248 SUPERSTIMULATION STRATEGIES FOR OVUM PICKUP IN HOLSTEIN DONORS

2016 ◽  
Vol 28 (2) ◽  
pp. 256
Author(s):  
L. M. Vieira ◽  
G. A. Bó ◽  
R. J. Mapletoft
Keyword(s):  
Oocyte Quality ◽  
Control Group ◽  
Limiting Factor ◽  
Total Dosage ◽  
In Vitro Production ◽  
Total Period ◽  
Expected Time ◽  
Follicular Wave ◽  
Vitro Production

In vitro embryo production (IVP) is an important tool to enhance genetic gain in cattle. However, oocyte quality is a limiting factor for the success of IVP programs in high-producing donors. A series of studies using protocols for follicular wave synchronization and superstimulation before ovum pickup were performed to improve the efficiency of ovum pickup and in vitro production in dairy cattle. The first study evaluated superstimulation with FSH (Folltropin-V®) before ovum pickup in lactating (n = 15) and non-lactating (n = 15) Holstein donors in a crossover design. Cows underwent synchronization of follicle wave emergence (FWE) and at the expected time of FWE, the FSH group received a total dosage of 200 mg of FSH in 4 decreasing doses 12 h apart; controls received no FSH, and ovum pickup was conducted 72 h after the expected FWE in all cows. The FSH-treated cows had a higher (P < 0.01) percentage of medium-sized follicles (6 to 10 mm) at the time of ovum pickup (55.1%) than control cows (20.8%) as well as lower cumulus‐oocyte complexes (COC) recovery rates (60.0 v. 69.8%, respectively; P = 0.002). However, FSH-treated cows had a higher blastocyst production rate (34.5 v. 19.8%; P < 0.01) and more transferable embryos per ovum pickup session (3.0 ± 0.5 v. 1.8 ± 0.4; P = 0.02). Subsequent trials evaluated plasma FSH profiles in 23 heifers and in vitro production following ovum pickup in 90 non-lactating Holstein donors superstimulated with a single IM injection of FSH in 0.5% hyaluronan (HA; MAP-5®, 50 mg). Controls received no treatment, while the F200 group received 200 mg of FSH in 4 decreasing doses 12 h apart. The F200HA and F300HA groups received 200 or 300 mg of FSH in 5 or 7.5 mL, respectively, of 0.5% HA by a single IM injection. Circulating FSH area under curve (AUC) in FSH-treated animals was greater than in the control group (P = 0.02). Although the AUC for F200 group did not differ from HA groups (P = 0.56), the total period of time plasma FSH levels were elevated was greater than in the HA groups (P < 0.01). In the IVP trial, FSH-treated cows had a greater proportion of medium-sized (6–10 mm) follicles than controls (P < 0.001). Also, numbers of follicles (P = 0.01) retrieved (control: 13.1 ± 1.0; F200: 16.5 ± 1.2; F200HA: 19.5 ± 2.1; F300HA: 15.4 ± 1.4; P = 0.01) and blastocysts produced per ovum pickup session (control: 2.4 ± 0.5; F200: 3.7 ± 0.7; F200HA: 4.7 ± 0.7; F300HA: 3.1 ± 0.6; P = 0.06) were greater in cows receiving FSH, regardless of treatment. Cows in the F200HA group had a greater recovery rate (P = 0.009), number of COC cultured (P = 0.04), and blastocysts per ovum pickup session (P = 0.06) than cows in the F300HA group. In conclusion, superstimulation of Holstein donors before ovum pickup increased the efficiency of in vitro production. Additionally, a single IM dose of FSH in 0.5% HA resulted in similar plasma FSH profiles to twice-daily FSH treatment. Non-lactating donors treated with FSH produced more embryos per ovum pickup session regardless of FSH treatment. Lastly, all in vitro-produced endpoints were greater following a single dose of 200 mg of FSH in 0.5% HA than 300 mg of FSH in 0.5% HA.

scholarly journals Hormonal protocols for in vitro production of Zebu and taurine embryos

