Influence of sire breed (Bos indicus versus Bos taurus) and interval from slaughter to oocyte aspiration on heat stress tolerance of in vitro-produced bovine embryos

Heat stress has been a challenge for bovine reproduction in tropical and subtropical environments. Although the role of the oocyte in thermotolerance has been studied, little attention has been paid to the contributions of sperm to embryo resistance to heat shock. The current study aimed to evaluate the development of fertilized and nonfertilized (parthenogenetic) bovine embryos undergoing heat stress during the pre-implantation stage. Cumulus–oocyte complexes obtained from ovaries collected from Bos indicus × Bos taurus crossbred cows at slaughter were in vitro matured with TCM-199 supplemented with 20 μg mL–1 of FSH, under 5% CO2 at 38.5°C for 24 h. Afterward, oocytes were randomly allocated into 2 groups: 1) IVF and 2) PART (chemical activation for parthenogenesis induction). In vitro-fertilized oocytes were cultured with 2.0 × 106 Holstein sperm mL–1 in Fert-TALP medium supplemented with heparin, for 20 h. For chemical activation, oocytes were activated with calcium ionomycin for 4 min, followed by 6-DMAP for 4 h, both in CR2aa medium supplemented with 0.1% BSA. Presumptive IVF (n = 1 262) or PART (n = 1 206) zygotes were denuded by vortexing and cultured in CR2aa medium with 2.5% of FCS under 5% CO2, 5% O2, and 90% N2 at 38.5°C. At 44 h post-insemination or chemical activation, embryos were exposed to 38.5 or 41°C for 12 h in an atmosphere of 5% CO2, 5% O2, and 90% N2. After that, embryos were cultured at 38.5°C under 5% CO2, 5% O2, and 90% N2 until Day 8 post-insemination. Blastocyst rates were evaluated at Day 7 and Day 8 post-insemination and were calculated based on the total number of presumptive zygotes. Blastocysts at 192 h post-insemination or activation were fixed and permeabilized for TUNEL assay (DeadEndTM Florimetric TUNEL System, Promega, Madison, WI) according to the manufacturer’s instructions. The effect of heat stress was compared within groups (IVF or PART) and the data were analysed by ANOVA. As expected, heat stress reduced the blastocyst rate of IVF embryos at Day 7 (24.3 ± 2.0% and 17.4 ± 2.2% for nonstressed and stressed IVF embryos; P < 0.05) and at Day 8 (32.4 ± 1.9% and 23.0 ± 2.1% for nonstressed and stressed IVF embryos; P < 0.01). However, the effect of heat stress on blastocyst rate of PART embryos was observed only at Day 8 post-insemination (30.0 ± 1.7% and 22.6 ± 2.0% for nonstressed and stressed PART embryos; P < 0.05), with no difference in blastocyst rate at Day 7 (21.6 ± 1.5% and 18.2 ± 1.8% for nonstressed and stressed PART embryos; P > 0.05). There was no difference in total cell numbers between nonstressed and stressed IVF or PART embryos. Apoptosis cell numbers and the apoptotic cell index were higher (P < 0.05) for stressed IVF (18.45 ± 1.24 and 0.16 ± 0.00) and PART (16.40 ± 5.20 and 0.17 ± 0.00) embryos than for nonstressed IVF (13.70 ± 0.75 and 0.13 ± 0.00) and PART (14.15 ± 0.86 and 0.13 ± 0.00) embryos. In conclusion, heat stress can induce apoptosis in both IVF and PART embryos, but its effect on pre-implantation development may occur at earlier stages in IVF embryos when compared with PART embryos. Financial support from Fapemig and CNPq.

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175 EFFECT OF HEAT STRESS ON GENE EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS (BOS TAURUS VS. BOS INDICUS)

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab175 ◽

2012 ◽

Vol 24 (1) ◽

pp. 199

Author(s):

