340 IN VITRO MATURATION OF PREPUBERTAL BOVINE OOCYTES ON A GRANULOSA CELL MONOLAYER FROM ADULT ANIMALS IMPROVES THEIR DEVELOPMENTAL COMPETENCE

2006 ◽  
Vol 18 (2) ◽  
pp. 277
Author(s):  
S. Ponebsek ◽  
C. Wrenzycki ◽  
K.-G. Hadeler ◽  
D. Herrmann ◽  
K. Korsawe ◽  
...  
Keyword(s):  
Granulosa Cell ◽  
Relative Abundance ◽  
Glucose Transporter ◽  
Cell Monolayer ◽  
Mrna Abundance ◽  
Developmental Competence ◽  
Hsp 70 ◽  
Developmental Capacity ◽  
Calf Oocytes

Oocytes from prepubertal calves have a decreased developmental competence compared with oocytes from adult animals. The goal of this study was to improve the developmental competence of juvenile oocytes by maturation on granulosa cell (GC) monolayers from adult animals. Oocytes were recovered by ovum pickup (OPU) from 48 Holstein Friesian calves at 7-8 months of age and 18 adult cows. Animals received intramuscular injections of 60 mg FSH 48 h prior to each OPU session. Follicles were punctured twice per week in six consecutive OPU sessions. Cumulus oocyte complexes (COCs) recovered from calves were divided into three quality groups (classes I-III) and were then randomly distributed into three maturation groups: COCs were matured for 24 h on either GC or fibroblasts or without co-culture. Cow oocytes were matured without co-culture. TCM-199 supplemented with BSA (0.1%), hCG (5 IU/mL), and eCG (10 IU/mL) served as the medium in all groups. After maturation, all COCs were fertilized in vitro; after 18 h, presumptive zygotes were cultured in SOF+BSA for 8 days (37�C, 5% CO2). On Day 3, cleavage rates and, on Day 8, blastocyst rates were determined. The relative mRNA abundance of the following transcripts, critically involved in early embryonic development was determined: growth differentiation factor-9 (GDF-9), heat shock protein 70 (Hsp-70), and glucose transporter-3 (Glut-3). Single immature and matured oocytes (for GDF-9 and Hsp-70) and 8-16-cell embryos and expanded blastocysts (for Hsp-70 and Glut-3) from calves and cows were examined by semiquantitative RT-PCR. Cleavage and blastocyst rates were similar in oocytes derived from cows and calves matured on GC (74.3% vs. 70.0% and 22.3% vs. 22.3%, respectively), but were significantly higher (P < 0.05; one way ANOVA, Student-Newman-Keuls Method) than in the group without co-culture on fibroblasts (55.2% vs. 53.6% and 11.7% vs. 5.5%, respectively). GDF-9 expression was similar in immature calf and cow oocytes. After maturation, a significant decrease in GDF-9 expression was observed in calf oocytes. Matured cow oocytes showed a significantly higher mRNA abundance of GDF-9 than matured calf oocytes. The relative abundance of Hsp-70 was decreased in matured oocytes of all groups. Expanded blastocysts derived from adult oocytes expressed Hsp-70 significantly higher than blastocysts derived from oocytes of the control calves. The relative abundance of Glut-3 mRNA was similar in 8-16 cell embryos and expanded blastocysts in all groups. Overall, mRNA expression pattern for Hsp-70 and Glut-3 in blastocysts from GC matured oocytes were similar to that of cow blastocysts. Results indicate that maturation of juvenile calf oocytes on granulosa cells from adult animals improves their developmental competence. These findings provide clues toward identification of factors critically involved in acquiring full developmental capacity at puberty.

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.

