Publications by authors named "José Carlos Ferreira-Silva"

10 Publications

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Evaluation of quality and gene expression of goat embryos produced in vivo and in vitro after cryopreservation.

Cryobiology 2021 May 5. Epub 2021 May 5.

Laboratory of Reproductive Biotechniques, Department of Veterinary Medicine, Federal Rural University of Pernambuco, Brazil. Electronic address:

In the present study, we aimed to identify morphological and molecular changes of in vivo and in vitro-produced goat embryos submitted to cryopreservation. In vivo embryos were recovered by transcervical technique from superovulated goats, whereas in vitro produced embryos were produced from ovaries collected at a slaughterhouse. Embryos were frozen by two-steps slow freezing method, which is defined as freezing to -32 °C followed by transfer to liquid nitrogen. Morphological evaluation of embryos was carried out by assessing blastocoel re-expansion rate and the total number of blastomeres. The expression profile of candidate genes related to thermal and oxidative stress, apoptosis, epigenetic, and implantation control was measured using RT-qPCR based SYBR Green system. In silico analyses were performed to identify conserved genes in goat species and protein-protein interaction networks were created. In vivo-produced embryos showed greater blastocoel re-expansion and more blastomere cells (P < 0.05). The expression level of CTP2 and HSP90 genes from in vitro cryopreserved embryos was higher than their in vivo counterparts. Unlikely, no significant difference was observed in the transcription level of SOD gene between groups. The high similarity of CPT2 and HSP90 proteins to their orthologs among mammals indicates that they share conserved functions. In summary, cryopreservation negatively affects the morphology and viability of goat embryos produced in vitro and changes the CPT2 and HSP90 gene expression likely in response to the in vitro production process.
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http://dx.doi.org/10.1016/j.cryobiol.2021.04.008DOI Listing
May 2021

Correlations of corpus luteum blood flow with fertility and progesterone in embryo recipient mares.

Trop Anim Health Prod 2021 Apr 22;53(2):280. Epub 2021 Apr 22.

Reproductive Biotechnics Laboratory, Federal Rural University of Pernambuco, Recife, PE, Brazil.

The aim of this study was to evaluate the correlation between the corpus luteum vascularization with the concentration of progesterone and the fertility of embryo recipient mares. Mangalarga Marchador mares (n = 33) were distributed into groups according to the days (D) after ovulation, as follows: D3 (n = 8), D4 (n = 8), D5 (n = 9), and D6 (n = 8). The evaluations of the corpus luteum, endometrium, and blood collection to quantify the progesterone concentration were carried out on D3, D4, D5, and D6. Among the parameters evaluated, only progesterone concentration on D6 differed from the other groups (P <0.05). A positive correlation (P <0.05) between the diameter and the area of the corpus luteum, and the objective and subjective methods of the corpus luteum vascular perfusion, was identified. Likewise, a positive correlation (P <0.05) was observed between the objective and subjective methods of the vascular perfusion in the corpus luteum and the progesterone concentration. The pregnancy rate obtained in this study (54.54%) was not affected (P> 0.05) by the day of embryo transfer, whose percentages were 37.50% (3/8) on D3, 50% (4/8) on D4, 66.70% (6/9) on D5, and 62.50% (5/8) on D6. It was estimated that with each increase on the day of embryo transfer, the pregnancy rate increases. The results allow to conclude that the corpus luteum vascularization in mares, evaluated by Doppler ultrasound, correlates with progesterone concentration and the embryo transfer day.
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http://dx.doi.org/10.1007/s11250-021-02583-9DOI Listing
April 2021

Occurrence, morphology, and morphometry of follicles containing multiple oocytes in FecG mutant Santa Inês ewes.

Anim Reprod Sci 2021 Mar 7;226:106690. Epub 2021 Jan 7.

Reproductive Biotechniques Laboratory, Federal Rural University of Pernambuco, Street Dom Manuel de Medeiros, s/n - Dois Irmãos, 52171-900, Recife, PE, Brazil.

