In vitro embryo production from early antral follicles of goats fed with a whole full-fat linseed based diet

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
J. Cadenas ◽  
N.A.R. Sá ◽  
A.C.A. Ferreira ◽  
V.M. Paes ◽  
F.L.N. Aguiar ◽  
...  
Keyword(s):  
Recovery Rate ◽  
Penetration Rate ◽  
Control Treatment ◽  
Follicular Growth ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Feed Supplementation ◽  
Antral Follicles ◽  
Sperm Penetration

Summary The present study aimed to use an in vitro follicle culture (IVFC) biotechnique as a tool to evaluate the influence of whole flaxseed as a feed supplementation in the diet on the in vitro development of caprine early antral follicles (EAFs) and further embryo production. In total, 18 adult goats were homogeneously allocated into two diet groups: Control and Flaxseed. EAFs from both experimental groups (300–400 µm) were isolated and cultured in vitro for 18 days. After IVFC, recovered cumulus–oocyte complexes were submitted to in vitro maturation, and subsequently to IVF and in vitro embryo culture. The endpoints evaluated were follicular growth and morphology, oocyte recovery rate and diameter, sperm penetration, pronuclei formation, embryo development, and estradiol production. The addition of the whole flaxseed in the diet did not affect (P > 0.05) follicular growth and diameter. A higher (P < 0.05) percentage of oocytes ≥ 110 µm was recovered from the flaxseed treatment. However, the sperm penetration rate was higher (P < 0.05) in the control treatment when compared with the flaxseed treatment, but no differences were found regarding the rate of fertilization nor cleaved embryos. In conclusion, dietary flaxseed increased the recovery rate of fully grown oocytes, but it did negatively affect the sperm penetration rate, even though there was no further effect on the cleavage rate.

233 HIGH NUMBERS OF ANTRAL FOLLICLES INFLUENCE THE IN VITRO EMBRYO PRODUCTION, BUT NOT THE CONCEPTION RATE OF FIXED-TIME ARTIFICIAL INSEMINATION IN NELORE CATTLE

2015 ◽  
Vol 27 (1) ◽  
pp. 206
Author(s):  
G. M. G. Santos ◽  
K. C. Silva-Santos ◽  
T. R. R. Barreiros ◽  
F. Morotti ◽  
B. V. Sanches ◽  
...  
Keyword(s):  
Artificial Insemination ◽  
Fixed Time ◽  
Device Removal ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Antral Follicles ◽  
Conception Rates ◽  
In Vitro Embryo Production ◽  
Embryo Grade

