP has also been applied to prepubertal ruminants of 2–6 months of age, including bovine and buffalo calves. In dairy cattle, the technology has gained momentum in the past few years stemming from the development of genetic marker selection that has allowed predicting the production phenotype of dairy females from shortly after birth. In Holstein calves, we obtained an average of ~22 viable oocytes and ~20% transferable blastocyst rate, followed by >50% pregnancy rate after transfer, declaring the platform ready for commercial application. The present and future of this technology are discussed with a focus on improvements and research needed.
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ABSTRACT The aim of this work is study the laparoscopic ovum pick-up (LapOPU) technique in spotted paca, describing surgery details, complications and oocyte recovery rate. Nine healthy adult non-pregnant captive females were used, in a total of 39 procedures. When the surgical plane of anaesthesia was achieved, the females were positioned at 20º Trendelenburg. Three 6mm trocars were placed on right and left inguinal and hypogastric regions. Abdomen was inflated with CO2 and the intra-abdominal pressure was stablished in 10mmHg. Follicular punctures were performed moving the ovaries with atraumatic forceps. For punctures, an 18-gauge 3.5 inch long needle attached to a vacuum system with pressure not exceeding 65mmHg was used. Oocytes were recovered into 50mL centrifuge tubes with media composed of PBS supplemented with 10 IU/mL of heparin and kept at 36°C. R Software was used for statistical analysis. Data normality distribution (Shapiro test) and variances homoscedasticity (Bartlett test) were tested and descriptive statistics (mean±SD) was used to present the results. It was only possible to perform LapOPU in 30 of 39 laparoscopies (76.92%). The surgical total time was 37.34 ± 18.53 minutes. The total number of visualized follicles, aspirated follicles, and retrieved oocytes were 502, 415, and 155, respectively. And the same parameters per animal were: 14.34 ± 12.23, 11.86 ± 10.03, and 4.43 ± 4.69 respectively. Oocyte recovery rate was 32.56 ± 27.32%. In conclusion, caudal positioning of portals with slight triangulation allows good viewing of the abdominal cavity and eases the manipulation of the ovaries. Thus this described LapOPU technique is feasible in spotted paca and easy to perform.
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A comparison of the amino acid sequences demonstrated that Siberian tiger gonadotropins are more homologous with those of porcine than any other commercially available preparation. The present study measured the efficacy of repeated ovarian stimulation with purified porcine gonadotropins on the follicular, hormonal, and immunogenic responses in Siberian tigers as well as on the ability of oocytes retrieved by laparoscopic follicular aspiration to fertilize and cleave in vitro. Controlled rate and vitrification cryopreservation methods were also compared for their ability to support ongoing cleavage following thawing of presumptive 2- to 4-cell tiger embryos generated in vitro. Vitrification supported continued embryonic cleavage in vitro while controlled rate freezing did not. Stereological microscopy indicated an excellent ovarian response with the recovery of quality cumulus-oocyte complexes that apparently fertilized and cleaved in vitro. However, ultrastructural and physiological examination revealed abnormal and unnatural responses such as the failure of some cumulus-oocyte complexes to reach maturity and progestagen levels to approach normalcy. At the same time, analyses of blood for antibodies failed to detect an immune reaction to these foreign gonadotropins in an assay that tested positive for the chorionic gonadotropin-stimulated domestic cat. Together, these observations suggest that porcine gonadotropins may be effective for the ovarian stimulation of tigers but that some modifications to administration protocols are needed to produce a more natural response.
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In mammals, recovery of oocytes by laparoscopic ovum pick-up (LOPU) coupled with in vitro production (IVP) of embryos represents a promising strategy for both amplification and genetic management of sparse animals from captive endangered wild species. As integrated technique developed mainly for domestic livestock, LOPU-IVP requires several studies to set up protocols for follicular stimulation or optimization of IVP before envisaging successful transposition to wild species. In deer, many endangered subspecies would be potentially concerned by applying such an approach using common subspecies for protocols optimization. The aim of the present study was to assess efficiency of follicle stimulation using ovine FSH (oFSH) for recovery of oocytes by LOPU in common sika deer (Cervus nippon nippon) before transposition of an optimized methodology for IVP of embryos from endangered Vietnamese sika deer hinds (Cervus nippon pseudaxis). In common sika deer, two doses of oFSH (0.25 and 0.5 U) and two frequencies of administration (12 and 24 h) were compared by monitoring of subsequent ovarian response, quality of oocytes recovered by LOPU, and in vitro developmental competence. In a first experiment, the dose of oFSH administered did not significantly affect the total number of follicles aspirated per hind per session (8.6 ± 1.0 vs. 8.2 ± 1.6 with 0.5 vs. 0.25 U oFSH, respectively; not significant). In a second experiment, frequency of 0.25 U oFSH administration did not affect ovarian response. Efficiency of IVP determined on blastocysts rates after in vitro maturation, fertilization, and development in oviduct epithelial cells coculture was increased when FSH was administered at 12-h intervals. Immune response after several follicular stimulations was detected against exogenous oFSH in plasma from the majority of sika deer hinds but was not associated with decreased ovarian response. When 0.25 U oFSH was administered at 12-h intervals to Vietnamese sika deer (N = 4), good quality cumulus oocyte complexes with complete and compact cumulus investments were recovered allowing a high cleavage rate after in vitro maturation and fertilization. Development to the blastocyst stage occurred in a high proportion (30% of oocytes) after coculture with ovine epithelial cells allowing cryobanking of transferable embryos from Vietnamese sika deer. These results confirm that LOPU-IVF after ovarian stimulation with oFSH may be a successful tool for cryobanking transferable embryos from endangered sika deer subspecies.