2014 ◽  
Vol 49 (10) ◽  
pp. 813-817 ◽  
Author(s):  
Carlos Antônio de Carvalho Fernandes ◽  
Tatimara Maria Miyauchi ◽  
Ana Cristina Silva de Figueiredo ◽  
Miller Pereira Palhão ◽  
Fabiana Cristina Varago ◽  
...  
Keyword(s):  
Bos Indicus ◽  
Follicular Development ◽  
Cold Season ◽  
Control Group ◽  
In Vitro Production ◽  
Follicular Wave ◽  
Different Seasons ◽  
Pre Treatment ◽  
Vitro Production

The objective of this work was to evaluate the effects of hormonal synchronization protocols, associated or not with follicular development stimulation, on the recovery of oocytes and on in vitro production of Bos indicus and B. taurus embryos, in different seasons. Ultrasound-guided follicular aspirations (n=237) were performed without pre-treatment (G1, control group) and after follicular wave synchronization (G2), or after follicular wave synchronization and follicle growth induction (G3). Bos indicus produced more oocytes and embryos than B. taurus (18.7±0.9 vs. 11.9±0.6 oocytes and 4.8±0.3 vs. 2.1±0.2 embryos). On average, oocyte and embryo yields were higher in G3 than in G2, and both were greater than in G1, which lead to a higher conversion of oocytes to embryos in these treatments. The hot or the cold season did not affect the B. indicus outcomes, whereas, in B. taurus, both oocyte recovery and embryo production were higher in the cold season. Follicular wave synchronization improves ovum pick-up and in vitro production of embryos in both cattle subspecies evaluated.

298 SPERM CAPACITATED WITH CALCIUM IONOPHORE AS A VECTOR FOR IN VITRO PRODUCTION OF BOVINE TRANSGENIC EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 256
Author(s):  
R. Simões ◽  
M. P. Milazzotto ◽  
C. Yamada ◽  
W. B. Feitosa ◽  
A. R. S. Coutinho ◽  
...  
Keyword(s):  
Cell Monolayer ◽  
Calcium Ionophore ◽  
Control Group ◽  
Sperm Cells ◽  
In Vitro Production ◽  
Successful Technique ◽  
Blastocyst Rate ◽  
Transgenic Embryos ◽  
Vitro Production

Production of transgenic mouse embryos by microinjection is a well established and successful technique. However, when microinjection protocols were used for bovine, the amount of the oocyte lipid content did not allow the production of bovine transgenic embryos. Sperm-mediated gene transfer (SMGT) is an alternative for this species because it has lower cost and does not require microinjection handling. One of the procedures to introduce exogen DNA into oocytes is by means of sperm capacitated with calcium ionophore (CaI). The aim of this work was to evaluate different CaI concentrations ([CaI]), sperm incubation times with CaI (tCa), and incubation times of sperm capacitated with DNA (tDNA) (EYFP; Clontech, Palo Alta, CA, USA) to establish a satisfactory method for IVP of bovine transgenic embryos. Slaughterhouse oocytes with compact cumulus and uniform ooplasm were in vitro maturated in TCM-199 medium + 10% FCS + FSH + hCG + estradiol (E2) + piruvate + gentamicin under 5% CO2 in air, at 39�C and high humidified atmosphere for 24 h. Semen was thawed in a water bath at 37�C for 30 s and separated by Percoll gradient (45/90%) at 600g for 30 min. After this procedure, sperm cells were washed in TALP-semen medium by centrifugation at 200g for 5 min at room temperature. Supernatant was removed and capacitation (5 � 106 spermatozoa/group) was induced with CaI (250 nM or 500 nM for 1 or 5 min). Capacitated sperm cells were incubated with 500 ng/mL DNA for 1 or 2 h. Nontreated spermatozoa were used as control group. Sperm cells (1 � 105) were used to inseminate 20 oocytes/90 mL microdroplets for 18 h. The presumptive zygotes were co-cultured in SOFaa medium with a granulosa cell monolayer under high humidified atmosphere, at 39�C and 5% CO2 in air. Blastocyst rates were analyzed by ANOVA. Independent variables were replicate, [CaI], tCa, tDNA, and the double and triple interactions among the last three variables; when appropriate, means were compared by orthogonal contrasts. There was [CaI] � tCa � tDNA interaction for blastocyst rate (P < 0.02). Treatments with 250 nM ([CaI]), 5 min (tCaI), and 1 h (tDNA) or 500 nM ([CaI]), 1 min (tCaI), and 1 h (tDNA) resulted in 36.1% and 37.4% blastocyst rates, respectively, similar to the control group (30.5%; P > 0.4). These results demonstrated that it is possible to capacitate spermatozoa with CaI to produce transgenic embryos, without alteration of blastocyst rate. This work was supported by FAPESP 03/08542-5 and 03/07456-8.