The aim of this study was to compare the conception rates to fixed-time artificial insemination (FTAI) and in vitro embryo production between Nelore cows with high or low antral follicle counts (AFC). First, multiparous Nelore cows (Bos indicus, n = 547, 40–60 days postpartum) were subjected to synchronization of ovulation. Randomly during their oestrous cycle (Day 0), cows received an intravaginal device containing 1.9 g of P4 (CIDR®) and 2 mg of oestradiol benzoate (Estrogin®), intramuscularly. At device removal (Day 8), cows received 500 µg of PGF2α (Ciosin®), 300 IU of eCG (Novormon®), and 1 mg of oestradiol cipionate (ECP®), intramusculary. All cows were inseminated 48 h after P4 device removal. Antral follicles = 3 mm were counted using an intravaginal microconvex transducer (Day 0), and cows were assigned to groups of high (G-High, = 25 follicles, n = 183), intermediate (G-Intermediate, 16–20 follicles, n = 183), or low AFC (G-Low, = 10 follicles, n = 181). In another study to compared the in vitro embryo production, Nelore cows (n = 66, 72–96 months) were subjected to ultrasound-guided follicular aspiration using an intravaginal microconvex array transducer (7.5 MHz). The COC were selected and cows were assigned to groups according to the oocyte production: G-High (n = 22, = 40 oocytes), G-Intermediate (n = 25, 18–25 oocytes), or G-Low (n = 19, = 7 oocytes). Previously tested semen from a single bull was used for IVF using a previously described protocol (Silva-Santos et al. 2014 Reprod. Domest. Anim. 49, 228–232). The oocyte and embryo production (viable embryo: grade I, II, III; vitrifiable embryo: grade I, II) were evaluated. The number of follicles was evaluated by Kruskal-Wallis, and the chi-square test was used for data on oocyte and embryo production (P = 0.05). The average follicular population was 30.7 ± 5.7 (G-High), 18.6 ± 1.64 (G-Intermediate), and 7.8 ± 2.4 follicles (G-Low; P < 0.05), but there were no differences in the conception rates among groups (51.9 v. 48.6 v. 58.6%, respectively; P > 0.05). The total number of oocytes recovered were 1109 (G-High), 534 (G-Intermediate), and 101 (G-Low; P < 0.05). The mean number of viable oocytes was 40.4 ± 10.6 (G-High), 14.8 ± 3.0 (G-Intermediate), and 3.8 ± 1.1 (G-Low; P < 0.05) and the percentage of viable oocytes was 80% (G-High), 69% (371/534, G-Intermediate), and 71% (G-Low; P < 0.05). Cleavage rate was 79% (G-High), 74% (348/472, G-Intermediate), and 71% (G-Low; P < 0.05), and blastocyst rate was 42% (G-High), 32% (153/472, G-Intermediate), and 13% (G-Low; P < 0.05). The number of viable embryos was 18.4 ± 6.7 (G-High), 6.1 ± 3.6 (G-Intermediate), and 0.6 ± 0.7 (G-Low; P < 0.05) and the percentage of vitrifiable embryos was 81% (G-High), 77% (118/153, G-Intermediate), and 58% (G-Low; P < 0.05). Therefore, Nelore cows with high oocyte production had ~10-fold higher oocyte production and produced ~30-fold more embryos compared with the low AFC group. In conclusion, AFC had no influence on the conception rates to FTAI; however, Nelore cows with high oocyte production exhibited higher in vitro embryo production.

Early oocyte penetration can predict the efficiency of bovine embryo production in vitro

Zygote ◽  
2008 ◽  
Vol 16 (3) ◽  
pp. 203-209 ◽  
Author(s):  
M. Machatkova ◽  
J. Horakova ◽  
P. Hulinska ◽  
Z. Reckova ◽  
K. Hanzalova
Keyword(s):  
Strong Correlation ◽  
Predictive Value ◽  
Penetration Rate ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Fertilization Rates ◽  
Oocyte Fertilization ◽  
Embryo Cleavage

SummaryThe aim of this work was to characterize oocyte fertilization and embryo cleavage in nine AI bulls to find parameters suitable for prediction of in vitro fertility. According to the d8 blastocysts rate, they were categorized as high, medium and low productive (HP, MP and LP, mean: 25.4, 21.0 and 13.6% respectively) bulls. For these categories, oocyte penetration and fertilization efficiency were assessed at 6 and 18 hours post insemination (hpi), respectively. Some presumptive zygotes were cultured and cleaved and fast-cleaved embryo rates were checked at 44 hpi. The penetration rate was significantly higher for HP bulls than for MP and LP bulls (67.9 versus 50.3 and 33.1%; p < 0.01). The syngamy rate was significantly higher for HP bulls than for MP and LP bulls (21.4 versus 10.2 and 5.7%; p < 0.05). Conversely, no significant differences in fertilization rates were found among HP, MP and LP bulls. The cleavage rate was significantly higher for HP than LP bulls (82.4 versus 74.4%; p < 0.01). The fast cleavage rate was significantly higher for both HP and MP bulls, as compared with LP bulls (82.1 and 84.7 versus 73.5%; p < 0.01). A strong correlation was found between blastocyst production and penetration (r = 0.803), syngamy (r = 0.826), cleavage (r = 0.635) and fast cleavage (r = 0.709). In conclusion, all the evaluated parameters showed a predictive value, the most significant being early penetration and syngamy.