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A simple and inexpensive pipette for in vivo recovery of sheep oocytes by folliculocentesis was developed. Two experiments were conducted to assess the recovery rate. In Experiment 1, 20 Merino × Corriedale ewes were heat synchronized using intravaginal sponges containing 60 mg of medroxyprogesterone acetate (MPA) for a period of 12 days. In Experiment 2, 26 Merino × Corriedale ewes were synchronized with CIDR® devices for 12 days, with the original device being replaced by a new one on Day 10 (2 days before withdrawal). In both experiments, ewes were superovulated with a total dose of 16 mg of follicle stimulating hormone (FSH) given in six decreasing dosage injections, starting 48 h before sponge/CIDR removal. Folliculocentesis was performed 36–40 h after sponge removal, and 16–20 h after CIDR removal. Overall, an average of 12.5 follicles per ewe were punctured and 10.2 oocytes per ewe were recovered (recovery rate 81.9%). The differences between the two experiments in terms of follicles punctured per ewe (10.8 vs. 13.7) and oocytes recovered per ewe (8.5 vs. 11.5) were not statistically significant (P>0.05). The follicle size did not significantly (P>0.05) affect the recovery rate, although there was a tendency for higher rates of recovery from 1–5 mm follicles (88.5%) compared with follicles over 5 mm (79.5%).
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Most current protocols of in vitro fertilization in ruminants are based on in vitro maturation of oocytes derived from abattoir material. For application of IVF technology to captive endangered species, however, noninvasive techniques are required which allow repeated collection of oocytes from live females. The aim of this study was to develop a method for embryo production from mature oocytes collected laparoscopically from red deer hinds. Follicular development was synchronized in red deer hinds by the insertion of intravaginal progesterone-releasing devices for 10 d, and ovarian stimulation was induced with 1000 IU, i.m. PMSG 48 h before progesterone device removal. Oocytes were harvested by laparoscopy under xylazine/ketamine sedation 24 h after progesterone device removal and then co-incubated with frozen-thawed red deer spermatozoa for 24 h. In Experiment 1, oocytes and embryos were fixed and stained at different developmental timepoints. Their external morphological changes (cumulus expansion, extrusion of the second polar body and cytokinesis) paralleled their nuclear developmental changes (formation of the 2nd metaphase spindle of meiosis, pronuclear formation and nuclear division, respectively). In Experiment 2, embryos were maintained in vitro until they ceased to undergo cell division. A total of 39 aspiration procedures was carried out on 14 red deer hinds. Forty-four cumulus-oocyte complexes (COC) were aspirated from 95 large Graafian follicles; of these, 27 were classed as mature/nondegenerated on the basis of cumulus/cytoplasmic morphology. Seventeen oocytes cleaved following in vitro fertilization, yielding six 2-cell embryos, six 4-cell embryos, four 8-cell embryos and one 16-cell embryo. The results indicate that laparoscopic aspiration of mature oocytes from hormone-treated females offers a valuable source of genetic material for assisted deer breeding programs.
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Embryo production by in vitro techniques has increased steadily over the years. For cattle where this technology is more advanced and is applied more, the number of in vitro produced embryos transferred to final recipients was over 30,000 in 1998. An increasing proportion of in vitro produced embryos are coming from oocytes collected from live donors by ultrasound-guided follicular aspiration (ovum pick up, OPU). This procedure allows the repeated production of embryos from live donors of particular value and is a serious alternative to superovulation. Ovum pick up is a very flexible technique. It can be performed twice a week for many weeks without side effects on the donor's reproductive career. The donor can be in almost any physiological status and still be suitable for oocyte recovery. A scanner with a sectorial or convex probe and a vacuum pump are required. Collection is performed with minimal stress to the donor. An average of 8 to 10 oocytes are collected per OPU with an average production of 2 transferable embryos. The laboratory production of embryos from such oocytes does not differ from that of oocytes harvested at slaughter as the results after transfer to final recipients. For other species such as buffalo and horses OPU has been attempted similarly to cattle and data will be presented and reviewed. For small ruminants, laparotomy or laparoscopy seems the only reliable route so far to collect oocytes from live donors.
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With an increased interest in transgenic animal production, the caprine species offers many advantages, and the prepubertal goat is a potential source of large numbers of oocytes for in vitro embryo production. The aim of the present study was to evaluate the follicular response and recovery of oocytes from prepubertal and adult goats following ovarian stimulation and laparoscopic recovery, and their developmental competence following culture in semi-defined media. Oocytes were collected over a 15-week period from prepubertal goats (3-7 months old) and adult controls (2-4 years old) that had been subjected to estrus synchronization and ovarian stimulation. Following insemination, zygotes were cultured for 96h in G1.2 followed by an additional 120h in G2.2. Morulae and blastocysts were scored using light microscopy on Days 7 and 9 followed by fluorescent staining for cell counts on Day 9 (216h postinsemination). The mean numbers of follicles aspirated and oocytes recovered were significantly greater for prepubertal than for adult goats (P<0.01). The number of oocytes recovered from prepubertal goats was observed to decline significantly with increasing age of the animals (P<0.05). The proportion of oocytes that matured and cleaved did not differ significantly between prepubertal and adult goats. Furthermore, no significant differences in morulae development (percentage of those cleaved), 5% versus 4%, or blastocyst development, 6% versus 7%, were observed for prepubertal and adult derived oocytes (P>0.1), respectively. Mean cell number per blastocyst also did not differ significantly. In conclusion, higher yields of oocytes were obtained from gonadotrophin-primed, prepubertal does than from adults, while in vitro development was similar.