292 EXPRESSION PROFILES OF STRESS AND METABOLIC MARKER GENES DURING IN VITRO PRODUCTION OF BUFFALO (BUBALUS BUBALIS) EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 253
Author(s):  
R. Rajhans ◽  
G. S. Kumar ◽  
G. T. Sharma
Keyword(s):  
Bubalus Bubalis ◽  
Marker Genes ◽  
Hsp 70 ◽  
In Vitro Production ◽  
Glucose Transporter 1 ◽  
Total Rna ◽  
Gene Transcripts ◽  
Vitro Production ◽  
Glut1 Gene

An increased understanding of the pre-implantation embryo developmental stage, with respect to physiological interaction of embryo with its micromilieu both in vivo and in vitro, is imperative to comprehend the events of pre-implantation development.The objective of the present study was to examine the temporal expression of heat shock protein 70 (Hsp-70) and glucose transporter 1 (Glut1) genes in pre-implantation-stage buffalo embryos produced under the standard in vitro production (IVP) system. Embryos were produced from slaughterhouse ovaries employing standard in vitro embryo production protocol, and presumptive zygotes produced following IVM/IVF were cultured in vitro in mSOF under mineral oil; FCS (10%) was added at 48 h post-insemination (hpi).The time series of development at stages being zygote (18-20 hpi), 2-1 cell (48 hpi), 8-16 cell (94-96 hpi), morula (120-144 hpi), and blastocyst (168-192 hpi), pre-implantation embryos conforming to the above developmental pattern were considered as 'fast-cleaving embryos', and all the embryos that did not conform to the above developmental timing were regarded as 'slow-cleaving embryos'. Pools of immature oocytes (IM, 120), Matured oocytes (MO, 120), 8-16 cell stages (8-16, 70), morula (M, 28), and blastocyst (B, 9) were collected and prepared for total RNA isolation and RT-PCR for the specific transcripts, with �-actin as loading control. The total RNA content ranged from 2.5 to 5.0 ng per oocyte/embryo. Presence of Hsp 70 and Glut1 gene transcripts was assessed in different stages of buffalo pre-implantation embryos using primers designed from bovine Hsp 70 and Glut1 by using the OLIGO program. RT was standardized using the embryo equivalent of 1-10 oocytes/embryo as the template as described by Arcellana-Panlilio and Schultz 1993 (Methods Enzymol. 225, 303-328) with PCR conditions being 59�C and 62�C for 45 s with 39 cycles for Glut1 and Hsp 70 gene transcripts, respectively. Amplicons were subjected to restriction digestion and sequencing (Acc. No. AJ812563, AJ812564). The expression of Hsp 70 throughout pre-implantation development in the fast-cleaving embryos indicated their maternal and zygotic origin, but transcripts of the Hsp 70 gene, represented by a single 488-bp amplicon, were not detected in slow-cleaving embryos, suggesting altered zygotic expression. Glut1 expression was prominent from the 8-16 cell stage, indicating a metabolic shift from pyruvate to glucose after the pre-compaction stage. For slow-cleaving embryos, transcripts of the Glut1 gene, represented by a single 327-bp amplicon, were absent during morula- and blastocyst-stage embryos, indicating the poor developmental competence of these embryos, which morphologically appeared normal. These transcription patterns reflect the embryonic response to the in vitro culture conditions and also correlate with the embryo quality and the speed of development of the pre-implantation buffalo (Bubalus bubalis) embryos.

102 DEVELOPMENTAL COMPETENCE OF BUFFALO (BUBALUS BUBALIS) OOCYTES VITRIFIED AT DIFFERENT STAGES OF MATURATION IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 159
Author(s):  
K. Loganathasamy ◽  
R. Rajhans ◽  
G. SaiKumar ◽  
G. T. Sharma
Keyword(s):  
Expression Pattern ◽  
Glucose Transporter ◽  
Storage Period ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Control Group ◽  
Hsp 70 ◽  
Glucose Transporter 1 ◽  
Total Rna