Treatment of aromatase (CYP19A1) inhibitor reduces fertility in porcine sperm

Zygote ◽  
2015 ◽  
Vol 24 (1) ◽  
pp. 98-106 ◽  
Author(s):  
Jong-Nam Oh ◽  
Jae Yeon Hwang ◽  
Kwang-Hwan Choi ◽  
Chang-Kyu Lee
Keyword(s):  
Penetration Rate ◽  
Quantitative Polymerase Chain Reaction ◽  
Aromatase Activity ◽  
Cleavage Rate ◽  
Western Blots ◽  
Sperm Penetration ◽  
Mrna And Protein Expression ◽  
Polymerase Chain ◽  
Fresh Semen

SummaryTo ascertain whether aromatase (CYP19A1) expression is linked to sperm fertility of pigs, the present study determined the expression of the CYP19A1 gene in porcine sperm and its relationship with fertilization in vitro. First, to investigate its role in fertility, the presence of CYP19A1 of mRNA and protein expression in porcine sperm were confirmed by real-time (RT) or quantitative polymerase chain reaction (q-PCR) and by western blots. The expression levels were determined quantitatively using two sperm groups recovered by a Percoll gradient, which revealed that the sperm group with a low density had a higher penetration rate than that of the high-density group (P < 0.05). However, the expression level of CYP19A1 was not significantly different between the two groups. Secondly, to examine the effect of aromatase activity on fertilization, fresh semen was treated with a steroidal inhibitor, exemestane (50 μM for 0.5 h), followed by the dose- and time-dependent viability test. Our results clearly showed that an exemestane treatment effect (P < 0.05) was found for both the sperm-penetration rate and the oocyte cleavage rate. These results indicated that CYP19A1 could be involved in sperm fertility and its expression in sperm plays an important role in fertilization.

Cleavage Rate of in vitro Matured Oocytes Fertilized with Conventional Semen in Buffaloes

2021 ◽  
Author(s):  
M.H. Pitroda ◽  
K.P. Khillare ◽  
M.B. Amle ◽  
M.D. Meshram ◽  
A.B. Mali ◽  
...  
Keyword(s):  
Fertilization Rate ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Quick Recovery ◽  
Slaughter House ◽  
Maturation Rate ◽  
Aspiration Technique ◽  
Current Scenario ◽  
In Vitro Embryo Production

Background: In vitro embryo production in buffaloes has gained much importance in this current scenario due to ever increasing population and high demand of milk and meat. Slaughter house derived bubaline ovaries are a cheap and abundant source of cumulus oocyte complexes.Methods: Oocytes from the buffalo ovarian follicles were recovered by aspiration technique as it facilitates quick recovery. Total 155 ovaries were used in the present study. Surface follicles were measured using vernier calliper and categorized into three groups viz. less than 3 mm, 3-5 mm and greater than 5 mm based on follicular diameter and oocytes were processed for IVM, IVF and IVC using conventional non sorted semen.Result: Overall percentage of small, medium and large follicles in the ovaries were recorded as 16.29 ± 0.94%, 8.14±0.60%, 5.35 ± 0.76%, respectively. Overall recovery rate of COCs was 38%. The percentage of these oocytes were 16.74% (A), 15.25% (B), 25.26% (C), 18.33% (D) and 29.87% (E) respectively. Maturation rate of oocytes were 81.96 ± 2.70%. Fertilization rate was 74.98 ± 3.87%, Cleavage rate % was 40.84±2.51% and Blastocyst percentage was 21.57±1.75% respectively. Application of in vitro embryo production technique using slaughter house ovaries can salvage the genetic potential of bubaline species.

