Oocyte quality determines bovine embryo development after fertilisation with hydrogen peroxide-stressed spermatozoa

2012 ◽  
Vol 24 (4) ◽  
pp. 608 ◽  
Author(s):  
Mohammad Bozlur Rahman ◽  
Leen Vandaele ◽  
Tom Rijsselaere ◽  
Mahdi Zhandi ◽  
Dominiek Maes ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Small Diameter ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Oocyte Diameter ◽  
Bovine Embryo ◽  
Embryo Production ◽  
Subsequent Performance

Exposure of gametes to specific stressors at sublethal levels can enhance the gametes’ subsequent performance in processes such as cryopreservation. In the present study, bull spermatozoa were subjected to H2O2 for 4 h at 100-, 200- and 500-μM levels; computer-assisted sperm analysis (CASA) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay were used for evaluation of subsequent sperm motility and DNA integrity, respectively. Exposure of spermatozoa to H2O2 did not affect sperm motility but DNA integrity was negatively affected by 500 μM H2O2 compared with mock-exposed spermatozoa, whereas both motility and DNA integrity were affected compared with untreated spermatozoa. Nevertheless, insemination of oocytes with spermatozoa exposed to 200 μM H2O2 increased fertilisation, cleavage and blastocyst rates (P < 0.05). Furthermore, the higher blastocyst yield after fertilisation of oocytes with spermatozoa exposed to 200 μM H2O2 was related to oocyte diameter, with large–medium oocytes yielding higher blastocyst rates, while small-diameter oocytes consistently failed to develop into blastocysts. In conclusion, the results indicate that exposure of spermatozoa to 200 μM H2O2 before sperm–oocyte interaction may enhance in vitro embryo production in cattle. However, this increased embryo production is largely dependent on the intrinsic quality of the oocytes.

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

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

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

scholarly journals Effect of insulin–transferrin–selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 890-899 ◽  
Author(s):  
A.L.S. Guimarães ◽  
S.A. Pereira ◽  
M. N. Diógenes ◽  
M.A.N. Dode
Keyword(s):  
Ascorbic Acid ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Embryo Production ◽  
Embryo Size ◽  
Blastocyst Rate

SummaryThe aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

Inadvertent sex selection in a protocol of in vitro bovine embryo production

1997 ◽  
Vol 47 (1) ◽  
pp. 265 ◽  
Author(s):  
E. Behboodi ◽  
A. Gutiérrez-Adán ◽  
G.B. Anderson
Keyword(s):  
Sex Selection ◽  
Bovine Embryo ◽  
Embryo Production

scholarly journals The effects of conjugated linoleic acid isomers cis-9,trans-11 and trans-10,cis-12 on in vitro bovine embryo production and cryopreservation

2014 ◽  
Vol 97 (10) ◽  
pp. 6164-6176 ◽  
Author(s):  
V.A. Absalón-Medina ◽  
S.J. Bedford-Guaus ◽  
R.O. Gilbert ◽  
L.C. Siqueira ◽  
G. Esposito ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Bovine Embryo ◽  
Embryo Production

100 COMPARISON OF PREGNANCY RATES OF FRESH IN VITRO-PRODUCED BOS INDICUS EMBRYOS PRODUCED IN THE SAME LABORATORY BUT COLLECTED AND TRANSFERRED IN PANAMA OR THE DOMINICAN REPUBLIC

2014 ◽  
Vol 26 (1) ◽  
pp. 164
Author(s):  
L. F. Nasser ◽  
S. C. Feliú ◽  
E. Rodríguez ◽  
K. Mojica ◽  
E. G. Oliveira ◽  
...  
Keyword(s):  
Dominican Republic ◽  
Bos Indicus ◽  
Disease Status ◽  
Pregnancy Rates ◽  
Bovine Embryo ◽  
Department Of Agriculture ◽  
Embryo Production ◽  
Essential Variable ◽  
Laboratory Facility

