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.

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.

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.

123 In Vitro Embryo Production in Lyophilized In Vitro Culture Medium as a Method to Increase the Medium's Shelf-Life

2018 ◽  
Vol 30 (1) ◽  
pp. 201
Author(s):  
M. Rubessa ◽  
F. Salerno ◽  
D. Weisgerber ◽  
B. Gasparrini ◽  
B. Harley ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Shelf Life ◽  
Statistical Difference ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Freeze Dried ◽  
In Vitro Embryo Production ◽  
Α Level ◽  
The Impact

The worldwide production of livestock embryos requires stable medium with long shelf life. In this experiment, we evaluated the impact of the freeze-dried in vitro culture (IVC) medium (Mdry) on in vitro embryo production. We compared the standard IVC and Mdry media for cleavage rate and embryo production. Media solutions (10 mL) were aliquoted into 50-mL conical tubes and lyophilized to form a powder concentrate using a Genesis freeze-dryer (VirTis, Gardener, NY, USA). Lyophilization consisted of a constant cooling from 20°C to –10°C at a constant rate of 1°C/min with a 2-h hold at –10°C before sublimation at 0°C. Mdry medium were held at –80°C for 4 months. When the IVC medium was rehydrated, the pH were adjusted to 7.4. Abattoir-derived Holstein oocytes (n = 618, in 7 replicates) were in vitro matured and fertilized with sexed semen, according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium with 5% BS at 39°C with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed. All recorded parameters were subjected to a Chi-Square Test 2 × 2. The parameters compared were percent cleavage, blastocysts, and embryos/cleaved. The α level was set at 0.05. All data were expressed as quadratic means and mean standard errors. The results (Table 1) showed not a statistical difference between control and Mdry. The Mdry had a higher percentage of cleaved zygotes (65.4% v. 53.4%) but not enough for a statistical difference. However, when we compared embryo production, there was no difference between treatments. The ratio between blastocysts and cleaved embryos was higher in the control group but not significant according to our selected α level. These results indicate that it is possible lyophilize IVC medium without interfering with the potential quality of the medium. Further studies will be needed to better understand the positive effect of the lyophilization on the cleavage rate. Table 1.Mean (SD in parentheses) percentage cleavage and blastocysts

135 Use and dose of porcine follicle-stimulating hormone for ovarian superstimulation prior to ovum pickup and in vitro embryo production in pregnant Holstein heifers

2019 ◽  
Vol 31 (1) ◽  
pp. 192
Author(s):  
R. V. Sala ◽  
L. C. Carrenho-Sala ◽  
M. Fosado ◽  
E. Peralta ◽  
D. C. Pereira ◽  
...  
Keyword(s):  
Limited Information ◽  
Dominant Follicle ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Oocyte Competence ◽  
Treatment Groups ◽  
Oocyte Recovery ◽  
In Vitro Embryo Production