119 HETEROLOGOUS BOVINE IN VITRO FERTILIZATION USING CRYOPRESERVED BOTTLENOSE DOLPHIN SPERMATOZOA

2012 ◽  
Vol 24 (1) ◽  
pp. 172 ◽  
Author(s):  
M. J. Sanchez-Calabuig ◽  
P. Beltran-Brena ◽  
E. Martinez-Nevado ◽  
D. Rizos ◽  
J. F. Perez-Gutierrez ◽  
...  
Keyword(s):  
Bottlenose Dolphin ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Sperm Chromatin ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Sperm Penetration ◽  
Pronuclear Formation ◽  
Bovine Oocytes

Assisted reproductive technologies are of great importance for increasing genetic diversity in captive animals without displacing them. The development and improvement of these techniques require accurate methods to assess sperm function. The ability of the sperm to bind the zona pellucida and the formation of a male pronucleus have been shown to have a high predictive value for fertilization outcome. The use of zona-intact bovine in vitro–matured oocytes in heterologous fertilization with dolphin spermatozoa could provide valuable information on its fertilizing ability. The aim of the present study was to evaluate male pronuclear formation in zona-intact bovine oocytes after coincubation with frozen-thawed bottlenose dolphin spermatozoa. A total of 1546 immature cumulus oocytes complexes (COC) were obtained from bovine ovaries collected at slaughter. The COC were matured for 24 h in TCM-199 supplemented with 10 ng mL–1 of epidermal growth factor and 10% FCS. Matured COC were inseminated with frozen-thawed Bovi-pure (Nidacon International, Mölndal, Sweden) separated bovine (control) or dolphin spermatozoa. At 18, 20, 22, 24, 26 and 28 h post-insemination (hpi), half of the presumptive zygotes from each group were fixed and stained with Hoechst 33342 to examine sperm penetration, polyspermy and pronuclear formation and the remainder were cultured in synthetic oviduct fluid supplemented with 5% FCS for evaluating fertilization rates by cleavage on Days 2 and 4 (Day 0 = day of IVF). As expected, in the control a higher percentage of 2 pronuclear formation was observed at 18 hpi (74.5%), with a decrease at 20 and 22 hpi (57.4 and 43.2%, respectively) and was significantly lower (P ≤ 0.001) at 24 hpi (13.3%), reaching the lowest values at 26 and 28 hpi. However, in the heterologous group significantly less oocytes with both pronuclear formed (P ≤ 0.001) were observed at 18, 20 and 22 hpi (1.2, 3.4 and 3.0%, respectively) compared with 24, 26 and 28 hpi (22.5, 11.4 and 8.9%, respectively). No polyspermy was detected in oocytes coincubated with dolphin spermatozoa. Moreover, the cleavage rate at Day 2 and 4 in heterologous fertilization was 13.0 and 34.8%, respectively, whereas for the control it was 90.0%. In conclusion, these results indicate that dolphin spermatozoa can penetrate bovine oocytes and induce the block to polyspermy and the differences found regarding pronuclear formation times between the 2 species could be due to distinct sperm chromatin organisation or condensation. In conclusion, our preliminary results show that heterologous fertilization using bovine oocytes is useful for characterising the viability of dolphin thawed spermatozoa, which also could be helpful in performing a more complete sperm evaluation. Further studies are necessary to provide more consistent evidence of the efficiency of this test. The authors thank the staff at Zoo Aquarium Madrid for their dedicated work toward dolphin semen collection.

158 IMPACT OF MATURED CATTLE OOCYTES AT HIGHER INCUBATION TEMPERATURE ON IN VITRO EMBRYO PRODUCTION

2016 ◽  
Vol 28 (2) ◽  
pp. 209
Author(s):  
M. Nkadimeng ◽  
E. van Marle-Koster ◽  
K. P. M. Lekola ◽  
M. L. Mphaphathi ◽  
M. M. Seshoka ◽  
...  
Keyword(s):  
Incubation Temperature ◽  
Cell Types ◽  
Cell Number ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Significant Difference ◽  
In Vitro Embryo Production