This study was conducted to characterize the morphology and morphometry of follicles containing multiple oocytes (MOFs) and determine the association with the FecG mutation in Santa Inês ewes. Based on the genotypes, 65 ewes were characterized as being homozygous wild-type (n = 25; FecG), heterozygous mutant (n = 27, FecG), and homozygous mutant (n = 13, FecG). The variables evaluated were follicle developmental stage, number of oocytes per follicle, morphology, and morphometry of MOFs. The FecG mutation did not affect the frequency of MOFs (P > 0.05) (3.0 % in FecG; 3.3 % in FecG; and 3.5 % in FecG). The greater viability (P < 0.05) of MOFs was identified in transitory stage of the FecG (95.0 %) and FecG (90.9 %) when compared to the FecG genotype (73.3 %). Furthermore, the morphology of transitory follicles with two oocytes was the variable and when evaluated was the most reliable determinant for predicting which ewes had an FecG mutation. In conclusion, the FecG mutation did not affect the frequency of MOFs. The ewes with FecG mutation had a greater frequency of morphologically normal MOFs in the transitory stage. Furthermore, the ewes with the FecG mutation had a greater likelihood of having MOFs containing two morphologically normal oocytes.
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http://dx.doi.org/10.1016/j.anireprosci.2021.106690DOI Listing
March 2021

Housekeeping genes for RT-qPCR in ovine preimplantation embryos.

Zygote 2020 Jul 30:1-8. Epub 2020 Jul 30.

Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Brazil.

Housekeeping genes (HKG) are paramount for accurate gene expression analysis during preimplantation development. Markedly, quantitative reverse transcription polymerase chain reaction (RT-qPCR) in ovine embryos currently lacks HKGs. Therefore, we tested 11 HKGs for RT-qPCR normalization during ovine parthenogenetic preimplantation development. Seven HKGs reached the qPCR efficiency threshold (97.20-105.96%), with correlation coefficients ranging from -0.922 to -0.998 and slopes from -3.22 to -3.59. GeNorm ranked glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TATA-binding protein (TBP) as the best HKG pair, while H3 histone, family 3A (H3F3A) was the third HKG. Relative gene expression was measured for zinc finger protein X-linked (ZFX) and developmental pluripotency-associated 3 (DPPA3) transcripts during ovine parthenogenetic preimplantation development. ZFX did not show any transcript abundance fluctuation among oocytes, cleavage-stage embryos, and morulae. DPPA3 transcript abundance was also similar among all developmental stages, therefore suggesting that it may not display a maternal gene expression profile. In silico analysis of ovine DPPA3 mRNA and protein showed high conservation to bovine orthologues. However, DPPA3 orthologues differed in regulatory motifs. In conclusion, GAPDH, TBP and H3F3A are stable HKGs in ovine parthenogenetic embryos and allow accurate RT-qPCR-based gene expression analysis.
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http://dx.doi.org/10.1017/S0967199420000295DOI Listing
July 2020

Application of platelet-rich plasma in the in vitro production of bovine embryos.

Trop Anim Health Prod 2020 Nov 23;52(6):2931-2936. Epub 2020 Jun 23.

Laboratório de Biotécnicas Reprodutivas, Universidade Federal Rural de Pernambuco, Recife, PE, Brazil.

The aim of this study was to replace fetal bovine serum (FBS) with platelet-rich plasma (PRP) for in vitro production of bovine embryos. The maturation media (TCM-199 medium) for the cumulus-oocyte complexes (COCs) was supplemented with 5% (G5) and 10% (G10) PRP or 10% FBS (GC). After fertilization, the presumed zygotes were randomly distributed in culture medium supplemented with 5% (G5) and 10% (G10) PRP or 10% FBS (GC) for 7 days. Cumulus cell (CC) expansion was greater (P < 0.05) in the GC (88.9%) group than in G5 (34.1%) or G10 (50.0%). Nevertheless, the expansion of CCs in group G10 was greater than in G5 (P < 0.05). Cleavage was higher in group G5 (86.0%) than in G10 (79.0%) (P < 0.05) and did not differ from group GC (82.0%). The percentage of blastocysts in group G5 (50.0%) was higher than in CG (40.2%) and G10 (34.2%) (P < 0.05). In addition, the number of blastomeres was higher in G5 (159.0 ± 4.18) than in GC (132.4 ± 4.11) and in G10 (127.1 ± 5.88) (P < 0.05). The addition of PRP into the oocytes maturation medium is not beneficial. On the other hand, the PRP addition into the embryo culture medium at 5% concentration is recommended where it increased the quantity and quality of in vitro-produced bovine embryos.
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http://dx.doi.org/10.1007/s11250-020-02307-5DOI Listing
November 2020

Ovarian and follicular variables used to determine ewes with different FecG genotypes.