129 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES AFTER IN VITRO MATURATION AND IN VITRO CULTURE UNDER COMPARATIVE OXYGEN TENSION

2011 ◽  
Vol 23 (1) ◽  
pp. 169
Author(s):  
J. T. Kang ◽  
M. Atikuzzaman ◽  
D. K. Kwon ◽  
S. J. Park ◽  
S. J. Kim ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Oxygen Tension ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Mrna Abundance ◽  
Developmental Competence ◽  
Multiple Genes ◽  
Oxygen Concentrations

The in vitro developmental abilities of porcine oocytes are generally increasing steadily at a similar ratio to those of in vivo embryos. However, it has been suggested that the in vitro culture system for the development of porcine embryos is not optimal. In this study, we investigated the effect of 2 oxygen concentrations (5 and 20%) on porcine embryo development during in vitro maturation and in vitro culture and analyzed differences in gene expression of resulting blastocysts. Oocytes were recovered by aspiration of slaughterhouse ovaries and then matured in tissue culture medium (TCM) 199 supplemented with 10% porcine follicular fluid (pFF), epidermal growth factor (EGF), insulin, pyruvate, cystine, and gonadotropin. Matured oocytes were then activated parthenogenetically, cultured in PZM-3 media for 7 days. In vitro maturation (M group) of oocytes was carried out under two oxygen concentration (5 and 20%) in terms of nuclear maturation (polar body extrusion; Exp. 1). The developmental differences between 5% oxygen culture group and 20% oxygen culture group during in vitro culture (C group) of embryos after parthenogenetic activation was investigated in terms of first cleavage and blastocyst formation (Exp. 2). Relative mRNA abundance of multiple genes in blastocysts was analyzed for transcript abundance of genes related with metabolism (GLUT1, LDHA), oxidative response (MnSOD, GPX1), apoptosis (BAX, Bcl2), and developmental competence (CCNB1, IGF2R; Exp. 3). The results show there were no significant differences in maturation rate between 2 oxygen concentrations during in vitro maturation (83 v. 86%). It was thought that cumulus cells surrounding oocytes might have attenuated oxidative stress, but number of resulting blastocysts were (P < 0.05) increased in 5% IVC group when compared with 20% IVC group (18.67 v. 14.09%, respectively). Moreover, the M20C5 group (23.01%) had a beneficial effect on in vitro culture compared with M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. Total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each group altered the expression of genes in various patterns. Therefore, it could be concluded that high oxygen tension during in vitro maturation and low oxygen tension during in vitro culture might alter the expression of multiple genes related to oocyte competence and improve (P < 0.05) embryo development, but not blastocyst quality. This study was supported by MKE (#2009-67-10033839, #2009-67-10033805), NRF (#M10625030005-508-10N25), BK21 for Veterinary Science, IPET (#109023-05-1-CG000), and Hanhwa L&C.

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.

286 DEVELOPMENTAL CAPACITY OF PREPUBERTAL BOVINE OOCYTES CULTURED WITH CYCLIC AMP MODULATORS

2015 ◽  
Vol 27 (1) ◽  
pp. 232 ◽  
Author(s):  
S. M. Bernal ◽  
J. Heinzmann ◽  
D. Herrmann ◽  
U. Baulain ◽  
K.-G. Hadeler ◽  
...  
Keyword(s):  
Oocyte Retrieval ◽  
Developmental Competence ◽  
Vitro Fertilization ◽  
Lactating Cows ◽  
Developmental Capacity ◽  
Commercial Breeding ◽  
Bovine Oocytes ◽  
One Way Anova ◽  
Dmso Vehicle Control