scholarly journals Use of Melatonin in the In Vitro Production of Bovine Embryos

2020 ◽  
Vol 21 ◽  
Author(s):  
Alan da Silva LIRA ◽  
Ricardo de Macedo CHAVES ◽  
Felipe de Jesus MORAES JUNIOR ◽  
Sergio Henrique COSTA JUNIOR ◽  
Brenda Karine Lima do AMARAL ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Significant Difference ◽  
Maturation Rate ◽  
Vitro Production

ABSTRACT We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

scholarly journals Synchronization with estradiol benzoate in the presence of the corpus luteum in Wagyu cows increases the number of medium follicles but does not interfere with in vitro production of embryos

2021 ◽  
Vol 42 (3) ◽  
pp. 1147-1158
Author(s):  
Maria Fernanda Zamai ◽  
◽  
Fábio Luiz Bim Cavalieri ◽  
Marcia Aparecida Andreazzi ◽  
Fabio Morotti ◽  
...  
Keyword(s):  
Corpus Luteum ◽  
Estradiol Benzoate ◽  
Breeding Systems ◽  
In Vitro Production ◽  
Embryo Production ◽  
Follicular Wave ◽  
In Vitro Embryo Production ◽  
Oocyte Donors ◽  
Vitro Production

Reproductive biotechnologies are emerging as an important element for livestock; however, some strategies must be modified to adapt to different breeding systems, such as the use of follicular synchronization protocols. This study aimed to evaluate follicular synchronization using estradiol benzoate (EB), in the presence of the corpus luteum (CL) from Wagyu oocyte donors in in vitro embryo production (IVEP). Rounds of IVEP were performed in heifers and cows (n=19) that were classified into three groups: G1/CL - animals with CL, G2/WCL - animals without CL, and G3/CL + EB - animals with CL that were subjected to follicular synchronization with EB at D0. The groups G1/CL and G2/WCL were considered the control and undertook the natural process of follicular dynamics. The results showed that the synchronization of the follicular wave with the application of EB in the presence of CL, presented a smaller number of small (6.05 ± 0.55) and large follicles (0.45 ± 0.15), but increased (P < 0.05) the number of medium-sized follicles (16.20 ± 0.90). However, the results of ovum pick up showed that regardless of whether or not EB was applied, and regardless of the presence or absence of CL in the Wagyu donor, there was no difference among the groups (P > 0.05) concerning the number of viable oocytes and the viability rate. It was concluded that follicular synchronization using EB in Wagyu oocyte donors that presented a CL, increased the number of medium-sized follicles. However, there was no improvement in the efficiency of ovum pick up, in vitro embryo production, and pregnancy rate.

278 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND SUPERSTIMULATION ON THE NORMALITY OF BOVINE EMBRYOS PRODUCED IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 296 ◽  
Author(s):  
K. Imai ◽  
T. Somfai ◽  
M. Ohtake ◽  
Y. Inaba ◽  
Y. Aikawa ◽  
...  
Keyword(s):  
Cell Division ◽  
Development Program ◽  
Time Lapse ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Cell Stage ◽  
Follicular Wave ◽  
Significant Difference