Anim Reprod Sci 2019 Sep 3;208:106117. Epub 2019 Jul 3.

Reproductive Biotechniques Laboratory, Federal Rural University of Pernambuco, Rua Dom, R. Manuel de Medeiros, s/n- Dois Irmãos, 52171-900, Recife-PE, Brazil.

Based on ovarian and follicular variables, there was determination of ewes with different FecG genotypes. Based on the FecG genotype, 65 Santa Inês ewes were assigned to three experimental groups: homozygous wild-type (n = 25; FecG), mutant heterozygous (n = 27; FecG) and mutant homozygous (n = 13; FecG). The ewe's ovaries were weighed and measured, then the follicles (oocyte, nucleus and nucleolus) were histologically evaluated for morphometry and morphology. Morphologically normal follicles, in the primordial and transitional stages, explained 70.18% of the variability morphological characteristics between mutant and wild-type ewes. Conducting the morphometric evaluation resulted in a more precise determination of the genotype groups when there was assessment of the primordial and secondary follicular developmental stages. The diameter of the oocyte and the oocyte nucleus of the primordial follicles explained 36.76% of the variability in follicular morphology between ewes with the mutation and those with the wildtype group. Similarly, the core diameter of oocytes in secondary follicles explained 10.63% of the variability in follicular morphology among FecG, FecG and FecG ewes. Thus, morphologically normal follicles in the primordial and transitional stages of development are the variables that allow for a more precise differentiation of Santa Inês ewes with the FecG mutation. These variables may be evaluated to make more efficient the adoption of biotechniques that when conducted there is utilisation of follicles in the initial developmental stages as a physiological basis for classifying whether specific follicles are useful when conducting the techniques.
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http://dx.doi.org/10.1016/j.anireprosci.2019.106117DOI Listing
September 2019

Freezing of Stallion Semen: Evaluation of Motility and Acrosin Activity in Sperm Cells Cryopreserved Using Different Semen Extenders.

Biopreserv Biobank 2018 Dec 30;16(6):439-443. Epub 2018 Jul 30.

Laboratorio de Biotécnicas Reprodutivas, Universidade Federal Rural de Pernambuco, Recife, Brazil.

The work described here aimed to verify the efficiency of different extenders for cryopreservation of equine semen using sperm motility and acrosin activity as spermatic parameters. The semen was fractioned into two equal parts and resuspended in an 11% lactose solution in a 1:1 proportion, where it remained for 20 minutes at room temperature. The semen was centrifuged at 600 for 10 minutes, and after the second centrifugation, each pellet received the freezing extender (Merck or Zorlesco) and was loaded into 4 mL straws. Each straw was placed in liquid nitrogen vapor steam for 15 minutes and further immersion in liquid nitrogen at -196°C for long-term storage. After thawing, semen samples were initially evaluated for sperm motility, both total and progressive, and acrosin activity. Moreover, semen was incubated at 37°C and further assessed at 60 and 120 minutes in a thermoresistance test (TRT) for sperm motility and acrosin activity. Immediately after thawing, both progressive and total motility, and acrosin activity were lower ( < 0.05) in thawed semen than in fresh semen. During the TRT, total sperm motility and acrosin activity after 60 minutes were lower ( < 0.05) than those obtained after thawing. Similarly, total sperm motility and acrosin activity were lower ( < 0.05) after 120 minutes than at 60 minutes of the TRT. The analysis of motility and acrosin activity allowed the conclusion that both extenders have a similar capacity to preserve the integrity of sperm cells subject to freezing and thawing.
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http://dx.doi.org/10.1089/bio.2018.0022DOI Listing
December 2018

Conceptus loss in Santa Inês ewes carrying twin pregnancies by natural mating or embryo transfer.

Theriogenology 2018 Jul 1;115:94-98. Epub 2018 May 1.

Laboratório de Biotécnicas Reprodutivas, Universidade Federal Rural de Pernambuco, Recife, PE, Brazil.