Prepubertal bovine donors are currently used for commercial breeding to accelerate the genetic gain and decrease the generation interval. Nevertheless, it has been reported that their oocyte developmental competence is lower than in adult females. Addition of cAMP regulators during in vitro maturation (IVM) has been suggested to enhance blastocysts rates (Albuz et al. 2010 Hum. Reprod. 25, 2999–3011). Here, we evaluated the effects of the cAMP modulators forskolin, 3-Isobutyl-1-methylxanthine (IBMX), and cilostamide during extended IVM on the developmental capacity of oocytes from prepubertal and adult bovine females. A total of 1851 oocytes from 24 lactating cows (>2 lactations) and 24 prepubertal donors (6–10 mo old) were collected by transvaginal oocyte recovery (OPU) twice per week and divided into 3 experiment groups: (1) TCM24 (OPU medium: PBS; 24 h of IVM; standard protocol/control); (2) cAMP30 [OPU medium: PBS-IBMX (500 μM); 2 h pre-IVM culture using forskolin (100 μM)-IBMX (500 μM) and 30 h of IVM adding cilostamide (20 μM)], and (3) DMSO30 [cAMP modulators are diluted in DMSO)/vehicle control; OPU medium: PBS-DMSO (46.3 mM); 2 h pre-IVM culture (280 mM DMSO) and 30 h IVM (5.6 mM DMSO)]. Following IVM, oocytes were either submitted to in vitro fertilization and embryo culture or fixed in 1% glutaraldehyde at 9, 20, 24, and 30 h after IVM and stained with Hoechst to evaluate their nuclear status. One-way ANOVA was implemented to evaluate recovered oocytes and meiotic stages. The Glimmix procedure from SAS/STAT was performed to compare blastocyst and cleavage rates. Total number of oocytes and IVM-suitable oocytes per donor per OPU session were similar in adult and prepubertal donors (total number/IVM suitable; prepubertal donors: 6.7/4.2, 6.4/4.0, 6.5/3.8; cows: 6.2/4.7, 6.2/4.4, 6.2/4.5 for TCM24, cAMP30 and DMSO30, respectively). At 9 h, cAMP regulators were able to maintain meiotic arrest in prepubertal and adult donors (GV: 80.0 and 40.9%, respectively) compared to standard IVM (GV: 61.1 and 31.2%) and DMSO30 (GV: 40.0 and 26.6%) protocols (P < 0.05). Using the cAMP30 protocol, the percentage of oocytes that reached MII stage at 20 h was lower in adult (4.5%) and prepubertal donors (5.26%) compared to the DMSO30 (50.0 and 42.8%, respectively) and TCM24 (56.2 and 44.4% respectively) protocols. Metaphase II rates after either 24 or 30 h were similar among treatments (prepubertal donors: 88.2, 70.5, and 84.2%; cows: 71.4, 85.7, and 81.2% for TCM24, cAMP30, and DMSO30, respectively; P > 0.05). Cleavage rates (prepubertal donors: 63.4, 54.9, and 52.1%, cows: 56.1, 57.8, and 51.6% for TCM24, cAMP30, and DMSO30, respectively) and blastocysts/presumptive zygotes rates (prepubertal donors: 26.2, 19.6, and 16.2%; cows: 27.5, 28.1, and 21.5% for TCM24, cAMP30, and DMSO30, respectively) did not show significant differences (P > 0.05). Although cAMP modulators delayed the progression through meiosis in adult and prepubertal oocytes, similar blastocysts rates were obtained. Our results suggest so far that oocyte retrieval and competence in prepubertal donors can be similar to that of the adult donors with and without addition of cAMP modulators.

148 EFFECTS OF CAFFEINE SUPPLEMENTATION ON BOVINE OOCYTE DEVELOPMENTAL CAPACITY

2017 ◽  
Vol 29 (1) ◽  
pp. 182
Author(s):  
S. M. Bernal-Ulloa ◽  
A. Lucas-Hahn ◽  
P. Aldag ◽  
D. Herrmann ◽  
U. Baulain ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Mammalian Species ◽  
Cell Mass ◽  
Phosphodiesterase Inhibitor ◽  
Developmental Competence ◽  
Differential Staining ◽  
Final Phase ◽  
Cell Numbers ◽  
Developmental Capacity

Oocyte culture in the presence of the nonspecific competitive phosphodiesterase inhibitor caffeine has been reported to increase developmental capacity of oocytes in different mammalian species. Here, we evaluated the effects of caffeine supplementation during the final phase of in vitro maturation (IVM) on developmental rates and blastocyst cell numbers. Bovine ovaries were collected from a local abattoir. A total of 1142 cumulus-oocyte-complexes were obtained by slicing. Cumulus-oocyte complexes were either in vitro matured for 24 h (Standard) or matured for 20 h followed by additional culture for 6 h in fresh IVM medium supplemented with 10 mM caffeine (Caffeine 6 h). In vitro fertilization was performed for 19 h using frozen-thawed sperm from 2 different bulls. After IVF, presumptive zygotes were cultured in vitro for 8 days until the blastocyst stage. Cleavage and blastocyst rates were evaluated 3 and 8 days after IVF, respectively. Expanded blastocysts from the different treatments were submitted to differential staining. SAS/STAT software (SAS Institute Inc., Cary, NC, USA) was used to evaluate cleavage and blastocyst rates using the Glimmix procedure and blastocyst cell numbers were compared using the linear model procedure. Cleavage rates were lower using caffeine for bull B and blastocyst production decreased for bull A. Caffeine treatment increased inner cell mass (ICM) number for bull B and decreased trophectoderm (TE) and total cell numbers for bull A. However, similar TE and total cells were obtained for bull B (Table 1; P < 0.05). Results show that developmental competence can be affected by caffeine supplementation at the final phase of IVM probably due to oocyte-sperm interaction changes. Table 1. In vitro developmental competence of oocytes cultured with caffeine at the end of IVM

