177 PERFORMANCE OF Gyr PREPUBERTAL HEIFERS IN IN VITRO EMBRYO PRODUCTION

2016 ◽  
Vol 28 (2) ◽  
pp. 219
Author(s):  
P. M. S. Rosa ◽  
A. J. R. Camargo ◽  
R. V. Serapião ◽  
L. S. A. Camargo ◽  
C. S. Oliveira
Keyword(s):  
Cleavage Rate ◽  
Embryo Production ◽  
Exact Test ◽  
Follicular Wave ◽  
Lactating Cows ◽  
In Vitro Embryo Production ◽  
Blastocyst Rate ◽  
Oocyte Donors ◽  
La Jolla

Bovine in vitro embryo production is highly relevant for dairy systems in Brazil, and Gyr dams are commonly used as oocyte donors. The aim of this study was to evaluate the use of prepubertal Gyr heifers as oocyte donors, an alternative to anticipate reproduction of those animals. For that, 11 Gyr [4 prepubertal (PP) donors and 7 adult cows © donors] were used in ovum pickup (OPU) sessions. The PP cows presented an average of 282.5 kg and 26.75 months, and had never displayed oestrous. Non-lactating cows presenting an average of 492 kg and 136 months were selected for C. Five replicates were performed, totaling 27 OPU sessions (C-17, PP-10) and 2–3 sessions per animal. Follicular wave was synchronised by aspiration of follicles larger than 8 mm 96 h before OPU. Cumulus-oocyte complexes (COC) were classified accordingly to their quality in viable (G1, G2, and G3) or non-viable (G4). Viable oocytes were matured and fertilized, and the presumptive zygotes were cultured in SOF medium at 38.5°C and 5% CO2 in air. Cleavage rate was assessed 48 to 72 h post-insemination (hpi) and blastocyst rate at 168 hpi. Mean number of structures was analysed by t-test, and percentage of viable, G1, G2, G3, G4, cleavage, and blastocyst rates were compared among groups by Fisher’s exact test (GraphPadInstat, La Jolla, CA, USA; P = 0.05). Results are followed by standard error values. All procedures were approved by a local ethics committee. We found that despite higher (P < 0.05) numbers for both viable oocytes (PP: 15 ± 2.6; C: 6.11 ± 0.76) and total oocytes (PP: 23.70 ± 2.83; C: 8.82 ± 1.19) in the PP group, the rate of viable oocytes was similar (P > 0.05) among PP and C groups (PP: 61.5 ± 6.51%, C: 66.79 ± 3.79%). Mean numbers of G1, G2, G3, and G4 oocytes were higher (P < 0.05) in the PP group (G1 = 7.1 ± 1.18; G2 = 4.9 ± 1.74; G3 = 3.9 ± 1.09; G4 = 7.8 ± 1.38) than in the C group (G1 = 2.70 ± 0.740; G2 = 2.47 ± 0.44; G3 = 1.11 ± 0.31; G4 = 2.52 ± 0.39). However, the proportion was similar (P > 0.05) among PP and C groups (PP: G1 = 29.5 ± 4.21%; G2 = 19.5 ± 2.85%; G3 = 15.9 ± 13.5%; G4 = 35.1 ± 6.33%; and C: G1 = 27.24 ± 4.44%; G2 = 29.60 ± 5.08%; G3 = 12.34 ± 3.01%, G4 = 30.79 ± 4.93%). Cleavage rate (PP: 91.3 ± 17.94%; C: 74.09 ± 4.65%), mean blastocyst number per OPU session (PP: 3.3 ± 1.29; C: 1.76 ± 0.28), and blastocyst rate (PP: 19.74 ± 7.40%; C: 27.03% ± 4.07%) were similar (P > 0.05) among groups. We conclude that prepubertal heifers presented increased numbers of viable oocytes per OPU session, but blastocyst yield was similar to adult cows. This data suggests that prepubertal Gyr heifers can be used as oocyte donors. Support from FAPERJ and Embrapa is acknowledged.

140 A comparison of in vitro embryo production between heifers and lactating Holstein donors without superstimulation

2019 ◽  
Vol 31 (1) ◽  
pp. 195
Author(s):  
E. P. Silva ◽  
M. K. Sermersheim ◽  
P. V. Marchioretto ◽  
R. Della Mea ◽  
L. M. Naves ◽  
...  
Keyword(s):  
Donor Age ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Exact Test ◽  
Lactating Cows ◽  
Cumulus Oocyte Complex ◽  
Sexed Semen ◽  
In Vitro Embryo Production ◽  
Blastocyst Rate