Heat stress during IVF is associated with reduced fertility in cattle oocytes. It may, however, enhance thermo-tolerance or cause detrimental effects on a variety of cell types or organisms, depending on the duration and intensity of the thermal challenge. The aim of this study was to evaluate the developmental potential of cumulus-oocyte complexes (COC) matured for 18 or 24 h and incubated at 39°C or 41°C. A total of 1000 immature oocytes were collected at slaughter from indigenous South African cow ovaries. The COC were randomly allocated (100/treatment) into 2 maturation times (18 or 24 h) and cultured in M199 + FSH-LH-estradiol medium under oil at 100% humidity and 5% CO2 at 39°C or 41°C. Post maturation, oocytes were subjected to normal subsequent embryo conditions. The Bracket and Oliphant medium was used for IVF. All matured oocytes were fertilised for 6 h with frozen-thawed Nguni bull semen at a concentration of 265 × 106. The presumptive zygotes from each treatment were cultured into SOF-BSA medium under oil and incubated at 39°C for assessment of cleavage rate 48 h post IVF. After Day 7 of culture, blastocyst were stained (Hoechst 33323) for nuclei cell count. Statistical analyses was performed using Genstat® software of SAS (SAS Institute, Cary, NC, USA; P < 0.05). Oocytes that were matured for 18 h in 41°C resulted in more 8-cell embryos (41%) compared with those incubated at 39°C (21.6%). However, no difference was observed for cleavage rate at both maturation times and incubation temperatures (41 or 39°C). There was more morula formation from oocytes matured for 18 h (19.6%) and 24 h (19.0%) at 41°C compared to 39°C (8.4%) group. The results further showed more blastocyst formation during 18 h at 41°C (15.2%) than at 39°C (7.4%) and during 24 h at 41°C (11.2%), 39°C (11.4%). However there was no difference in the nuclei cell number during 18 h at 41°C (45.2), 24 h (45.8), and 18 h at 39°C (43.4) of maturation. Thus, there was a significant difference in the nuclei cell numbers at 24 h on 39°C (n = 133.2) and 41°C (n = 45.8). In conclusion, oocytes that were matured for 18 and 24 h at 41°C or for 18 h at 39°C developed further to blastocyst stage on in vitro embryo production, however, with low nuclei cell numbers due to accelerated maturation temperature or shortened maturation period.

149 EFFECTS OF FSH ADDED IN DEFINED MEDIUM MATURATION ON THE PRE-IMPLANTATION DEVELOPMENT AND EXPRESSION OF Bax, Bcl-2, AND CONNEXIN 43 GENES IN BOVINE IVP BLASTOCYSTS

2010 ◽  
Vol 22 (1) ◽  
pp. 233
Author(s):  
L. V. M. Gulart ◽  
L. Gabriel ◽  
L. P. Salles ◽  
G. R. Gamas ◽  
D. K. Souza ◽  
...  
Keyword(s):  
Internal Control ◽  
Connexin 43 ◽  
Calf Serum ◽  
Defined Medium ◽  
Culture Conditions ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Blastocyst Rate