Cryopreservation of unfertilized oocytes at very low temperature (-196�C) is carried out to ensure their continuous availability during different assisted reproductive techniques. However, various problems associated with the freezing of oocytes influence their developmental competence, resulting in suboptimal embryo production. The present study was planned to assess the developmental competence of buffalo oocytes vitrified at different meiotic stages of maturation. Expression profile of developmentally important genes, viz, heat shock protein 70 (HSP 70) and glucose transporter 1 (Glut1), was verified in these vitrified warmed oocytes. Buffalo cumulus-oocyte complexes were collected from slaughterhouse ovaries and divided into six groups: control (no vitrification); 0 h group (vitrified before maturation), and 6-, 12-, 18-, and 24-h groups [vitrified respectively at 6, 12, 18, and 24 h post-in vitro maturation (IVM)]. Vitrification solution consisted of propylene glycol (40% w/v), and trehalose (0.25 mol/L) in PBS + BSA (4% w/v) and vitrification was carried out by directly plunging 0.25-mL French mini-straws into liquid nitrogen. After a minimum storage period of 7 days, the straws were thawed at 37�C for 30 sec. In all groups, the oocytes completed 24-h of maturation. After 24 h maturation, a few oocytes from each of the six groups were stained with ethidium bromide to reveal their nuclear status. The remaining oocytes were co-incubated with frozen thawed buffalo semen in fertilization TALP with 6 mg/mL fatty acid free BSA and 10 �g/mL heparin sodium salt for 18 h. Presumptive zygotes were cultured in mSOF for 8 days. Vitrified warmed oocytes were subjected to total RNA isolation and RT-PCR for detection of mRNA transcripts of HSP 70 and Glut1 genes. Data were analyzed by one-way ANOVA and F-test analysis. Differences of P < 0.05 were considered significant. The percentage of oocytes recovered from all five vitrification groups varied from 89 to 92 out of which 84-91% of oocytes were morphologically normal. A higher proportion of nonvitrified control oocytes (72.8%; 40/55) reached the metaphase II stage than for the oocytes vitrified at 24 (60%; 36/60), 18 (54.4%; 31/57), 12 (42.3%; 22/52), 6 (33.3%; 20/60), and 0 (31.7%; 19/60) h of IVM. The cleavage rate of nonvitrified control oocytes was higher (36.8%) than that of oocytes vitrified at 0 (1.6%), 6 (2.0%), 12 (3.2%), 18 (5.3%), and 24 (5.2%) h of IVM. With regard to subsequent development, 0- and 6-h oocytes were blocked at 8 cells, whereas in other groups development reached the late morula (4.8%) and blastocyst (3.5%) stages, confirming that the stage of maturation at which oocytes are vitrified influenced the nuclear maturation and developmental competence. Total RNA content was 2.24 � 0.40 ng/oocyte in the control group and 2.11 � 0.22 ng/oocyte in the group vitrified after 24 h of IVM. The expression pattern of HSP 70 and Glut1 was identical in control and vitrified groups, indicating that the vitrification protocol did not alter the expression pattern of these genes.

204 PERIPHERAL PROGESTERONE CONCENTRATION AFFECTS BOVINE OOCYTE QUALITY AT THE MOLECULAR LEVEL

2013 ◽  
Vol 25 (1) ◽  
pp. 250
Author(s):  
N. Schlüter ◽  
A. Hanstedt ◽  
H. Stinshoff ◽  
K. Knauer ◽  
S. Wilkening ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Relative Abundance ◽  
Clinical Signs ◽  
Follicular Phase ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Glucose Transporter 1 ◽  
Vitro Production

The developmental competence of cumulus–oocyte complexes (COC) used for in vitro production is dependent on several factors including the stage of the oestrus cycle. In a recent study, we were able to show that circulating progesterone (P4) had no effect on follicle number, size, recovery rate, or in vitro production suitability of recovered COC (Schlüter et al. 2012 Reprod. Fertil. Dev. 24, 175–176). The aim of the present study was to determine the influence of circulating P4 concentrations on the molecular quality of bovine COC collected during repeated OPU sessions. The COC were aspirated twice per week for 5 to 6 weeks from 12 Holstein Friesian heifers. The first OPU session took place on Day 7 of the oestrous cycle after spontaneous ovulation (ovulation = Day 0). Blood samples were taken at the time of each OPU session, and P4 concentrations were determined using a radioimmunoassay. All animals showed clinical signs of oestrus and large follicles (≥8.5 mm) during the course of the OPU sessions. Following the aspiration of a large follicle, a CL-like structure (induced CL) could be detected. According to the P4 concentrations, the cycle was divided into 3 phases: CL phase after spontaneous ovulation (oCL; P4: ≥1 ng mL–1), follicle phase 1 (Fp; P4 <1 ng mL–1), and induced CL phase (iCL; P4: ≥1 ng mL–1). The length of the cycle after spontaneous ovulation did not differ significantly from that after induced ovulation (22.4 ± 3.1 days v. 23.8 ± 1.8 days, respectively). During the oCL-phase, blood P4 concentrations were significantly higher than during the iCL-phase (4.9 ± 2.3 ng mL–1 v. 3.0 ± 1.6 ng mL–1). For mRNA analysis, denuded COC were individually frozen at –80°C to analyse the relative transcript abundance using RT-qPCR. The transcripts studied play important roles during oocyte development [growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), glucose transporter 1 (SCL2A1), hypoxia inducible factor 2α (HIF2α), progesterone receptor (PGR), progestin and adipoQ receptor 5 (PAQR5), progesterone receptor membrane component 1 and 2 (PGRMC1, PGRMC2)]. Data were tested using analysis of variance (ANOVA) followed by multiple pairwise comparisons using Tukey’s test. A P-value of ≤0.05 was considered significant. The relative abundance of all transcripts except SCL2A1 was significantly increased in oocytes collected from follicles of the oCL phase compared with that from oocytes that had been aspirated during the iCL phase. A significant increase in the relative amount of PGR, PGRMC1, PGRMC2, and BMP15 transcripts was detected in oocytes stemming from the follicular phase to those from the iCL phase. No differences in the relative abundance of all transcripts were seen comparing oocytes from oCL phase and oocytes from the follicular phase. In summary, circulating P4 concentrations had an effect on the molecular quality of COC recovered during repeated OPU session, which might affect further development. The financial support of the FBF (Förderverein Biotechnologieforschung) e.V. is gratefully acknowledged.