The benefit of superstimulation with exogenous FSH before ovum pickup for in vitro embryo production has been the subject of significant controversy. In addition, there is limited information on different dose regimens. Thus, the objective of the present study was to evaluate the effect of dose of porcine (p)-FSH during superstimulation before ovum pickup (OPU) on in vitro embryo production in pregnant heifers. Pregnant Holstein heifers (n=36) were assigned to a complete 3×3 crossover design. Three treatment groups were evaluated as follows: p-FSH 0mg (FSH0), p-FSH 160mg (FSH160) and p-FSH 300mg (FSH300). Three sessions of OPU were performed on each animal at 48, 62 and 76 days of gestation, with a washout interval between sessions of 14 days. Follicular wave emergence was synchronized by dominant follicle removal. Heifers in the FSH0 group received no further treatment, whereas the remaining groups received a total of 4 injections 12h apart as follows: FSH160 (48.0, 42.7, 37.3 and 32.0mg) or FSH300 (90.0, 80.0, 70.0 and 60.0mg), beginning 36h after dominant follicle removal. Ovum pickup was performed in all heifers 40h after the last p-FSH injection. Heifers were subjected to OPU for oocyte recovery, and number of follicles was determined. Recovered oocytes were processed and in vitro embryo production performed. Differences between treatment groups were evaluated by generalized linear mixed models. Data are presented (Table 1) as mean±standard error of the mean. There was no effect of days in gestation for any of the outcomes evaluated (P&gt;0.05). Follicle numbers at the time of oocyte recovery were different (P&lt;0.01) between groups. Heifers in the FSH300 group had a greater (P&lt;0.05) number of medium, large and total follicles than heifers in the FSH0 group, whereas heifers in the FSH160 were intermediate. Total number of recovered, viable and cleaved oocytes were greater (P&lt;0.01) in FSH300- than in FSH160- and FSH0-treated heifers. Cleavage rate and blastocyst development rate were not different (P&gt;0.10) between groups. The number of grade 1 and 2 blastocysts was greater in FSH300- than in FSH160- and FSH0-treated heifers (P&lt;0.03). In summary, the use of 300mg of p-FSH before OPU in pregnant heifers increases the number of follicles, oocytes and blastocysts produced per heifer with no detrimental effect on oocyte competence. Table 1.Ovum pickup and in vitro embryo production in pregnant heifers treated with different doses of porcine FSH

133 Test of minimum-intervention protocols for optimizing in vitro embryo production in bison

2019 ◽  
Vol 31 (1) ◽  
pp. 192
Author(s):  
M. L. Zwiefelhofer ◽  
E. M. Zwiefelhofer ◽  
S. X. Yang ◽  
S. Maeda ◽  
J. Singh ◽  
...  
Keyword(s):  
Control Group ◽  
Nominal Data ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Chi Squared ◽  
In Vitro Embryo Production ◽  
Categorical Distribution ◽  
Long Acting ◽  
Parks Canada

The study was done to determine whether minimal handling protocols for ovarian synchronization and ovarian superstimulation may be used to increase in vitro embryo production in bison. Ultrasound-guided cumulus-oocyte complex (COC) collection was done in a group of bison (n=23; random start) during the anovulatory season to synchronize new follicular wave emergence. The COC were classified morphologically (compact-good and -regular, expanded, denuded, degenerate) but not processed further. At the time of COC collection (Day 0), bison were assigned randomly to 3 groups and given 5mL of saline IM (non-superstimulated controls; n=11), 10 Armour units of pFSH (Antrin R10, Kyoritsu Seiyaku Corp., Tokyo, Japan) in 5mL of saline IM once per day from Day 0 to 2 (regular FSH; n=5), or 30 armour units of a sustained-release form of pFSH (Antrin R10Al, Kyoritsu Seiyaku Corp.) in 5mL of saline SC on Day 0 (long-acting FSH; n=7). On Day 4, a second COC collection was performed. Only compact COC were processed. The COC were matured in vitro for 25 to 28h at 38.8°C, fertilized (2×106 sperm mL−1) and co-incubated at 38.8°C in 5% CO2 for 18h. Presumptive zygotes were denuded and cultured at 38.8°C in 5% O2, 5% CO2 and 90% N2. Nominal data were compared by t-test and analysis of variance. Binomial data were compared among groups by chi-squared. There was no difference between the first (random) COC collection (n=23) and second collection (n=11 non-superstimulated controls) in the total number of follicles detected, but the distribution among size categories (3-4, 4-8, and &gt;8mm) differed, i.e. fewer in the 3 to 4mm category at the time of the second COC collection (12.2±1.0v. 8.1±1.4; P&lt;0.05). In the nonstimulated control group, there were no differences between the first and second COC collections in the number of follicles aspirated (12.7±1.0v. 10.4±1.5), number of COC collected (7.7±0.9v. 5.3±1.3), or in the categorical distribution of COC. At the second COC collection, the number of follicles in the &gt;8mm category was greater in the regular FSH group than in the control or long-acting FSH groups (2.8±0.5v. 1.1±0.3, and 1.9±0.4, respectively; P&lt;0.05), but no differences were detected in the number of follicles aspirated, COC collected, or in the categorical distribution of COC. The cleavage rate (of total oocytes submitted to in vitro maturation), recorded 2 days after IVF, was higher in the control group than in either the regular FSH or long-acting FSH groups [25/35 (71.4%), 7/28 (25.0%), 8/35 (22.8%); P&lt;0.0001]. The freezable embryo production rate, recorded 7 days after IVF, was greater in the control group than in the regular FSH or long-acting FSH groups [19/35 (54.3%), 5/28 (17.9%), 5/35 (14.3%); P&lt;0.01]. In conclusion, minimal-handling interventions used in the present study to increase embryo production in bison were not effective, likely as a result of the timing, frequency, and duration of superstimulation. A random start resulted in greater COC collection than collection 4 days after ovarian synchronization, and embryo production rates were greater in non-superstimulated bison. This work was supported by Parks Canada and Saskatchewan ADF. Antrin products donated by Kyoritsu Seiyaku Corp., Japan.