FSH at low concentrations affect embryo production. In vitro culture conditions also affect embryo production and embryonic expression of genes and alter oocyte competence to produce embryos. The search for better and less variable culture conditions simulating those in vivo has led to the development of several systems of oocyte in vitro maturation culture. To compare the efficiency of the systems of MIV we utilized 4 groups: (1) TCM-199 control; (2) α-minimal essential medium (MEM); 3) α-MEM + 1 ng of FSH; 4) α-MEM+ 10 ng of FSH. The medium of Group 1 is non-defined by the presence of fetal calf serum (10%). Groups 2, 3, and 4 are defined and polyvinyl alcohol (1%) was used as a macromolecule. Porcine FSH (1 IU mg-1) was used at 1 and 10 ng mL-1 and at 100 ng in defined and non-defined medium, respectively. Bovine ovaries were collected at an abbatoir. Oocytes (n = 1718) with homogeneous cytoplasm and with more than 3 layers of granulosa cells were used. Mature oocytes from the 4 treatments (11 replicates of each treatment) were inseminated with frozen-thawed, motile sperm separated by Percoll, using Sperm TALP HEPES medium. Presumptive zygotes with up to 2 or 3 layers of cumulus cells were cultured in 50-mL drops of SOF medium, supplemented with 10% FCS and 1 mg mL-1 BSA under mineral oil in a humid 5% CO2 atmosphere at 38.5°C after. Cleavage rate was evaluated 72 h post-insemination (hpi), and blastocyst rate was evaluated 168-192 hpi. Cleavage and blastocyst rates were calculated on the basis of number of presumptive zygotes. The expression of the following genes (Bax, Bcl-2, and conexin 43) was evaluated in blastocysts by RT-PCR. One-way ANOVA was used to compare blastocyst number. There was no difference in the proportion of embryos with more than 8 blastomeres in all groups tested, indicating that the rate of development during the first 72 hpi was similar for oocytes matured in chemically defined medium and for oocytes matured in medium containing serum. Bax is a pro-apoptotic marker and Bcl-2 an antiapoptotic marker. Connexin 43 (Cx43) may be a marker of embryo competence. Glyceraldehyde 3-phosphate dehydrogenase was used as internal control. The Bax gene was not expressed in any group. The Bcl-2 and Cx43 genes were expressed, mainly in the α-MEM 10. Although no differences were observed in blastocyst rate among the groups (30% to 40%), the strong expression of Bcl-2 and of Cx43 on the group containing 10 ng mL-1 of FSH may indicate that FSH could improve embryo quality under defined conditions. The authors thank FAP-DF, CNPq, FUNPE, FINATEC, CAPES, and Biovitro Tecnologia de Embrioes Ltda, for laboratory assistance and grants, and Frigorifico Ponte Alta, Brasília-DF, for supplying bovine ovaries.

206 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND SUPERSTIMULATION ON IN VITRO EMBRYO PRODUCTION

2008 ◽  
Vol 20 (1) ◽  
pp. 182 ◽  
Author(s):  
K. Imai ◽  
Y. Inaba ◽  
H. Yoshioka ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
...  
Keyword(s):  
Formation Rate ◽  
Cumulus Cell ◽  
Oocyte Quality ◽  
Annual Conference ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Follicular Wave ◽  
The Mean ◽  
In Vitro Embryo Production

We previously reported that follicular wave synchronization, by removal of the dominant follicle on Day 5 after ovum pickup (OPU), was effective in increasing oocyte quality in the developing follicles (Imai et al. 2006 32th Annual Conference of the IETS, poster presentation no. 277). The current study was designed to examine the effect of superstimulatory treatment to induce subsequent follicular wave synchronization on embryo production by OPU and IVM-IVF-IVC in Holstein dry cows. Cows were reared under the same feeding and environmental conditions, and 2 OPU sessions were conducted in each cow. In the first session, OPU was performed in 8 cows on arbitrary days of the estrous cycle by using a 7.5-MHz linear transducer with needle (Cova needle, Misawa Medical, Tokyo, Japan) connected to an ultrasound scanner (SSD-1200, Aloka, Tokyo, Japan). Follicles larger than 8 mm in diameter were then aspirated and a CIDR was inserted on Day 5 (the day of first OPU session = Day 0). Cows then received 30 mg of FSH (Antrin-R10; Kawasaki Mitaka Pharmaceutical Co., Tokyo, Japan) twice a day from Days 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2 mg) by i.m. injection. Cloprostenol (PGF; Clopromate C; Sumitomo Pharmaceuticals Co., Tokyo, Japan; 0.75 mg) was administered in the morning of Day 9 (third day of superstimulation). 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. Collected oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. Grades 1 and 2 COC were matured, fertilized, and cultured as described by Imai et al. [2006 J. Reprod. Dev. 52(Suppl.), S19–S29]. Embryo development was assessed by the cleavage rate on Day 2 and by the blastocyst formation rate on Days 7 to 8 (the day of insemination = Day 0). Data were analyzed by Student's t-test. There were no differences in the mean (� SD) number of aspirated follicles or collected oocytes between the first (32.5 � 6.8 and 26.0 � 12.7, respectively) and second (29.3 � 10.4 and 19.0 � 9.4, respectively) OPU sessions (P > 0.1). The percentage of Grade 1 and 2 oocytes for the second OPU session (90.5 � 13.8%) was significantly higher (P < 0.01) than for the first OPU session (63.1 � 6.3%), and significant differences were found for cleavage (79.4 � 14.1, 61.8 � 25.1, P < 0.01) and blastocyst rates (68.1 � 16.7, 24.2 � 22.3, P < 0.001) between sessions. The mean numbers of blastocysts obtained per session were 4.3 � 2.9 and 12.8 � 8.7 in the first and second sessions, respectively (P < 0.01). These results indicate that superstimulatory treatment and subsequent follicular wave synchronization were effective on in vitro embryo production by increasing the oocyte quality.