Laboratory production of buffalo (Bubalus bubalis) embryos

1998 ◽  
Vol 10 (5) ◽  
pp. 379 ◽  
Author(s):  
P. Palta ◽  
M. S. Chauhan
Keyword(s):  
In Vitro Maturation ◽  
Large Scale ◽  
Follicle Stimulating Hormone ◽  
Bubalus Bubalis ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Nuclear Maturation ◽  
Fertilization Rates ◽  
Vitro Production

There is an increasing interest in large-scale in vitro production (IVP) of buffalo embryos through in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes for faster multiplication of superior germplasm. The recovery of total and usable quality oocytes from slaughterhouse ovaries is low in this species. The nuclear maturation rates of buffalo oocytes matured in the presence of follicular fluid or serum and hormones like luteinizing hormone, follicle-stimulating hormone and oestradiol vary from 70 to 80% and are comparable to those reported for cattle oocytes. However, with fertilization rates of 40–55%, and the yield of blastocysts at around 10–15%, the efficiency of IVP is much lower than that in cattle. The in vitro sperm preparation procedures and the systems employed for performing IVF and culture of zygotes up to blastocyst stage are suboptimal and need substantial improvements. The quality and viability of blastocysts produced need to be checked by cell count, and after transfer to synchronized recipients, for development of quality control standards.

scholarly journals Establishing an in vitro production program for buffalo embryos (Bubalus bubalis) in Colombia

2015 ◽  
pp. 4495-4504
Author(s):  
Felipe Gamarra P ◽  
Viviana Rendón V ◽  
Aldemar Chávez R ◽  
Leonardo Perez S ◽  
Walter Cardona-Maya ◽  
...  
Keyword(s):  
Bubalus Bubalis ◽  
Fertilization In Vitro ◽  
Natural Cycle ◽  
Ultrasound Guided ◽  
In Vitro Production ◽  
Production Program ◽  
Frozen Semen ◽  
Sexed Semen ◽  
Vitro Production

Objective. Evaluate the results of the standardization of the in vitro production program of buffalo embryos, using oocytes obtained by ultrasound guided oocyte puncture during the 2012 breeding season in Colombia. Materials and methods. Fifty seven buffalo females were selected for ultrasound guided transvaginal aspiration of follicles, oocytes were identified within follicular fluid, classified and transported to the laboratory and matured in vitro for 18 to 20 hours. Frozen semen of seven Mediterranean bulls were used, motile sperm was obtained using the Percoll technique and oocytes were inseminated with 2 million sperm/ml. Presumptive zygotes were cultured for 6 days, grade 1 embryos obtained were frozen using ethylene glycol. Embryos were transferred to females on day 5 during natural cycle. Results. 97 aspirations were performed on the 57 animals, in 8.2% of the aspirations no oocytes were found. 8 oocytes/aspiration were obtained. Of the 783 oocytes, 92% were classified as viable (721/783) and were fertilized. The cleavage and blastocyst rate were 23% and 19% respectively. 37 embryos were transferred and 11 pregnancies were obtained, confirmed by rectal palpation 60 days after transfer, with a pregnancy rate of 29.7%. Conclusions. The results presented here are comparable with others in literature and shows the feasibility of producing in vitro embryos and pregnancies after the standardization of current protocols, with normal and sexed semen and transfer during natural cycle in buffalo.Key words: Buffaloes, embryo, fertilization in vitro, reproduction (Source: MeSH).

scholarly journals Differences in Parameters of an Embryo In Vitro Production Program between Cattle (Bos Indicus) and Buffaloes (Bubalus bubalis)