C. F. Silva ◽

A. C. S. Castilho ◽

R. A. Satrapa ◽

R. Z. Puelker ◽

E. M. Razza ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Mrna Expression ◽

Reverse Transcription ◽

Target Genes ◽

Bos Taurus ◽

Bos Indicus ◽

Mrna Levels ◽

Bovine Embryos

Heat stress (HS) reduces the production of bovine embryos, especially taurine embryos, which are not adapted to heat. However, little is known about the competence of embryos produced under HS in breeds adapted or not adapted to heat. The aim of this study was to compare the gene expression of PLAC8, HSF1, COX2 and CDX2, related to competence and implantation, in bovine in vitro-produced embryos (Bos taurus vs Bos indicus), submitted or not submitted to HS. Oocytes from Nelore (zebu) and Jersey (taurine) cows were aspirated by ovum pickup, in vitro-matured in TCM-199 medium with bicarbonate containing 10% FCS, 2 μg mL–1 of pyruvate, 75 μg mL–1 of gentamicin, 20 μg mL–1 of FSH and 10 IU mL–1 of LH for 22 h at 38.5°C in 5% CO2 in air. Matured oocytes were fertilized with semen from Nelore (n = 6) and Jersey (n = 6) bulls, respectively, at 38.5°C in 5% CO2 in air. The fertilization medium was TALP-IVF supplemented with 6 mg mL–1 of fatty acid-free BSA, 2 μL mL–1 of pyruvate, 75 μg mL–1 of gentamicin, 11 μg mL–1 of heparin and 44 μL mL–1 of penicillamine, hypotaurine and epinephrine. The day of fertilization was considered Day 0. Twelve hours post-insemination, presumptive zygotes were denuded and randomly divided into 2 groups, nonstressed or stressed and both were in vitro cultured at 38.5°C in 90% N2, 5% CO2 and 5% O2 in SOFaaci medium supplemented with 5% FCS, 5% BSA and 0,2% sodium pyruvate. In the stressed group, 96-h post-insemination embryos were subjected to HS of 41°C for 6 consecutive hours and then returned to 38.5°C. On Day 7, pools with 5 blastocysts [Nelore (n = 9); Nelore HS (n = 7); Jersey (n = 5); Jersey HS (n = 5)] were subjected to RNA extraction (RNeasy, Qiagen Inc., Valencia, CA, USA). The expression of target genes was analysed by real-time reverse transcription PCR with oligo-dT in reverse transcription and bovine specific-primers in PCR. The expression of cyclophilin A was used as an internal control. The mean mRNA levels of target genes among groups were compared by parametric ANOVA, followed by orthogonal contrast. Heat stress reduced (P < 0.05) mRNA expression of CDX2 and PLAC8 in both breeds; additionally, the expression of these genes was higher in the zebu breed when compared with the taurine breed. Messenger RNA expression of COX2 did not differ between groups, under HS or not, in both the Jersey and Nelore breeds. Moreover, HS reduced the mRNA expression of HSF1 (P < 0.05) in Nelore groups, but not in Jersey groups. The highest levels of PLAC8 and CDX2 in nonstressed Nelore embryos indicate better competence and a higher capacity of implantation of these embryos when compared with Jersey and HS embryos in both breeds. Moreover, low HSF1 levels in stressed Nelore embryos indicate the thermotolerance ability of this breed. In conclusion, the data indicate that HS alters the pattern of gene expression in Nelore and Jersey in vitro-produced bovine embryos. This research was supported by FAPESP.

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In vitro tuberization and tuber proteins as indicators of heat stress tolerance in potato

American Potato Journal ◽

10.1007/bf02853487 ◽

1989 ◽

Vol 66 (1) ◽

pp. 35-45 ◽

Cited By ~ 21

Author(s):

J. Nowak ◽

D. Colborne

Keyword(s):

Heat Stress ◽

Stress Tolerance ◽

In Vitro Tuberization ◽

Heat Stress Tolerance

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Effects of heat stress on development, quality and survival of Bos indicus and Bos taurus embryos produced in vitro

Theriogenology ◽

10.1016/j.theriogenology.2012.10.003 ◽

2013 ◽

Vol 79 (2) ◽

pp. 351-357 ◽

Cited By ~ 44

Author(s):

C.F. Silva ◽

E.S. Sartorelli ◽

A.C.S. Castilho ◽

R.A. Satrapa ◽

R.Z. Puelker ◽

...

Keyword(s):

Heat Stress ◽

Bos Taurus ◽

Bos Indicus

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22 INFLUENCE OF SIRE AND SIRE BREED (GIR VS. HOLSTEIN) ON EARLY PREGNANCY AND EARLY EMBRYONIC LOSS RATES IN HOLSTEIN COWS DURING SUMMER HEAT STRESS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab22 ◽

2005 ◽

Vol 17 (2) ◽

pp. 161 ◽

Cited By ~ 2

Author(s):

M.F. Pegorer ◽

J.L.M. Vasconcelos ◽

P.J. Hansen ◽

C.M. Barros

Keyword(s):