Bovine oocyte donor age seems to have an important role on in vitro embryo production. Therefore, the aim of this study was to compare cumulus-oocyte complex (COC) recovery, cleavage, and blastocyst rates between heifers and lactating Holstein donors. A total of 89 animals (heifers: n=60, 11 to 17 months of age; lactating cows: n=29, 60 to 180 days in milk) were used in this experiment. Ovum pickup (Day −1) was performed on a random day of the oestrous cycle without superstimulation by a single technician. Conventional or sexed semen were used for IVF (Day 0). Cleavage and blastocyst rates were evaluated on Days 3 and 7, respectively. Cleavage was determined when structures had 4 or more cells and blastocyst rate included grades I and II blastocysts. Continuous data were analysed by t-test, and binomial data were analysed by Fischer’s exact test. A total of 1,289 COC were recovered from lactating cows (n=509) and heifers (n=777). Total and viable COC per donor were higher (P&lt;0.05) in lactating cows (total=17.55±13.08; viable=11.24±8.9) than heifers (total=12.95±5.39; viable=7.88±4.08). The percentage of viable COC was similar (P&gt;0.05) in lactating cows (64.04%; 326/509) and heifers (60.87%; 473/777). Cleavage rate was higher (P&lt;0.0001) in lactating cows (58.74%; 299/509) than in heifers (45.05%; 350/427). Cleavage rate was similar (P&gt;0.05) with conventional and sexed semen in heifers (conventional=37.68%; 52/138; sexed=46.63%; 298/639) and cows (conventional=55.05%; 49/89; sexed=59.52%; 250/470). Blastocyst rate was higher (P&lt;0.0001) in lactating cows (19.84%; 101/509) than in heifers (8.49%; 66/777). Conventional semen had a higher (P&lt;0.0001) blastocyst rate (18.11%; 24/138) than sexed semen (6.57%; 42/639) in heifers. However, there was no difference (P&gt;0.05) in blastocyst rate between conventional (24.71%; 22/89) and sexed semen (18.8%; 79/420) in lactating cows. Blastocyst/cleaved structures ratio was higher (P&lt;0.05) in heifers using conventional (46.15%; 24/52) compared with sexed (14.06%; 42/298) semen. However, in lactating cows there was a tendency (P&lt;0.1) for higher blastocyst/cleaved structures ratio using conventional (44.89%; 22/49) compared with sexed semen (31.6%; 79/250). In conclusion, a higher number of total and viable COC were aspirated from Holstein lactating cows than heifers. Furthermore, cleavage and blastocyst rates were higher in lactating cows than in heifers. Conventional semen gave more embryos than did sexed semen in heifers but not in cows. Overall, oocytes from Holstein lactating donors are more suitable for in vitro embryo production than are those from heifer donors.

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.

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.

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.

176 Synchronisation of follicle wave emergence prior to superstimulation with purified FSH for ovum pickup affects blastocyst rate in pregnant Holstein heifers

2020 ◽  
Vol 32 (2) ◽  
pp. 215
Author(s):  
L. Carrenho-Sala ◽  
M. Fosado ◽  
R. Sala ◽  
E. Peralta ◽  
D. Pereira ◽  
...  
Keyword(s):  
Mixed Models ◽  
Ovarian Response ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Treatment Groups ◽  
Follicular Wave ◽  
The Mean ◽  
Blastocyst Rate ◽  
Oocyte Donors ◽  
Timing Of Initiation

The timing of initiation of superstimulatory treatments relative to follicle wave emergence has been shown to affect ovulatory response and invivo embryo production. The significant increase of invitro embryo production (IVP) and the possibility of using pregnant animals as oocyte donors has created the need to optimise superstimulatory treatments for IVP in pregnant cattle. Thus, the objective of the present study was to evaluate the effect of synchronisation of follicle wave emergence before superstimulation for ovum pickup (OPU) and IVP in pregnant heifers. Pregnant (47-62 days of gestation) Holstein heifers (n=28) 19.5±0.3 months of age were assigned in a completely randomised design to one of two groups: synchronisation of follicular wave emergence by dominant follicle removal (DFR; all follicles &gt;6mm) or untreated control (no DFR). Superstimulatory treatments were initiated 36h after DFR or at random stages of the follicular wave in the no-DFR group and consisted of the administration of 160mg of purified FSH (Folltropin-V, Vetoquinol) over four injections 12h apart as follows: 48.0, 42.7, 37.3, and 32.0mg. Ovum pickup was performed in all heifers 40h after the last purified FSH injection. Heifers were subjected to OPU for oocyte recovery, and the number of follicles was determined. Recovered oocytes were processed in groups by treatment, and IVP was performed. Differences between treatment groups were evaluated using generalised linear mixed models. Results are presented in Table 1 and are expressed as means±s.e.m. for data collected at the time of OPU or as proportions for embryo production results. The number of small follicles (&lt;6mm) at the time of OPU was greater in the no-DFR group than in the DFR group (P=0.04). Conversely, there were no differences between treatments in the number of medium follicles (6-10 mm; P=0.17), large follicles (&gt;10 mm; P=0.11), total follicles (P=0.93), total number of recovered oocytes (P=0.4), or number of viable oocytes (P=0.53). The mean oocyte percentage recovery rate was not different between heifers in the DFR (53.6±4.7%) and no-DFR (56.5±4.7%) groups (P=0.52). Both cleavage and blastocyst rate were greater (P&lt;0.008) in the DFR group than in the no-DFR group; as a result, the number of transferable embryos per animal was 5.6 in the DFR group and 2.8 in the no-DFR group. In summary, initiation of superstimulatory treatments at the time of follicle wave emergence improves cleavage and blastocyst rates, thus leading to greater embryo production. Table 1.Ovarian response and embryo production in pregnant heifers superstimulated with or without synchronisation of follicle wave emergence Variable DFR No DFR Small follicles, n 8.1±1.2A 12.1±1.8B Medium follicles, n 18.3±1.3 13.7±2.0 Large follicles, n 2.4±0.6 1.4±0.4 Total follicles, n 28.8±1.4 27.2±2.2 Total oocytes, n 15.4±1.5 16.0±1.9 Viable oocytes, n 13.7±1.5 13.4±1.8 Cleavage rate,% (n) 77.1 (192)A 64.4 (188)B Blastocyst rate,% (n) 40.6 (192)A 20.7 (188)B A,BMeans within a row with different superscripts differ (P&lt;0.05).