We previously reported that follicular wave synchronization by dominant follicle removal on Day 5 and the start of a superstimulatory treatment on Day 7 after ovum pick-up (OPU) was effective to increase oocyte quality (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). The present study was designed to examine the effect of superstimulatory treatment-induced follicular wave synchronization on quality of embryos obtained by OPU and in vitro production. Japanese Black cows were reared under the same feeding and environmental conditions and 2 OPU sessions were conducted in each cow. The first OPU session was performed in 7 cows at arbitrary days of estrous cycle using a 7.5-MHz linear transducer with needle connected to an ultrasound scanner. Then, follicles larger than 8 mm in diameter were aspirated and CIDR was inserted on Day 5 (the day of first OPU session = Day 0). The cows then received 30 mg of FSH twice a day from Days 7 to 10 in decreasing doses (4, 4, 3, 3, 2, 2, 1, 1 mg per shot) by i.m. injections. Cloprostenol (PGF; 0.75 mg) was administered in the morning of Day 9. The second OPU session was performed 48 h after PGF administration (Day 11) and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Grade 1 and 2 cumulus oocyte complexes were in vitro matured, fertilized (IVF), and cultured as described by Imai et al. (2006 J. Reprod. Dev. 52, Suppl. S19-29). Some zygotes were fixed and stained to check their sperm penetration. Embryo development was monitored by time-lapse cinematography for 168 h after IVF. Cleavage pattern of embryos was classified morphologically into normal and abnormal (including those with multiple fragments, protrusions, 3 to 4 blastomeres, and uneven cell division) groups at their first cleavage. Normal penetration rate of second OPU session was significantly (P < 0.05) higher than that of the first OPU session. There were no differences in the mean percentage of total blastocyst and grade 1 blastocyst rates between the first (45.2 and 46.9%, respectively) and second (47.5 and 41.8%, respectively) OPU sessions. However, the rates of blastocysts developing from embryos that were beyond the 4-cell stage at 48 h after IVF was significantly (P < 0.05) higher after the second OPU session (81.2%) than after the first OPU session (67.4%). Furthermore, a significant difference (P < 0.05) was found in the rates of normal cleavage at the first cell division in embryos that developed to the blastocyst stage between the first and second OPU sessions (53.3% and 73.9%, respectively). These results indicate that superstimulatory treatment-induced follicular wave synchronization improved the normality of fertilization and development of cattle oocytes obtained by OPU. This work was supported by the Research and Development Program for New Bio-industry Initiatives.

scholarly journals Effect of Continuous Administration of Enalapril Maleate on the Oocyte Quality and In Vitro Production of Parthenote Embryos in Nulliparous and Multiparous Goats Undergoing Serial Laparoscopic Ovum Pick-Up

Animals ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 868
Author(s):  
Pamela A. Bravo ◽  
Maria E. Moreno ◽  
César C.L. Fernandes ◽  
Rafael Rossetto ◽  
Camila M. Cavalcanti ◽  
...  
Keyword(s):  
Ovarian Response ◽  
Oocyte Quality ◽  
In Vitro Production ◽  
Continuous Administration ◽  
Enalapril Maleate ◽  
Vitro Production ◽  
To Receive ◽  
Intravaginal Device

The aim of this work was to determine the effect of enalapril maleate administration, during oocyte recovery by serial laparoscopic ovum pick-up (LOPU), on the ovarian response and in vitro embryo production (IVP). Twenty cross-bred goats were allocated equally into two groups: Nulliparous and Multiparous. In each group, five animals were selected to receive daily doses of enalapril maleate during the hormonal protocol. Estrus was synchronized by a PGF2α analog, followed 48 h later by insertion of an intravaginal device with progesterone. Forty-eight hours after, a single dose of FSH/eCG was administered. The FSH/eCG doses were repeated three times, on every four day. Oocytes were recovered by LOPU 24 h after each FSH/eCG dose. Viable oocytes were matured in vitro, to be parthenogenetically activated and cultured for 72 h to the cleavage stage. The drug treatment increased the proportion of total follicles observed at LOPU (p < 0.01) in multiparous goats. In both parity groups, enalapril administration had no effect on the proportion or quality of oocytes recovered. Furthermore, the number of embryos cleaved was similar between the groups. Thus, enalapril maleate affected the ovarian response in multiparous animals only and had no effect on the oocyte quality or IVP.

scholarly journals Effect of immunocytotherapy on the state of the immune system of women with idiopathic habitual miscarriage

2020 ◽  
Vol 22 (4) ◽  
pp. 751-764
Author(s):  
L. V. Krechetova ◽  
V. V. Vtorushina ◽  
E. V. Inviyaeva ◽  
L. V. Vanko ◽  
M. A. Nikolaeva ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Th17 Cells ◽  
Peripheral Blood Lymphocytes ◽  
Inflammatory Factors ◽  
Control Group ◽  
In Vitro Production ◽  
Th17 Response ◽  
Low Level ◽  
Vitro Production