Heat Stress ◽

Milk Production ◽

Early Pregnancy ◽

Pregnancy Loss ◽

Bos Taurus ◽

Bos Indicus ◽

Fixed Time ◽

Holstein Cows ◽

Estrus Detection ◽

Sire Breed

Heat stress has negative effects on pregnancy rates of lactating dairy cattle. There are genetic differences in tolerance to heat stress, and it has been shown that Bos indicus cattle are more resistant to high temperatures than Bos taurus. In the present work, effect of sire and sire breed on conception rate of Holstein cows during the Brazilian summer (January and February, 2003 and 2004) was determined. Lactating Holstein cows (n = 1113), producing an average of 25.2 kg milk/day and at 247 ± 136 days postpartum, were AI approximately 12 h after estrus detection (n = 433) or fixed-time AI (FTAI) after hormonal induction of ovulation (n = 680), using semen from 4 Gir (Bos indicus dairy bull, 657 AI) and 3 Holstein sires (Bos taurus, 456 AI). Pregnancy diagnosis was performed by ultrasonography or rectal palpation at 28–45 and 70–84 days after AI. Cows diagnosed pregnant at the first examination and open at the time of the second were considered as having lost their embryo. The results were analyzed by logistic regression using PROC GENMOD: the model considered the effect of sire, sire breed, inseminators, milk production 7 days before AI, period of lactation, and AI type (FTAI vs. AI after estrus detection). There was no significant effect of sire breed on early pregnancy rate (EPR: Gir = 78/657, 11.9%; and Holstein = 30/456, 6.6%, P = 0.34) or early pregnancy loss (EPL: Gir = 25/78, 32%; and Holstein = 10/30, 33.3%, P = 0.73). However, there was a sire effect on pregnancy loss (43.7, 35.3, 10, and 40% for Gir bulls 1, 2, 3, and 4, respectively) and (50, 18.2, and 38.5% for Holstein bulls 5, 6, and 7, respectively), with bull 3 having the lowest rate of pregnancy loss (P = 0.03). There was no effect of the AI type on EPR (FTAI = 65/680, 9.5%; and AI after estrus = 43/433, 9.9%; P = 0.8). Surprisingly, there were higher pregnancy losses in cows AI after estrus detection (17/43, 39.5%) as compared to FTAI cows (18/65, 27.7%; P = 0.03). The variables days at postpartum, milk production 7 days before AI, and inseminator did not significantly influence EPR or EPL. It is concluded that use of Bos indicus sires did not improve EPR of lactating Holstein cows during summer as compared to use of Bos taurus sires. However, some bulls, either indicus or taurus, were more effective in decreasing EPL and selection of bulls by this criterion may result in higher parturition rates in lactating Holstein cows. This work was supported by USDA IFAFS Grant # 2001-52101-11318.

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296 INFLUENCE OF SHORT-TERM HEAT STRESS ON THE BLASTOCYST RATE OF BOVINE EMBRYOS (INDICUS VS. TAURUS) FROM OOCYTES OBTAINED BY OVUM PICKUP

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab296 ◽

2006 ◽

Vol 18 (2) ◽

pp. 255

Author(s):

E. Sartorelli e Sartorelli ◽

A. C. Z. Barcelos ◽

R. A. Satrapa ◽

D. F. Martins ◽

M. F. G. Nogueira ◽

...

Keyword(s):