Because of Panama's stricter sanitary status, a specialised protocol was developed with the Department of Agriculture in the Dominican Republic to legalize the exchange of biological materials (oocytes/embryos). This protocol allows the team of specialised technicians, currently working in Born® Animal Biotechnology's Panamanian facility, to operate using the same in vitro bovine embryo production system (IVP, In vitro Brasil®) to service Dominican producers. Because the donors are not located at a specific centre with controlled sanitary management, a special protocol was developed in which blood tests were done to certify that the entirety of the herd at each client's farm was free of infectious bovine rhinotracheitis, DBVD, leptospirosis, leucosis, brucellosis, and tuberculosis. As timing during IVP is an essential variable that can have detrimental effects on the final results, precautions were taken to ensure that the oocytes arrived at the Panamanian laboratory facility within 24 h of aspiration. A portable incubator was used to transport oocytes and embryos during the import and export portions of the procedure. A comparison of pregnancy rates based on oocyte source and recipient transfers from September 2012 until May 2013 was analysed with ?2 (Table 1). The number of embryos produced in Panama was significantly higher than in the Dominican Republic, which was likely due to the larger number of donors and oocytes from the Panama herd. However, pregnancy rate was higher in the Dominican Republic likely because of the health status of the Dominican recipients, which were free of the diseases mentioned above. Recipients were the same type and breed and under similar management conditions in both countries. The disease status aspect will be examined with greater numbers of animals in the future. The data suggest that the present IVP and recipient management protocols could serve as a model for other Central American and Caribbean countries under similar management systems. Table 1.In vitro embryo production and pregnancy rates of Bos indicus embryos transferred in Panama or the Dominican Republic (September 2012 through May 2013)

242 EFFECTS OF TWO COMMERCIAL BOVINE SEMEN EXTENDERS FOR SHORT-TERM STORAGE ON MOTILITY PATTERN OF CHILLED BISON SEMEN

2011 ◽  
Vol 23 (1) ◽  
pp. 219
Author(s):  
B. M. Toosi ◽  
G. Gratton ◽  
C. Lessard ◽  
G. P. Adams
Keyword(s):  
Sperm Motility ◽  
Repeated Measures ◽  
Test Tube ◽  
Computer Assisted ◽  
San Jose ◽  
Short Term ◽  
Development Fund ◽  
Straight Line ◽  
Wood Bison

Difficulties of adequate cryopreservation of bison semen has limited the success of artificial insemination and in-vitro embryo production in bison. We evaluated the effects of short-term cooling on motility of bison sperm using two commercial semen extenders (Triladyl® and Andromed®; Minitube, Ingersoll, ON, Canada). Semen was collected by electroejaculation of mature wood bison (n = 3) and plains bison (n = 3) twice a week for 2 wk. Upon collection, the ejaculate was divided into 3 equal aliquots, which were then diluted 1:2 (vol/vol) in Triladyl or Andromed, or were not extended (n = 24 samples per treatment). Samples were maintained at 37°C until transfer to the laboratory (≤2 h). One millilitre of each sample was then placed into a test tube (15 mL, BD Falcon, BD Biosciences, San Jose, CA, USA) and were kept in water bath set at 5°C inside a walk-in refrigerator (4°C). Characteristics of sperm motility were evaluated before cooling (Day 0) and every 24 h after cooling for 5 days using a computer-assisted semen analyzer. Total motility (TM), progressive motility (PM), velocity curved line (VCL), velocity average path (VAP), and velocity straight line (VSL) were compared among treatments by ANOVA for repeated-measures. Values are expressed as mean ± SEM. After collection, the PM of the raw semen and semen extended in Triladyl or Andromed were not significantly different (63.1 ± 4.4%, 63.3 ± 3.1%, and 56.9 ± 4.5%, respectively). Cooling semen for a period of 24 h resulted in a decrease (P < 0.05) in PM in all 3 groups (4.4 ± 2.0%, 22.7 ± 2.9%, and 28.7 ± 4.3%, respectively). The PM of semen extended in Tryladyl or Andromed was greater than that of raw semen on Day 1 (P < 0.05). Semen extended in Triladyl and Andromed maintained PM on Day 2 (24.7 ± 3.3% and 21.8 ± 3.8%, respectively), but PM declined progressively to 1.1 ± 0.6% and 6.3 ± 2.1% by Day 5. A similar pattern was observed for the TM. The VCL, VAP, and VSL parameters for semen extended with Triladyl and Andromed decreased gradually between Day 0 and Day 5 (P < 0.05). From Day 1 to 4 after cooling, these velocity parameters were not significantly different between semen extended with Triladyl or Andromed; however, they were greater than those of raw semen (P < 0.05). All sperm velocity parameters for the raw semen declined by more than 60% between Days 0 and 2 (P < 0.05). On Day 0, VCL for semen extended with Andromed (152.2 ± 4.3) was greater than that of semen extended with Triladyl and raw semen (P < 0.05; 122.5 ± 7.0 and 122.4 ± 6.6, respectively). The VCL then decreased to 98.9 ± 12.9, 100.5 ± 10.8, and 18.6 ± 6.8 in Andromed, Triladyl and raw groups respectively on Day 2 (P < 0.05), followed by a further decline to 51.8 ± 14.3, 19.9 ± 10.0, and 9.0 ± 5.0, respectively, observed on Day 5. In conclusion, both Triladyl and Andromed improved characteristics of sperm motility of chilled bison semen. Despite an initial decrease within the first 24 h, bison sperm extended with Triladyl or Andromed maintained an acceptable degree of motility for up to 2 days after chilling to 5°C. Supported by Agriculture and Agri-Food Canada, Agriculture and Development Fund, and Canadian Animal Genetic Resources program.