2020 ◽  
Vol 9 ◽  
pp. 29-37
Author(s):  
Jesús Alfredo Berdugo ◽  
Ariel Marcel Tarazona-Morales ◽  
José Julian Echevererry ◽  
Jose Luis Konrad ◽  
Gustavo Angel Crudeli ◽  
...  
Keyword(s):  
Bos Indicus ◽  
Bubalus Bubalis ◽  
In Vitro Production ◽  
Production Program ◽  
Vitro Production

scholarly journals Differences in Parameters of an Embryo In Vitro Production Program between Cattle (Bos Indicus) and Buffaloes (Bubalus bubalis)

2020 ◽  
Vol 9 ◽  
pp. 29-37
Author(s):  
Jesús Alfredo Berdugo ◽  
Ariel Marcel Tarazona-Morales ◽  
José Julian Echevererry ◽  
Jose Luis Konrad ◽  
Gustavo Angel Crudeli ◽  
...  
Keyword(s):  
Bos Indicus ◽  
Bubalus Bubalis ◽  
In Vitro Production ◽  
Production Program ◽  
Vitro Production

Pregnancy and calving rates following transfer of in-vitro-produced river and F1 (river × swamp) buffalo (Bubalus bubalis) embryos in recipients on natural oestrus or synchronised for ovulation

2007 ◽  
Vol 19 (5) ◽  
pp. 670 ◽  
Author(s):  
Xianwei Liang ◽  
Xiufang Zhang ◽  
Bingzhuang Yang ◽  
Mingtang Cheng ◽  
Fenxiang Huang ◽  
...  
Keyword(s):  
Bubalus Bubalis ◽  
Sperm Cells ◽  
In Vitro Production ◽  
In Vitro Development ◽  
Antral Follicles ◽  
Swamp Buffaloes ◽  
Swamp Buffalo ◽  
Vitro Development ◽  
Vitro Production

The main objective of this study was to compare pregnancy and calving rates following transfer of in-vitro-produced fresh river and F1 (river × swamp) buffalo embryos in recipients synchronised by Ovsynch protocol or following natural oestrus. River embryos were produced from cumulus–oocyte complexes (COCs) derived by ovum pick up (OPU) on 40 Murrah and Nili-Ravi donor buffaloes over a twice-weekly collection schedule for 120 single OPUs. F1 embryos were produced by fertilisation of swamp COCs recovered from abattoir ovaries coincubated with river sperm cells. Both groups of embryos were produced following the same protocol for in vitro production. With regard to the OPU source of COCs, 923 antral follicles were punctured and 647 COCs were recovered (70%). From 462 selected COCs for IVM, 257 (55.6%) cleaved zygotes were recorded leading to 93 blastocysts (20.1%). In total, 590 swamp COCs were aspirated from abattoir ovaries and 476 were selected for IVM leading to 270 (56.7%) cleaved zygotes and resulting in 137 blastocysts (28.8%). River and F1 embryos were transferred between Day 6 to 7 of in vitro development, corresponding to blastocyst–expanding blastocyst, into F1 recipients synchronised by Ovsynch and swamp buffaloes following natural oestrus, respectively, each of them receiving two embryos. According to palpation per rectum of the ovaries at the time of embryo transfer, 26 of the 47 (55.3%) F1 recipients synchronised by Ovsynch were considered suitable for transfer, resulting in seven pregnancies (26.9%) and four calvings (15.3%) owing to three abortions occurring between 2 and 3 months of pregnancy. In total, 29 swamp recipients following natural oestrus were judged suitable as recipients, resulting in 12 pregnancies (41.4%) and 10 calvings (34.5%) owing to two abortions at 2 and 3 months of gestation respectively. Pregnancy and calving rates following transfer of river and F1 embryos were similar. Likewise, weight at birth of calves derived from transfer of river and F1 embryos was not different: 30.5 ± 1.4 and 32.9 ± 2.4 respectively. Pregnancy and calving rates following AI in a group of river and swamp buffaloes considered for reference in this study were similar to recipients carrying in-vitro-produced embryos. Collectively, no apparent postnatal complications were recorded in resulting live calves.

THE IN VITRO PRODUCTION OF ANDROGENS BY FOLLICULAR TISSUE OF RABBITS

1964 ◽  
Vol 47 (2) ◽  
pp. 306-313 ◽  
Author(s):  
Denis Gospodarowicz
Keyword(s):  
In Vitro Production ◽  
Vitro Production

ABSTRACT Incubation in vitro of rabbit follicles in separate experiments with dehydroepiandrosterone-14C (DHEA-14C), progesterone-14C and pregnenolone-3H in the presence of FSH gave the following results: 39 % of the radioactivity of DHEA-14C is converted to androstenedione and testosterone, while only 3 % of the radioactivity of either progesterone-14C or pregnenolone-3H is found in the androgen fraction. From the ratio of testosterone to androstenedione formed from the three precursors, the results are interpreted to mean that DHEA and pregnenolone, and not progesterone, are precursors of androgens in the follicle.

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