293 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION ON IN VITRO EMBRYO PRODUCTION IN COWS

2006 ◽  
Vol 18 (2) ◽  
pp. 254
Author(s):  
A. F. Ramos ◽  
R. Rumpf ◽  
M. R. Mollo ◽  
J. U. Câmara ◽  
I. Pivato ◽  
...  
Keyword(s):  
Latin Square ◽  
Estradiol Benzoate ◽  
Continuous Variables ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Follicular Wave ◽  
The Mean ◽  
In Vitro Embryo Production

In order to achieve the ideal time of ovum pickup (OPU) for in vitro embryo production (IVP) in cows regarding number and quality of oocytes recovered, this study investigated the effect of synchronization of wave emergence with estradiol benzoate (EB) injected 7 days prior to follicular aspiration. In a Latin square design, 12 crossbred beef cows were randomly divided into three groups, with three replicates each. Cows were synchronized with a norgestomet ear implant for 7 days followed by an i.m. prostuglandin F2� (PGF2�) injection and aspiration of all ovarian follicles larger than 3 mm in diameter. After that, follicles from cows in group 2X were aspirated twice a week with 4- and 3-day intervals, and follicles from groups 1X and 1X-EB were aspirated once a week. Cows from group 1X-EB also received an im injection of 2 mg of EB immediately after OPU. Throughout the study cows were kept with an ear norgestomet implant that was replaced every 2 weeks. Ultrasound evaluations of numbers of follicles greater than 3 mm in diameter and size of the largest follicle at the time of OPU were performed. Recovered oocytes were evaluated for quality, and the viable ones (Grades I, II, and III) were in vitro-fertilized on Day 0. Cleavage rate was evaluated on Day 2 and blastocyst production on Day 7. Continuous variables were compared by ANOVA and binomial data were compared by chi-square. For the 2X group, only data from the OPU performed 3 days after the last OPU were used for analysis. Results are presented as percentages or mean � SEM. Size of the largest follicle was greater (P < 0.05) in 1X coes (12.9 � 0.2 mm) than in 1X-EB cows (11.1 � 0.3 mm), which was greater than in 2X (9.6 � 0.4 mm) cows. The 1X cows had more follicles at OPU than 2X cows (17.5 � 0.7 vs. 14.1 � 0.9), whereas the 1X-EB group (15.9 � 0.7) was intermediate and not different from the others. There was no difference in the mean number of recovered oocytes among 2X (9.6 � 0.6), 1X (12.7 � 0.8) and 1X-EB (12.3 � 1.0) cows, and the mean number of viable oocytes among groups (5.8 � 0.5, 7.3 � 0.5, and 7.0 � 0.6) for 2X, 1X, and 1X-EB cows, respectively). The rate of viable oocytes was also similar among groups [58.8% (191/325) for 2X, 58.4% (267/457) for 1X, and 57.2% (231/404) for 1X-EB cows]. Cleavage [68.6% (131/191), 65.2% (174/267), and 68.4% (158/231)] and blastocyst [38.7% (74/191), 43.8% (117/267), and 44.2% (102/231)] rates were also not different among 2X, 1X, and 1X-EB groups, respectively. Although the use of 2 mg of EB in association with a norgestomet implant 7 days prior to OPU altered the follicular wave profile, it was not enough to improve number and quality of the oocytes recovered. Moreover, this study failed to demonstrate a positive effect of OPU earlier after wave emergence, when the effect of dominance should be less pronounced, on IVP in cows. The first author was supported by the fellowship 141077/2004-2 of CNPq, Brazil.

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.

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.

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