112 EFFECTS OF GUAIAZULENE ON IN VITRO CULTURE OF BOVINE ZYGOTES, AND ON mRNA TRANSCRIPTS RELATED TO EMBRYO QUALITY

2009 ◽  
Vol 21 (1) ◽  
pp. 156
Author(s):  
E. Dovolou ◽  
M. Clemente ◽  
G. S. Amiridis ◽  
I. Messinis ◽  
A. Kalitsaris ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Early Embryo ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Early Embryo Development ◽  
Mrna Transcripts ◽  
Oviductal Fluid ◽  
Maximum Humidity

We have previously shown that follicular and oviductal fluid provide greater total protection against lipid peroxidation than the respective media used for the in vitro embryo production. Reactive oxygen species (ROS) production has been implicated as a major cause for the reduced in vitro bovine embryo production; it is believed that they participate in meiotic arrest of oocytes, embryonic block and cell death. The aim of this study was to determine whether guaiazulene (G), an exogenous antioxidant, added in the post fertilization culture medium would affect the early embryo development and the quality of the produced blastocysts in terms of mRNA expression of several important genes. In a previous study we had shown that media modified with 0.01 mm of G provided the same antioxidant protection as the respective in vivo environments (i.e. the follicular and the oviductal fluid). Bovine cumulus–oocyte complexes (COC) were aspirated from ovaries derived from slaughtered cows and matured in groups of 50 in 500 μL in TCM199 with 10% fetal calf serum and 10 ng mL–1 Epidermal Growth factor at 39°C in an atmosphere of 5% CO2 in air and maximum humidity. Twenty-four hours later matured oocytes were inseminated with frozen/thawed bull semen and co-incubated in the same conditions as maturation. Presumptive zygotes were divided into 4 groups and cultured in groups of 25 in 25 μL of SOF with 5% FCS (Control–, n = 355), supplemented with 0.01 mm of G (n = 344) or 0.1 mm of G (n = 345) or 0.05% DMSO – the G diluent–(Control+, n = 347) at 39°C in an atmosphere of 5% CO2, 5% O2 and maximum humidity. Blastocyst yield was recorded on Days 6, 7, 8 and 9; Day 7 blastocysts from each group were snap frozen and stored at –80°C for mRNA extraction. Quantification of transcripts for aldose reductase mRNA (AKRIBI), prostaglandin G/H synthase-2 (PGHS-2, COX-2), glyceraldehyde 3-phosphate dehydrogenase (GADPH), facilitated glucose/fructose transporter, member 5 (GLUT-5) genes related to metabolism, glutathione peroxidase 1 (GPX1), glucose-6-phosphate dehydrogenase (G6PD) antioxidant enzymes and placenta-specific 8 (PLAC8) related to implantation was carried out by real-time quantitative RT-PCR. Data for embryo development and on transcript abundance were analyzed by chi square and ANOVA, respectively. Cleavage rate tended to be higher in 0.01 mm group than in Control– (77.87% v. 71.41%, P = 0.07). Barring that, no other differences were detected in cleavage rate (Control+: 71.32%; 0.1 mm: 72.75%) or in the overall blastocyst yield on Day 9 (Control–: 25.50%; Control+: 26.71%; 0.1 mm: 25.75%; 0.01 mm: 29.58%). The relative abundance of genes studied varied among groups, but these differences were not significant. We infer that under the current culture conditions, G as an antioxidant has no serious direct effect on early embryo development or on embryo quality at least on the mRNA transcripts studied. Further studies using the same antioxidant in different atmospheric conditions are planed. ED and GSA were sponsored by COST (FAO702) and OECD fellowships, respectively.