293 EFFECT OF GUAIAZULENE ON IN VITRO BOVINE EMBRYO PRODUCTION

2007 ◽  
Vol 19 (1) ◽  
pp. 262 ◽  
Author(s):  
I. Dimitriadis ◽  
E. A. Rekka ◽  
E. Vainas ◽  
G. S. Amiridis ◽  
C. A. Rekkas
Keyword(s):  
Lipid Peroxidation ◽  
Embryo Development ◽  
Antioxidant Properties ◽  
Thiobarbituric Acid ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Embryo Production ◽  
Reactive Material

The substrates used in in vitro embryo production (IVP) mimic the in vivo fluids in which oocytes mature, oocytes are fertilized, and the early embryos develop (follicular and oviductal fluid). It is well established that oxidative stress negatively affects in vitro culture (IVC) outcomes. Guaiazulene (G) is a component of chamomile species oil with known antioxidant properties. In the present study, all IVP media were modified by the addition of G solutions so that the former exhibited a total protection against induced lipid peroxidation (TPaLP) similar to that of the respective in vivo environment. The IVP outcomes were then compared between G-processed and control oocytes. Bovine preovulatory follicular (BF) and oviductal (BO) fluid samples were collected from 10 Holstein 4- to 5-year-old cows in estrus. TPaLP was assessed according to the samples&apos; ability to inhibit rat hepatic microsomal lipid peroxidation, by determination of the 2-thiobarbituric acid reactive material. TPaLP (mean % � SEM) of the BF and BO were 70.63 � 10.03 and 16.33 � 4.33, respectively, whereas those of the IVP [in vitro-matured (IVM), in vitro-fertilized (IVF), and IVC] media were lower (17.94 � 1.66, -1.82 � 0.78, and 14.57 � 1.26, respectively). TPaLP of the 0.1 mM G-modified IVP medium increased to 67.2 � 5.85, 19.98 � 2.49, and 69.19 � 6.22, respectively. A total of 2041 class A oocytes were used. The proportion of cleavage, early embryo development (embryos with more than 4 cells), or both after IVP (18 h IVM–5% CO2 in air, and 18 h IVF, 48 h IVC–5% CO2, 10% O2, 85% N) in the presence of G (n = 1237) during each of the IVP phases or any possible combination of IVP phases was compared with the respective control (C, n = 804). Statistical analysis was performed by a chi-squared test; P &lt; 0.05 was considered significant. G improved cleavage and embryo development rates when present during IVM (79.4 and 57.8% vs. 64.5 and 38.2% for C) or both IVM and IVC (78.0 and 60.7% vs. 57.8 and 36.5%, respectively). When present only during 18 h of IVF, G had no effect on embryo production. However, an increased embryo development rate resulted from the combined exposure to G during IVF and IVM (56.4 vs. 29.6%), during IVF and IVC (55.3 vs. 35.5%), or at all IVP phases (56.6 vs. 34.9%). The latter effect resembled the one obtained after G addition only to the IVC medium (62.5 vs. 39.7%, respectively). We concluded that the addition of G to IVP substrates, at concentrations that mimic the in vivo TPaLP conditions, could promote bovine IVP efficiency.

238 KNOCKING DOWN OF THYROID HORMONE RECEPTORS INHIBITS DEVELOPMENT OF EARLY BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 267
Author(s):  
N. Y. Rho ◽  
F. A. Ashkar ◽  
T. Revay ◽  
P. Madan ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Reference Genes ◽  
Messenger Rna ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Embryo Production ◽  
Mrna Transcripts ◽  
In Vitro Embryo Production