PSX-A-23 Late-Breaking: Supplementation of Nerve Growth Factor (β-NGF) in the maturation medium and efficiency of in vitro embryo production in cattle

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 365-365
Author(s):  
Lucas Gonçalves ◽  
Muller C Martins ◽  
Natalia Arle ◽  
Rafaela T Torres ◽  
Luisa Migilo ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Oocyte Maturation ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Nerve Growth ◽  
Maturation Medium ◽  
In Vitro Embryo Production ◽  
Blastocyst Rate

Abstract The aim of this study was to evaluate the supplementation of Nerve Growth Factor (β-NGF) in the maturation medium in in vitro embryo production routines. Antral follicles were aspirated from ovaries of cows obtained from slaughterhouses and then oocytes were selected for quality (grades I and II) for in vitro maturation and subjected to 4 successive in vitro embryo production routines (IVEP). Supplementation of 100 ng of β-NGF was performed in the oocyte maturation medium 22 hours before in vitro fertilization. 48 hours after fertilization of the oocytes, an analysis was made of their cleavage rate by counting blastomeres with the aid of a stereoscopic microscope (cleavage rate = number of embryos / number of initial oocytes). Seven days after fertilization, the blastocyst rate was determined through the relation to the number of oocytes that started cleavage and reached this stage of development (blastocyst rate = number of blastocyst / number of oocytes that started cleavage). To verify the existence of a difference between the supplemented and the non-supplemented groups, the paired T test was applied, using the Excel / Action software (Microsoft). In vitro embryo production routines supplemented with β-NGF in the maturation medium had, on average, a higher cleavage rate (P = 0.0072) and a higher blastocyst rate (P = 0.0033) compared to non-supplemented routines with β-NGF. In this study was demonstrated that Nerve Growth Factor supplementation in the maturation medium improves the efficiency of in vitro embryo production in cattle, and this protein has a probable action in the oocyte maturation process.

scholarly journals Effect of deslorelin acetate treatment in oocyte recovery and in vitro embryo production in domestic cats

2016 ◽  
Vol 19 (10) ◽  
pp. 1091-1095
Author(s):  
Camila Louise Ackermann ◽  
Eduardo Trevisol ◽  
Leticia Ferrari Crocomo ◽  
Tatiana da Silva Rascado ◽  
Rodrigo Volpato ◽  
...  
Keyword(s):  
Treated Group ◽  
Control Group ◽  
Domestic Cats ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Oocyte Recovery ◽  
Significant Difference ◽  
In Vitro Embryo Production ◽  
Blastocyst Rate