145 Comparison of single to multiple injections of follicle-stimulating hormone before ovum pickup in Holstein heifers: Oocyte recovery and embryo production

2021 ◽  
Vol 33 (2) ◽  
pp. 180
Author(s):  
D. G. B. Demetrio ◽  
J. F. Hasler ◽  
M. Oliveira ◽  
C. G. B. Demetrio ◽  
J. C. Fonseca ◽  
...  
Keyword(s):  
Time Synchronization ◽  
Random Effect ◽  
Oocyte Quality ◽  
Follicular Growth ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Multiple Injections ◽  
Oocyte Recovery ◽  
Blastocyst Rate ◽  
Stimulation Of

The demand for invitro-produced embryos from heifers with high genetic merit has increased over time. Synchronization and stimulation of follicular growth before ovum pickup (OPU) has been used to improve oocyte quality and, consequently, embryo production. Multiple injections involve extra labour and stress for both personnel and cattle. The release of FSH can be prolonged by using 0.5% hyaluronan (HA) as a diluent, allowing a decrease in the number of injections. The objective of this study was to compare oocyte recovery and embryo production between single or multiple injections of FSH before OPU of Holstein heifers. During April and May 2020, 20 Holstein heifers (8 to 15 mo old) from Ruann Dairy (Riverdale, CA) were randomly divided and submitted to two different treatments (crossover design). Gonadotrophin-releasing hormone (GnRH; Fertagyl®, Merck, 129µg, IM) was given to synchronize the follicular wave emergence. Treatment 1×FSH consisted of a single intramuscular (IM) injection of 100mg of FSH (Folltropin®, Vetoquinol) 36h after GnRH. The FSH consisted of a 2.5-mL injection of 400mg of FSH diluted in 10mL of 0.5% HA. OPU was performed 48 to 50h after FSH. Treatment 5×FSH consisted of 100mg of FSH divided into 5 equal IM injections (10-14h intervals) 36h after GnRH. The FSH consisted of 5×1-mL injections of 400mg of FSH in 20mL of saline. OPU was performed 18 to 20h after the last FSH injection. All donors received both treatments at a 14-day interval and the recovered oocytes were fertilized with the same sexed female-sorted semen in both rounds. OPU, oocyte classification, IVM, IVF, and culture (IVC) were performed as described by Demetrio et al. (2020 Anim. Reprod. 17, e20200053). All oocytes went into IVM, except for degenerated oocytes. The number of 4-cell (or more) embryos on Day 3 of IVC divided by the number of oocytes in IVC after IVF is defined as the cleavage rate. The number of blastocysts (early to hatched) on Day 7 of IVP divided by the number of oocytes in IVC after IVF is defined as the blastocyst rate. Poisson-normal (count data) and Logistic-normal (proportion data) models were used to analyse the data. Treatment, donor (random effect), and sire were included in the models. The results are summarized in Table 1. There were no differences between the two treatments on the number of oocytes recovered per OPU (total and grade 1 and 2), percentage of grade 1 and 2 oocytes, cleavage rate, blastocyst rate and number of embryos (total and grade 1). Oocyte recovery and embryo production are highly donor dependent. Stimulation of the follicular growth before OPU with one single injection of FSH diluted in 0.5% HA 36h after GnRH can be efficiently used for IVP in Holstein heifers, without decreasing the number of oocytes recovered and/or embryos produced with the advantage of reducing labour and stress of handling cattle. Table 1. Number and quality of oocytes and cleavage and blastocyst rates Treatment OPU Oocytes per donor Grade 1 and 2 oocytes (%) Cleavage rate (%) Blastocyst rate (%) Total embryos per OPU Grade 1 embryos per OPU 1×FSH 20 17.0 45.7 84 39.8 6.2 3.8 5×FSH 20 19.9 46.5 82 35.6 6.3 4.0

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