Thyroid hormones (TH) play an important role in the physiology of vertebrates, ranging from the regulation of metabolic processes to cell proliferation, differentiation, and embryo development. We have previously shown a beneficial effect of supplementing TH in in vitro embryo production media. Recently, detection of TH receptors (TR) in oocytes and early stages of pre-implantation embryos indicated a possible regulatory role for TH in these stages (unpublished data). The objective of this study was to investigate the importance of TR expression in the pre-attachment bovine embryo in vitro. Bovine embryos, produced by standard in vitro embryo production procedures, were microinjected at the zygote stage with small interfering RNA (siRNA) specifically designed for knocking down either TR-α or TR-β. In addition, groups of zygotes were microinjected with scrambled siRNA (SI) or were not injected (NI), and these groups served as controls. Embryo developmental rates were assessed using light microscopy for blastocyst formation rates and expression of TR messenger RNA (mRNA) transcripts at the blastocyst stage was assessed by quantitative PCR across all groups. Expression of TR mRNA was normalized against glyceraldehyde 3-phosphate dehydrogenase, H2a, and 18S as reference genes. There was a significant decrease in blastocyst formation rates in both embryo groups injected with either TR-α (P < 0.002) and TR-β (P < 0.001) siRNA compared with the NI and SI groups. Moreover, the TR-β knockdown group exhibited a lower developmental rate than the TR-α knockdown group, which indicates a stronger inhibitory role for TR-β. Quantification of the level of TR mRNA expression in four groups normalized with three different reference genes shows a consistent significant reduction in the levels of TR-α (P < 0.05) and TR-β (P < 0.02) mRNA transcripts compared with the NI and SI groups. However, TR-β expression was inhibited more than was TR-α expression. In conclusion, the results indicate that knocking down either TR-α or TR-β restrains embryo development. This suggests that TH play a vital role in the regulation of embryo development through their receptors during bovine early embryogenesis. The specific role of each of these receptors and their mechanism of action in mediating development needs to be further elucidated. Funding was provided by CRC, NSERC, and the EmbryoGENE network.

17 SPERM FERTILITY RATE ASSESSED BY EMBRYO PRODUCTION IN VIVO AND IN VITRO IN SOUTH AFRICAN BULLS

2017 ◽  
Vol 29 (1) ◽  
pp. 116
Author(s):  
M. H. Mapeka ◽  
F. V. Ramukhithi ◽  
C. M. Pilane ◽  
D. Norris ◽  
C. Banga ◽  
...  
Keyword(s):  
South African ◽  
Sperm Motility ◽  
Developmental Stages ◽  
Fertility Rate ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Frozen Semen ◽  
Recovery Methods

The aim of this study was to determine the sperm fertility rate by embryo production in vivo and in vitro in South African bulls and further compare the embryo quality developed from different oocyte recovery methods. A total of 15 frozen semen straws (5 Bonsmara; 5 Nguni; 5 Boran) were thawed and evaluated for sperm motility characteristics using sperm class analyzer. The fertilizing ability of frozen–thawed semen was assessed by performing AI and in vitro fertilization. For AI, 6 cows were superovulated and inseminated with frozen–thawed semen followed by flushing on Day 7 post-insemination and then evaluated for embryo developmental stages. For IVF, oocytes were retrieved using two recovery methods namely ovum pick-up (OPU) and ovary aspiration. A total of 383 (106, OPU; 277, ovary aspiration) oocytes were matured in M199 + 10% fetal bovine serum (FBS) maturation medium at 38.5°C for 24h. Oocytes were washed in Bracket and Oliphant’s fertilization medium, co-incubated with frozen–thawed (Boran) semen at 38.5°C for 6 h, and then cultured in SOF-BSA medium, incubated at 38.5°C, 5% CO2 for 7 days, and further evaluated for embryo development. Data were analysed by ANOVA. Total sperm motility was >70% in all breeds. Boran had a significantly (P < 0.05) higher total post-thaw sperm motility (93.2 ± 3.6) compared with Nguni (75.1 ± 4.2) and Bonsmara (80.7 ± 6.9). Furthermore, Boran had higher (P < 0.05) progressive motility (39.7 ± 3.4) and rapid motility (36.1 ± 5.9) compared with other breeds. Interestingly, Boran produced significantly (P < 0.05) higher blastocyst rate (56.34%) compared with Bonsmara (38.03%) Nguni (31.08%). Superovulation and OPU resulted in a significantly higher (P < 0.05) number of blastocysts (10.5 ± 3.3 and 10.5 ± 3.3) respectively, compared with aspiration (1.3 ± 3.3). Moreover, the OPU method yielded a significantly higher (P < 0.05) number of grade 2 blastocyst (3.0 ± 0.1) compared with aspiration (0.50 ± 0.1). However, there was no significant (P > 0.05) difference in the number of grade 1 and grade 3 blastocysts obtained when the 3 recovery methods were used. In conclusion, the Boran breed showed better a sperm fertility rate following in vivo and in vitro embryo production. The superovulation and OPU methods resulted in higher numbers and better quality blastocysts compared with aspiration.

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