Objectives The present study investigated the effect of contraceptive treatment with deslorelin acetate on in vitro embryo production and oocyte recovery in domestic queens. Methods Twenty-one mature domestic cats were used. Eleven queens (treated group) and one tom were kept in an experimental cattery, and 10 queens were privately owned (control group). When in interestrus or diestrus (day 0) a deslorelin acetate implant (Suprelorin, 4.7 mg/animal) was inserted into the subcutaneous tissue of the interscapular region in all queens in the treated group. After 6 months of treatment, all animals were ovariohysterectomized, and the ovaries were used for in vitro embryo production. Percentage of cleavage was determined 18 h after oocyte insemination and blastocyst formation was assessed on the eighth day of culture. The rate of cumulus-oocyte complexes (COCs) recovery was analyzed by an unpaired t-test. The cleavage and blastocyst rates were expressed as percentages and analyzed by Fisher’s exact test. All analyses were performed using GraphPad Prism v5.0, with P <0.05 set as the level of significance. Results In the treated group, we recovered 8.3 ± 1.15 grade I COCs per queen; the cleavage rate was 60% and the blastocyst rate was 36%. In the control group, we recovered 18.4 ± 3.21 grade I COCs per queen; the cleavage rate was 55.97% and the blastocyst rate was 34%. Forty percent of treated females did not produce any blastocysts. In the treated group, we observed a significant decrease in COC recovery. Although there was no significant difference in cleavage and blastocyst rates between groups, 40% of treated females did not produce any blastocysts. Conclusions Recovery of grade I COCs is negatively affected by deslorelin treatment in domestic cats. Regarding embryo production, new studies are still necessary to evaluate the success of this technique owing to the individual effect caused by deslorelin acetate.

365 QUALITY ASSESSMENT OF BOVINE CRYOPRESERVED SPERM AFTER SEXING BY FLOW CYTOMETRY AND ITS USE FOR IN VITRO EMBRYO PRODUCTION

2010 ◽  
Vol 22 (1) ◽  
pp. 339
Author(s):  
J. O. Carvalho ◽  
R. Sartori ◽  
G. M. Machado ◽  
G. B. Mourão ◽  
M. A. N. Dode
Keyword(s):  
Flow Cytometry ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Fertilization Rate ◽  
Computer Assisted ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Acridine Orange Staining ◽  
In Vitro Embryo Production

Several studies using sex-sorted sperm by flow cytometry have shown that its fertility is reduced. Therefore, this study evaluated structural and functional characteristics of sperm sexed by flow cytometry. In addition, in vitro embryo production (IVP) and development was assessed when frozen-thawed unsorted and sex-sorted sperm from 4 Nellore bulls. Each ejaculate was separated into three fractions: non-sexed (NS), sexed for X-sperm (SX), and sexed for Y-sperm (SY). After thawing, each sample was analyzed for sperm motility by computer-assisted semen analysis (CASA, Berkeley, CA), sperm head agglutination, sperm morphology, membrane integrity by propidium iodide (PI) and 6-carboxy-fluorescein diacetate (CFDA) staining, acrosome integrity by peanut agglutinin (PNA), capacitation by chlortetracycline (CTC), and chromatin integrity by acridine orange staining. Then, the samples were placed in 45 : 90% (NS90) or 45 : 60% (NS60, SX, and SY) Percoll™ gradients. After Percoll™ centrifugation, sperm pellets were analyzed or used for IVP. All analyses were replicated independently three times. For IVP, 2,271 in vitro matured oocytes were used. To assess fertilization rate, presumptive zygotes were fixed and stained with lacmoid at 18 h post-insemination (hpi). Cleavage was evaluated at Day 2 (48 hpi) and blastocyst development at Days 6, 7, 8, and 9 of culture. Data were analyzed using generalized linear models. No differences (P > 0.05) were observed between SX and SY groups for e sperm variables evaluated either before or after Percoll™. However, non-sexed sperm had higher sperm motility, greater percentage of sperm with intact membranes, and greater percentage of live sperm with intact acrosomes than sexed sperm (P < 0.05). An effect of Percoll™ was observed in the non-sexed samples, with those submitted to 45 : 90% gradient having higher motility, greater percentage of cells with intact membrane, and lower recovery rate than those submitted to a 45 : 60% gradient. No differences among groups were observed for fertilization rate, being 74.0 ± 5.7, 63.2 ± 5.1, 67.2 ± 5.7, and 55.4 ± 5.9% for NS90, NS60, SX, and SY, respectively. Group NS90 showed a greater cleavage rate than did the SY group, while groups NS60 and SX had similar rates to the others. Blastocyst development rates on Day 6 to Day 9 were greater for group NS90. For example, on Day 8 the blastocyst rate was 34.9 ± 3.6, 22.2 ± 3.2, 18.1 ± 3.3, and 14.8 ± 2.9% forNS90, NS60, SX, and SY groups, respectively. All groups showed similar embryonic developmental stages on Day 6 to Day 9. Although sex-sorting affected sperm characteristics, it did not cause a decrease with in vitro fertility. However, differences in blastocyst rates between groups NS60 and NS90 indicated that the sperm selection protocol affected embryo production. Financial support: Embrapa Genetic Resources and Biotechnology.