scholarly journals N-cadherin mediated cell contact inhibits germinal vesicle breakdown in mouse oocytes maintained in vitro

Reproduction ◽  
2006 ◽  
Vol 131 (3) ◽  
pp. 429-437 ◽  
Author(s):  
J J Peluso
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Monolayer ◽  
Germinal Vesicle ◽  
Cell Contact ◽  
Germinal Vesicle Breakdown ◽  
Cell Specificity ◽  
Specific Manner ◽  
A Site

The effect of granulosa cell contact on the ability of zona-free oocytes to undergo germinal vesicle breakdown (GVBD) was assessed using a granulosa cell co-culture system. Oocytes contacted granulosa cells in a site-specific manner such that their GV was away from the granulosa cells. Also contact with granulosa cells reduced the percentage of oocytes undergoing GVBD from about 40% to 15%. GVBD was inhibited by contact with granulosa cells but not a granulosa cell-secreted product, since oocytes cultured in the same culture, that were adjacent to the granulosa cell monolayer underwent GVBD at the same rate as controls. Similarly, media collected from granulosa cell cultures did not attenuate the rate of GVBD. The ability of granulosa cell contact to inhibit GVBD was equal to that of db-cAMP. Moreover, the ability of granulosa cells to inhibit GVBD was not mimicked by spontaneously immortalized granulosa cells. This cell specificity appeared to be related to N-cadherin, since granulosa cells and oocytes express N-cadherin and a N-cadherin antibody attenuates the effect of granulosa cell contact. The mechanism through which N-cadherin mediated cell contact maintains meiotic arrest is unknown. It is possible that homophilic N-cadherin binding between the granulosa cells and oocyte acts through a junxtacrine mechanism, which in part may lead in the activation fibroblast growth factor (FGF) receptors that are expressed by the oocyte. The involvement of FGF receptors is supported by the observations that FGF and a N-cadherin peptide known to activate FGF receptors inhibit GVBD.

scholarly journals Protein kinases influence bovine oocyte competence during short-term treatment with recombinant human follicle stimulating hormone

Reproduction ◽  
2005 ◽  
Vol 130 (3) ◽  
pp. 303-310 ◽  
Author(s):  
Atef Ali ◽  
Marc-André Sirard
Keyword(s):  
Protein Kinase ◽  
Follicle Stimulating Hormone ◽  
Term Treatment ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Short Term ◽  
Short Term Treatment ◽  
Developmental Capacity ◽  
Bovine Oocytes

The aim of this study was to investigate the effect of short-term treatment (first 2 or 6 h) with recombinant human follicle-stimulating hormone (r-hFSH) during in vitro maturation (IVM) on the developmental competence of bovine oocytes. The roles of protein kinase A (PKA) and protein kinase C (PKC) (possibly involved in FSH response), were investigated using activators (Sp-cAMPS, PMA) or inhibitors (Rp-cAMPS, sphingosine) of these two protein kinases, respectively. The developmental competence of bovine oocytes was measured by the rate of blastocyst formation after in vitro fertilization (IVF). Our results showed that when cumulus–oocyte complexes (COCs) were cultured with r-hFSH for the first 6 h, a highly significant (P < 0.0001) improvement is seen in blastocyst development rate as a proportion of oocytes in culture compared with those matured with r-hFSH for the first 2 or 24 h. A transient exposure (6 h) to the highest dose (100 μM) of forskolin (an activator of adenylate cyclase) increased (P < 0.05) the rate of blastocyst formation. But the PKA inhibitors (Rp-cAMPS) did not affect the stimulatory effects of r-hFSH on the blastocyst yield. However, stimulation of PKC by low doses of PMA (0.1–0.5 μM) during short-term treatment, enhanced (P < 0.0001) the developmental capacity of oocytes, while sphingosine (a specific inhibitor of PKC) inhibited (P < 0.05) the stimulatory effects of r-hFSH on the rate of blastocyst formation. Our results indicate that although the developmental capacity of bovine oocytes in vitro can be modulated by both the PKA, and the PKC pathways, the activation of PKC during short-term treatment can mimic the effect of r-hFSH on the cytoplasmic maturation in bovine oocytes in vitro.