We aimed  for assessing effects of immunocytotherapy upon  the subpopulations of CD4+CD25highFoxP3+ cellswithnaturalregulatoryactivityandactivatedTh17cellswiththeCD4+CD25highRORγt+ phenotype, as well as in vitro production of cytokines in mitogen-stimulated cells from  peripheral blood  in the patients with idiopathic habitual miscarriage (IHM). The study group consisted of 33 patients with IHM who became pregnant after a pre-gestational alloimmunization. In 27 patients, the pregnancy was prolonged to the  full term  and  ended  with the  birth  of viable babies,  in six cases it was terminated before  12 weeks of gestation. Before  administration of immunocytotherapy (ICT), 19 patients were examined, of them  16 after alloimmunization outside of pregnancy, 17 at 5-6 and 8-9 weeks of pregnancy. Eleven patients were immunized at 12 weeks of pregnancy. In the control group,  12 fertile women  outside  pregnancy and 10 women  at 12 weeks of physiological pregnancy were examined. The proportion of FoxP3+ and RORγt+ cells with the CD4+CD25high phenotype was evaluated among  T-lymphocytes from peripheral blood,  as well as content of proinflammatory cytokines (IFNγ,  TNFα, IL-1β, IL-2, IL-5, IL  -6,  IL-8, IL-12p70) and  anti-inflammatory factors  (IL-4, IL-10), as well as IL-17  amounts.We have found  that,  following pre-gestational alloimmunization, the women  who lost this pregnancy, had a  low  level  of  FoxP3+Тregs that  suppress  pro-inflammatory Th17-dependent  reactions, however, without changing levels of activated Th17  cells (CD4+CD25highRORγt+   lymphocytes). These  facts,  along  with  high in vitro production of IL-17  by peripheral blood cells at the terms of 5-6 weeks of gestation, suggest that,  after pre-gestational alloimmunization in women  with miscarriage, a predilection is formed  to pro-inflammatory cytokine production. However, at the 5-6 week-period, it is realized  not in the Th1 direction of, but towards Th17 response, and a low level of CD4+CD25highRORγt+ cells may reflect an increased migration of Th17 cells from peripheral blood to the uterine endometrium.Thus,  we have shown  the  effect of immunocytotherapy upon  subpopulational composition of peripheral blood  lymphocytes and  the  cytokine profile,  as well as upon  the  course  of first trimester and  outcomes of pregnancy in women  with idiopathic habitual miscarriage.

45 EFFECT OF OSMOLARITY IN CULTURE MEDIUM ON THE PRE-IMPLANTATION DEVELOPMENT OF PORCINE NT AND IVF EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 141
Author(s):  
I. S. Hwang ◽  
H. J. Moon ◽  
J. H. Shim ◽  
M. R. Park ◽  
D. H. Kim ◽  
...  
Keyword(s):  
Culture Medium ◽  
Developmental Rate ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Final Concentration ◽  
Control Group ◽  
In Vitro Production ◽  
Fetal Fibroblasts ◽  
Vitro Production