157 FIRST IN VITRO EMBRYO PRODUCTION IN ALPACAS (LAMA PACOS)

2009 ◽  
Vol 21 (1) ◽  
pp. 177 ◽  
Author(s):  
G. Gamarra ◽  
E. Huaman ◽  
S. León ◽  
M. Carpio ◽  
E. Alvarado ◽  
...  
Keyword(s):  
South American ◽  
Cleavage Rate ◽  
Embryo Production ◽  
South American Camelids ◽  
Nitrogen Vapor ◽  
Epidermal Growth ◽  
Thermos Flask ◽  
In Vitro Embryo Production ◽  
Lama Pacos

The objective was to produce alpaca embryos in laboratory due to its potential role for the multiplication of genetically superior animals and for conservation purposes. Ovaries were collected from an alpaca abattoir located in the Central Highlands of Peru and transported in a thermos flask with warm saline and antibiotics to the laboratory located 200 km away on the coast. Alpaca epididymal sperm to be used for fertilization was previously frozen by diluting in a TRIS-Fructose based extender with 10% glycerol and frozen as pellets in liquid nitrogen vapor. From 31 ovaries, 262 cumulus–oocyte complexes (COCs) were collected (mean of 8.5 COCs per ovary) which were matured in TCM-199 supplemented with 10% heat inactivated FCS plus epidermal growth factor (EGF), FSH, LH, estradiol, and cysteamine for 30 h incubation at 38.5°C, 5% CO2 and 90% humidity. The selected oocytes post-maturation were fertilized with the frozen/thawed sperm that was subjected post-thawing to Percoll gradient (90 and 45% Percoll), centrifugation and resuspension in TALP-IVF medium supplemented with 20 μm D-penicillamine, 10 μm hypotaurine, 1 μm epinephrine and 1.1 μg mL–1 of heparin. The oocytes were inseminated with a concentration of 10 × 106 spermatozoa per drop of 100 mL of fertilization medium containing 30 oocytes each and incubated for 24 h at 38.5°C, 5% CO2 and 90% humidity. The presumptive zygotes were transferred to 200 μL drops (30 zygotes per drop) of SOFaa media supplemented with 5% heat-inactivated FCS which was replaced by SOFaa plus 1% heat-inactivated FCS on day 5 after fertilization. The incubation period post-fertilization was up to day 7 at 38.5°C, 5% CO2 and 90% humidity, when the embryos were inspected and graded. The cleavage rate was evaluated at 72 h post-fertilization and embryo development was evaluated on day 5 and 7 post-fertilization. The cleavage rate was 27.1% (71/262) and the percentage of oocytes that reached the stage of morula and blastocyst was 8.0% (21/262). The percentage of blastocyst that hatched when incubated after day 7 was 14.28% (3/21). The in vitro embryo production in alpacas was successful and suggests the possibility for application in intensive reproduction for conservation of South American camelids and for genetic improvement. Research was partially funded by contributions of BIONICHE and SAIS TUPAC AMARU, Junin, Peru.

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.

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