Heat Stress ◽

High Temperatures ◽

Bos Indicus ◽

Bovine Embryos ◽

Short Term ◽

Cleavage Rate ◽

Blastocyst Rate ◽

The Difference ◽

Resistance To Heat

There is evidence that the deleterious effects of heat stress (HS) on fertility are less pronounced in Bos indicus than in B. taurus breeds, due primarily to differences in their thermoregulatory capacity. In the present work, the resistance to heat stress of Nelore embryos (B. indicus) was compared to either a breed not adapted (Angus; B. taurus) or adapted to high temperatures (Bonsmara; 5/8 B. indicus × 3/8 B. taurus). In Experiments (Exp.) 1 (Nelore vs. Angus) and 2 (Nelore vs. Bonsmara), oocytes obtained by ovum pickup OPU (during autumn) were matured (TCM-199), fertilized, and cultured (SOFaaci) in vitro. Ninety-six hours post-insemination (hpi), embryos with more than 16 cells were randomly allocated in two main groups: Group Control (embryos were maintained at 39°C all of the time) and Group HS (embryos were maintained at 41°C during 12 h and afterwards returned to 39°C). Blastocyst rates were determined on the 7th day of culture. In Exp. 1, 294 oocytes from Nelore and 144 from Angus cows had a cleavage rate of 67.9 and 59.4%, respectively. Ninety-six-hpi embryos (>16 cells) were distributed in four groups: Nelore Control (n = 97), Nelore HS (n = 95), Angus Control (n = 34) and Angus HS (n = 25). The blastocyst rates were 39/97 (40.2%), 23/95 (24.2%), 19/34 (55.9%), and e 8/25 (32.0%), respectively. The difference in rate of blatocyst formation caused by heat stress on Nelore (16.0%) and Angus (23.9%) was not significantly different (P < 0.05), and suggests, from oocytes obtained by OPU, that Nelore embryos may be more tolerant to HS than Angus embryos. However, it is necessary to increase the number of blastocysts per group in order to better characterize the effects of heat stress on these embryos. In Exp. 2, 294 oocytes from Nelore and 101 from Bonsmara cows had a cleavage rate of 41.2 and 51.2%, respectively. Ninety-six-hpi embryos (>16 cells) were distributed in four groups: Nelore Control (n = 44), Nelore HS (n = 49), Bonsmara Control (n = 22), and Bonsmara HS (n = 22). The blastocyst rates were 35/44 (79.5%), 30/49 (61.2%), 10/22 (45.5%), and 6/22 (27.3%), respectively. In spite of the fact that Bonsmara embryos had a lower blastocyst rate as compared to Nelore, the decline on blastocyst rate caused by HS was very similar in Nelore (18.3%) and Bonsmara embryos (18.2%). Additional OPU are underway to test the hypothesis that thermotolerance of Nelore embryos is similar to that in embryos from a breed adapted to high temperatures (Bonsmara), and superior to embryos from a non adapted breed (Angus). E. S. S., R. A. S., and M. F. G. N. were supported by a fellowship from FAPESP, and A. C. Z. B. by a fellowship from CAPES of Brazil.

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291 INFLUENCE OF THE BREED OF BULL (BOS INDICUS × BOS TAURUS) IN TOLERANCE TO HEAT SHOCK IN BOVINE EMBRYOS PRODUCED IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab291 ◽

2010 ◽

Vol 22 (1) ◽

pp. 302 ◽

Cited By ~ 1

Author(s):

T. Nabhan ◽

R. A. Satrapa ◽

R. A. L. Simões ◽

C. F. Silva ◽

E. M. Razza ◽

...

Keyword(s):

Heat Shock ◽

Embryo Development ◽

Bos Taurus ◽

Bos Indicus ◽

The Other ◽

Time Interval ◽

Time Intervals ◽

Group 4 ◽

Oocyte Aspiration

There is evidence that deleterious effects of heat shock (HS) on fertility are less pronounced in breeds tolerant to high temperatures, due mainly to differences in their thermoregulatory capacity. In vitro experiments have shown that Bos indicus embryos are more resistant to HS than Bos taurus. In order to better understand the differences related to HS resistance between Bos indicus and Bos taurus, the main objective of this study was to determine if tolerance to HS is caused by genetic contribution from the oocyte, spermatozoa, or both. Additionally, the influence of the time between collection of ovaries in the abattoir and oocyte aspiration in the laboratory on early embryo development was ascertained. In experiment 1, oocytes from Nellore and crossbreed Holstein cows (cHOL) were collected in a local abattoir, matured and fertilized using semen (n = 6 for each breed) from Nellore (NEL), Angus (ANG), Brahman (BRA,) and Gir (GIR) bulls. In experiment 2, oocytes from Nellore and Holstein (HOL) cows were collected in an abattoir and the oocytes were aspirated in the laboratory 4 (group 4 h) or 6.5 h (group 6.5 h) later, matured and fertilized using semen (n = 6 for each breed) from NEL, GIR, and HOL. In both experiments, 96 h post-insemination (hpi), embryos with > 16 cells were separated in 2 groups: control and HS. In the control group the embryos were cultured at 39°C, whereas in the HS group the embryos were submitted to 41°C for 12 h, and then returned to 39°C. In experiments 1 and 2 the results were analyzed by ANOVA (Proc MIXED, SAS Institute, Cary, NC, USA). In experiment 1, there was no effect of HS on blastocyst and hatched blastocyst rates in all breeds studied. The percentage of oocytes that cleaved and reached the morula stage was significantly lower (P < 0.05) in cHOL × GIR compared with the other breeds. Additionally, blastocyst rate was higher in cHOL × NEL than in cHOL × ANG and cHOL × GIR (P < 0.05). In experiment 2, cleavage, morula, and blastocyst rates in group 4 h were higher (P < 0.05) compared with group 6.5 h. The HS decreased blastocyst rates in all breeds (NEL × NEL, HOL × HOL, and HOL × GIR), and in both time intervals (4 and 6.5 h). The breed NEL × NEL had higher cleavage rate (P < 0.05) for both time intervals compared with HOL × HOL and HOL × GIR. In addition, Nellore oocytes fertilized with Nellore semen (NEL × NEL) originated higher blastocyst rates (P < 0.05) in control and HS group than the other breeds. We conclude that (a) embryos from Holstein are more susceptible to HS than embryos from crossbred Holstein; (b) the oocyte is more important than the spermatozoa for the development of thermotolerance, because the breed of the bull did not influence embryo development after HS; (c) in vitro early embryonic development was impaired by increasing (from 4 to 6.5 h) the time interval between ovary collection and oocyte aspiration. Fellowships to T. Nabhan from CAPES and to R. A. Satrapa, R. A. L. Simoes, and E. M. Razza from FAPESP. Funding from FAPESP (Sao Paulo, Brazil).