In vitro production of the pig embryo is very important as an initial step to improve its application in biotechnology. The in vitro production system for pig embryos, however, has been plagued by the high incidence of polyspermy and poor embryo quality. The present study was conducted to examine the relationship between apoptosis and osmolarity of culture medium in pre-implantation development of porcine NT and IVF embryos. Oocytes were aspirated from ovaries collected from a local abattoir, and then matured in TCM-199 for 40–44 h. Fresh semen was diluted and equilibrated at 16�C. The final concentration of motile spermatozoa was adjusted to 5 � 105 cells/mL in fertilization medium. Fetal fibroblasts were prepared from a 35-day-old porcine fetus for use as donor cells. The NT and IVF embryos were cultured in PZM-3 supplemented with 0.05 M sucrose or a final concentration of 138 mM NaCl (280–320 mOsmol) for the first 2 days, and then cultured in PZM-3 (250–270 mOsmol) for the remaining days. For the control, NT and IVF embryos were cultured in PZM-3 for whole culture period. After 6 days of culture, the developmental ability of embryos, total cell numbers, ratio of ICM/TE, and apoptosis of cells in blastocysts were examined. The developmental rate to the blastocyst stage of NT embryos was significantly higher (P &lt; 0.05) in the sucrose and NaCl groups than in the control [14.7% (21/153) and 21.7% (34/154) vs. 11.5% (18/152), respectively]. Also, the developmental rate to the blastocyst stage after IVF was slightly higher in embryos cultured in the medium supplemented with NaCl than in the control group [21.8% (49/235) and 26.4% (61/237) vs. 18.9% (44/247)]. For apoptosis, both NT and IVF blastocysts produced in the sucrose and NaCl groups showed slightly lower frequency of apoptosis compared to that of the control (2.2% and 2.8% vs. 3.1% for NT; 0.9% and 0.7% vs. 1.1% for IVF). These studies suggest that the high osmolarity in the early embryo culture stage could enhance the in vitro development of both porcine NT and IVF embryos to the blastocyst stage and could reduce the apoptosis of cells.

scholarly journals Effects of superstimulation with fsh on follicular population and recovery rate of oocytes in the growing phase of the first and second follicular wave

2012 ◽  
Vol 47 (No. 2 - 3) ◽  
pp. 33-37
Author(s):  
S. Čech ◽  
V. Havlíček ◽  
M. Lopatářová ◽  
M. Vyskočil ◽  
R. Doležel
Keyword(s):  
Czech Republic ◽  
Corpus Luteum ◽  
Recovery Rate ◽  
Dairy Farm ◽  
Cumulus Cells ◽  
Dominant Follicle ◽  
In Vitro Production ◽  
Follicular Wave ◽  
Vitro Production

Effectiveness of in vitro production of embryos (IVP) is limited among other factors by the recovery rate of oocytes. Gonadotropin superstimulation can improve the recovery rate of oocytes. The effect of FSH treatment on follicular population and recovery rate of oocytes at ovum pick-up (OPU) in the growing phase of the 1st as well as the 2nd follicular wave after superstimulation was the object of our experiment. Twelve unpregnant milking cows (15&ndash;20 kg milk per day) housed on a dairy farm were used in the experiment. The cows bearing corpus luteum were synchronized by PGF<sub>2 </sub>(day 0) and they were treated by FSH (Folicotropin inj. sicc. ad us vet., Spofa Prague, Czech Republic, single doses 80, 80, 80, 80, 40 and 40 UI) in 12 h intervals on days 12, 13 and 14. Transvaginal ultrasonographic puncture of oocytes in cows bearing a new corpus luteum was performed on day 7 (OPU 1, various phase of the follicular wave, removal of the dominant follicle) and it was repeated on days 10 (OPU 2, growing phase of the follicular wave &ndash; control), 16 (OPU&nbsp;3, growing phase of the first follicular wave after superstimulation) and 20 (OPU 4, growing phase of the second follicular wave after superstimulation). All follicles &gt; 2 mm were punctured. The ovarian follicles (ultrasonographically) and numbers and qualities of obtained oocytes (microscopically) were evaluated during and immediately after OPU. Follicular population was divided to small (FS, 2&ndash;5 mm), medium (FM, 5&ndash;9 mm) and large (FL, &gt; 9 mm) follicles. Oocytes were classified as 1st (intact cumulus, &gt; 3 layers of cumulus cells), 2nd (complete 1&ndash;3 layers of cumulus cells), 3rd (incomplete layers of cumulus cells, expanded cumulus mass) and 4th (absence of corona cells, degenerated oocytes) classes. Although we found the least of FS (x = 1.0) during OPU 3, significantly more FM (x = 24.7) and FL (x = 3.1) follicles were found at this procedure in comparison with others. Likewise a significantly higher number of oocytes (x = 8.1) was obtained at OPU 3 in comparison with OPU 1 and OPU 2. Significantly higher number of FM (x = 6.1) was found and non-significantly higher number of oocytes was obtained at OPU 4 in comparison with OPU 1 and 2. The results show that administration of FSH increases the number of follicles and the number of collected oocytes in the growing phase of the 1st follicular wave after superstimulation, nevertheless a higher number of follicles and a higher recovery rate of oocytes can be expected in the growing phase of the 2nd follicular wave after superstimulation as well.