Commercial application of reproductive biotechnologies such as multiple ovulation and embryo transfer depends on its overall efficiency. Sheep embryo transfer is gradually gaining wider adoption, but pregnancy rates after embryo transfer remain lower than those derived from natural mating for most breeds. The work was aimed to evaluate embryonic and fetal losses in Santa Inês ewes carrying twin pregnancies by natural mating or embryo transfer. Ewes were subjected to synchronized natural mating by ram effect or used as recipients for embryo transfer. Ewes diagnosed as carrying twin pregnancies at day 25 were used in the experiment (n = 42). Conceptus viability was monitored by ultrasonography on days 30, 35, 40, 45, 50, and 55 after conception. Conceptus loss was similar (P > 0.05) within natural mating 11/42 (26.19%) and embryo transfer 14/42 (33.34%). However, overall embryonic loss (80.0%) was greater (P < 0.05) than fetal loss (20.0%), with no difference within groups The results allow the conclusion that conceptus loss after embryo transfer is similar to natural mating and occurs predominantly during the embryonic stages.
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http://dx.doi.org/10.1016/j.theriogenology.2018.04.024DOI Listing
July 2018

Full-term potential of goat in vitro produced embryos after different cryopreservation methods.

Cryobiology 2017 04 27;75:75-79. Epub 2017 Jan 27.

Universidade Federal Rural de Pernambuco (UFRPE), Laboratório de Biotécnicas Aplicadas a Reprodução Animal, Recife, PE, Brazil.

Cryopreservation of preimplantation embryos represents a major challenge due to their shape and relatively large cells. Embryo source and cryopreservation method are key factors to cryotolerance efficiency and few reports have investigated more promising protocols for goat embryos. The study was aimed to compare different cryopreservation methods for goat in vitro produced (IVP) embryos. Goat blastocysts were subjected to conventional freezing (CF), Dimethyl sulfoxide vitrification (DMSO-V) and Dimethylformamide vitrification (DMF-V). Cryopreserved blastocysts were assessed for re-expansion, cell viability and in vivo development rates. Blastocyst re-expansion after cryopreservation was similar between groups, but cell viability was lower for DMF-V (32%) than CF (68%) and DMSO-V (60%). Pregnancy and delivery rates were similar for CF (60% and 50%) and DMSO-V (50% and 45%) and higher then DMF-V (20% and 15%), respectively. Finally, kidding rates were also indistinguishable for CF (40%) and DMSO-V (35%), but higher then DMF-V (12.5%). In conclusion, conventional freezing and vitrification using DMSO have similar efficiencies for cryopreservation of goat IVP embryos and cryoprotectant for vitrification affects its outcome.
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http://dx.doi.org/10.1016/j.cryobiol.2017.01.009DOI Listing
April 2017

Influence of biostimulation and temporary weaning on follicular dynamics and pregnancy rates in Nelore cows (Bos taurus indicus).

Trop Anim Health Prod 2015 Oct 14;47(7):1285-91. Epub 2015 Jun 14.

Department of Animal Reproduction, Federal University of Pernambuco, Recife, Permanbuco, Brazil.

The objective of this study was to evaluate the effect of biostimulation and temporary weaning on the follicular dynamics and pregnancy rates in Nelore cows. We used three groups of 75 cows: a control group without biostimulation and suckling calves (WB), a group that was biostimulated and had suckling calves (BE) and a group that was biostimulated and subjected to temporary weaning for 56 h (BETW). Ovarian dynamics were examined using ultrasonography. All groups showed follicular atresia. The interval between beginning of the treatment and wave emergence was 3.25 ± 0.30 days for BE, 3.40 ± 0.27 days for BETW and 3.37 ± 0.50 days for WB. The time between implant removal and ovulation was 64.50 ± 1.88 h for BE, 66.75 ± 1.35 h for BETW and 60.85 ± 3.10 h for WB. Eight cows were submitted to ultrasound analysis, and the percentages of cows that had ovulatory follicles of the new follicular wave with maximum diameters greater than 0.80 cm were 100 % (8/8) in BE (1.28 ± 0.12 cm), 100 % (8/8) in BETW (1.52 ± 0.07 cm) and 87.5 % (7/8) in WB (1.21 ± 0.10 cm). The pregnancy rate was 52 % (39/75) in BE, 69.3 % (52/75) in BETW and 37.3 % (28/75) in WB. The association of biostimulation and temporary weaning increased follicular development, ovulation synchronisation and, consequently, the pregnancy rate in beef cows.
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http://dx.doi.org/10.1007/s11250-015-0861-0DOI Listing
October 2015