272 DEVELOPMENTAL COMPETENCE OF OOCYTES OBTAINED FROM BOS TAURUS AND BOS INDICUS DAIRY COWS RAISED IN A TROPICAL CLIMATE

2006 ◽  
Vol 18 (2) ◽  
pp. 243
Author(s):  
L. S. A. Camargo ◽  
J. H. M. Viana ◽  
W. F. Sa ◽  
A. M. Ferreira ◽  
A. A. Ramos ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Calf Serum ◽  
Developmental Competence ◽  
Tropical Region ◽  
Humid Atmosphere ◽  
Vitro Fertilization ◽  
Developmental Capacity

The effects of heat stress on Bos taurus reproductive performance in tropical and subtropical regions are well known, and have been associated with lower oocyte developmental capacity. The aim of this study was to evaluate the developmental competence of oocytes from Bos taurus (Holstein) and Bos indicus (Gyr) dairy cows raised in a Brazilian tropical region, located at 21°35′S latitude, 43°51′W longitude, and 435 meters altitude. Cumulus–oocyte complexes (COCs) were recovered by oocyte pickup (OPU) from mature non-lactating Holstein (n = 9) and Gyr (n = 13) donor cows between the end of spring and the beginning of autumn, with at least two OPU sessions/cow. COCs were in vitro-maturated in TCM-199 (GIBCO, Grand Island, NY, USA) with 10% inactivated estrus cow serum for 24 h under 5% CO2 at 38.5°C in air. Bos taurus and Bos indicus semen with similar cleavage rates, previously evaluated by in vitro fertilization with oocytes obtained from slaughterhouse ovaries, were used to reduce bull effect. Holstein and Gyr spermatozoa were obtained through swim-up method and co-incubated with Holstein (n = 390) and Gyr (n = 505) oocytes, respectively, in Fert-TALP medium (Parrish et al. 1988 Biol. Reprod. 38, 1171–1180) supplemented with 10 μg/mL heparin (Sigma-Aldrich, Sao Paulo, Brazil) and 6 mg/mL fatty acid-free bovine albumin (Sigma) for 18 h in 5% CO2 at 38.5°C in air. Presumptive zygotes were co-cultured with their own cumulus cells in CR2aa medium (Wilkinson et al. 1996 Theriogenology 45, 41–49) supplemented with 10% fetal calf serum in humid atmosphere of 5% CO2 at 38.5°C in air. On Day 7 to 8 of co-culture, Gyr and Holstein blastocysts were assessed and those classified as grade 1 (IETS Manual) were transferred to synchronized Bos indicus × Bos taurus crossbred recipients managed under the same nutritional and environmental conditions. Pregnancy diagnosis was performed between 35 and 50 days after estrus. Cleavage, blastocyst, and pregnancy rates were analyzed by chi-square test. Cleavage and blastocyst rates were greater (P < 0.05) in Gyr than in Holstein (66.7% vs. 53.1% for cleavage and 19.6% vs. 10.8% for blastocyst, respectively), but the pregnancy rate was similar (P > 0.05; 44.5% vs. 60% for Gyr and Holstein, respectively). These results show that Gyr oocytes obtained in a tropical region have greater developmental capacity than Holstein oocytes, suggesting an interaction between genotype and environment that influences the success of an in vitro embryo production program; nevertheless, the blastocyst viability after transferring to recipients is similar for both breeds.

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