155 EFFECTS OF CULTURE WITH OVALBUMIN IN ABSENCE OF FETAL BOVINE SERUM AND BOVINE SERUM ALBUMIN ON IN VITRO PRODUCTION OF CATTLE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 236
Author(s):  
T. A. D. Tetzner ◽  
N. Z. Saraiva ◽  
C. S. Oliveira ◽  
S. C. Méo ◽  
M. M. Souza ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Cell Number ◽  
Differential Staining ◽  
Control Group ◽  
Cortical Granule ◽  
In Vitro Production ◽  
Nuclear Maturation ◽  
Fetal Bovine ◽  
Vitro Production

Embryo quality is influenced by culture conditions, which affect IVM, IVF, and IVC rates. One of the most efficient ways to evaluate the embryonic quality of IVP blastocysts is by differential staining of inner cell mass (ICM) and trophoblast (TF). Bovine embryos of superior quality should present the total number of cells close to the number of cell cycles (Neuber et al. 2002 Theriogenology 57, 2193-2202). In this study, we analyzed the effects of fetal bovine serum (F) and bovine serum albumin (B) replacement for ovalbumin (O) on nuclear maturation, cortical granule migration, pronuclear development, blastocyst rates, and differential staining of ICM and TF in Day 7 blastocysts. The treatment groups were named as follows: the first letter is the protein source used for IVM, the second for IVF, and the third for IVC. When 2 protein sources were used in the same step, the plus symbol (+) was used. The oocytes were IVM in TCM-199, supplemented with the following: 10% F, or 4 mg mL-1 B, or 4 mg mL-1 O, and 1.0 Âμg mL-1 of FSH, 50 Âμg mL-1 of hCG, 1.0 Âμg mL-1 of estradiol, 0.2 mM sodium pyruvate, and 83.4 Âμg mL-1 of amikacin. IVF was accomplished in TALP-IVF medium, with 0.2 mM pyruvate, 83.4 Âμg mL-1 of amikacin, and 6 mg mL-1 B or O. IVC was in SOF, with F, B, or O. The control group (CONT) consisted of the treatment FBF + B. Pronuclear development was compared by the chi-square test, whereas the other results were analyzed by ANOVA followed by the Tukey test, using SAS at 5% significance level (SAS Institute Inc., Cary, NC, USA). For IVM, the treatments F, B, O, and B + O did not affect (P > 0.05) nuclear maturation (73.92 to 78.78%) and cortical granule migration rates (58.89 to 66.76%). Regarding pronuclear development, the treatment FO (76.67%) was similar (P > 0.05) to the control group (82.95%), which was superior (P < 0.05) to the treatments BB (56.98%), BO (39.02%), OB (37.36%), and OO (39.24%). Blastocyst rates in FBF (42.8%) and control (45.0%) groups were superior (P < 0.05) to treatment OOO (26.0%) but similar (P > 0.05) to FOF, BBB, BOB, and OBO (32.0 to 35.8%). The average of blastocyst ICM cells of the group OOO (16.79) was inferior (P < 0.05) to the other groups. However, the average of TF cells on blastocysts of the group OOO (38.25) was similar (P > 0.05) to the groups BBB (45.74) and BOB (45.60) and inferior (P < 0.05) to the groups CONT (57.59), FBF (54.41), FOF (56.74), and OBO (47.35). The total average cells in the blastocysts of the group OOO (56.04) was inferior (P < 0.05) to the groups CONT (84.86), FBF (78.96), FOF (81.32), BBB (68.11), BOB (69.55), and OBO (69.82). The total cell number in the treatments, with several sources of protein supplementation, varied from 56.04 to 84.86. Considering the evaluation interval, this average cell number was discreetly inferior to that expected for the chronological age of the blastocysts. We concluded that it is possible to produce bovine embryos in the absence of F and/or B, with the protein source O, although it reduced blastocyst rates when used in all 3 steps of embryo in vitro production and resulted in blastocysts of inferior quality. Financial support: FAPESP 05/60389-2 and CNPq.

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