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214 INFLUENCE OF THE BREED OF BULL (GIR v. HOLSTEIN) ON THE RESISTANCE OF BOVINE EMBRYOS TO HEAT SHOCK AT EARLY STAGES OF IN VITRO DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab214 ◽

2008 ◽

Vol 20 (1) ◽

pp. 186 ◽

Cited By ~ 1

Author(s):

D. Y. Saito ◽

R. A. Satrapa ◽

R. M. Romão ◽

T. Nabhan ◽

R. A. L. Simões ◽

...

Keyword(s):

Heat Shock ◽

Bos Taurus ◽

Bos Indicus ◽

Holstein Cows ◽

Bovine Embryos ◽

In Vitro Development ◽

Early Stages ◽

Holstein Bulls ◽

Vitro Development

Heat shock (HS) has negative effects on pregnancy rates in lactating dairy cows. There are genetic differences in tolerance to HS, with Bos indicus cattle and embryos being more resistant to elevated temperatures than Bos taurus. Recently, Pegorer et al. (2007 Theriogenology 67, 692–697) observed that Gir semen (Bos indicus), when compared with Holstein semen (Bos taurus), increased pregnancy rates of lactating Holstein cows during the Brazilian summer. The objective of the present study was to evaluate the influence of breed of bull (Gir or Holstein) on the resistance of embryos to HS during the early stages of in vitro development. Oocytes from Holstein ovaries obtained from a local abattoir were matured in TCM 199 medium, fertilized with semen from 1 of 12 bulls (6 Gir and 6 Holstein), and cultured in synthetic oviduct fluid to the blastocyst stage. Ninety-six hours after insemination (96 hpi), embryos with less than 16 cells were discarded; half of the embryos (e16 cells) that were fertilized by each bull were submitted to HS (41�C for 12 h) and the other half (e16 cells) were maintained at 39�C (control group). After the HS period, embryos were maintained at 39�C until the end of the experiment. Six semen straws from different Gir bulls (Bos indicus) and 6 straws from Holstein bulls (Bos taurus) were used, in a total of 6 assays, to minimize the bull effect. Each breed of bull (Gir or Holstein) was used to fertilize a total of at least 300 Holstein oocytes per treatment (control v. HS). Cleavage (48 hpi), blastocyst (144 hpi), and hatched blastocyst (216 hpi) rates of embryos submitted or not submitted to HS were analyzed by PROC GLM of SAS (SAS Institute Inc., Cary, NC, USA) to adjust the model of ANOVA, with each combination of assays used as a block. The proportion data were arcsine transformed to study the effects of breed, treatment, and their interaction. There was no difference in cleavage rate when Gir semen was used to fertilize oocytes from Holstein cows (Gir � Holstein; 76.6%) as compared with Holstein � Holstein (75.5%). However, HS significantly decreased (P < 0.01) blastocyst rates (in relation to embryos e16 cells) from both Gir (74 and 62.6%, control and HS, respectively) and Holstein bulls (69 and 55%, respectively). The same was observed for hatched blastocysts from Gir (42 and 28.3%) and Holstein bulls (37 and 25%). Nevertheless, there was no interaction of breed of bull � treatment (HS) for blastocysts (P > 0.09) and hatched blastocysts (P > 0.1). It was concluded that the breed of bull used in the present experiment (Gir v. Holstein) did not increase the resistance of bovine embryos to HS at early stages of in vitro development.

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