Erratum to: Daily vaginal temperature in Girolando cows from three different genetic composition under natural heat stress.

[This corrects the article DOI: 10.1093/tas/txab138.].

Oocyte origin affects the in vitro embryo production and development of Holstein (Bos taurus taurus) - Gyr (Bos taurus indicus) reciprocal cross embryos.

A reciprocal crossbred embryo production approach was used to assess effects of maternal breed on embryo development in tropical conditions (average temperature 22.0 °C and 77.9% relative humidity). Oocytes were recovered by ovum pick-up (OPU) from Gyr and Holstein donors (n = 90 Holstein and 83 Gyr OPUs). Female F1 embryos were produced by fertilization with sperm bearing X-chromosomes from Holstein semen (n = 615 Gyr oocytes) or Gyr semen (n = 255 Holstein oocytes). Blastocysts were transferred to recipients 168 h post-insemination (h.p.i.) (n = 70-144) and there were assessments of pregnancies until birth. Oocyte number per OPU (Gyr 10.0 ±â€¯0.7 compared with Holstein 6.3 ±â€¯0.4) and percentage viable oocytes (Gyr 78.8 ±â€¯1.9% compared with Holstein 71.2 ±â€¯2.2%) were less for Holstein donor animals. There was a 2.8 fold fewer total number of F1 blastocysts when Holstein donors were used (Gyr: 260, Holstein: 91). Pregnancy assessment during the different stages of gestation indicated the percentage pregnancy was less when embryos were produced from Holstein oocytes (Gyr and Holstein respectively: early pregnancy, 47.9% compared with 38.6%; mid-pregnancy, 44.4% compared with 31.4%; late pregnancy, 41.0% compared with 22.9%). Pregnancy length was also affected by maternal breed (Gyr: 280.8 ±â€¯0.6, Holstein: 286.3 ±â€¯0.7). It is concluded that in a tropical environment the maternal breed affects crossbred embryo development with pregnancy rates during the latter stages of gestation being greater when Gyr oocytes are used for production of embryos.

Easy In Vitro Synthesis of Optimised Functioning Reporter mRNA from Common eGFP Plasmid.

The extensive growth in number and importance of experiments and clinical-aimed techniques based solely or majorly on the activity of RNA strands, e.g. CRSPR/Cas9 and siRNA, has put emphasis on the necessity of standardisation of experiments with RNA. Considering RNA degradation during its handling seems to be a major hindrance in all RNA-based tools, the assessment of its integrity is of utmost importance. Furthermore, evaluating whether the RNA to be transfected is intact requires time-consuming electrophoresis protocol. In view of the RNA lability and the necessity for controlling experiments performed with this molecule, the transfection of a reporter mRNA may be of aid in optimising experiments. Nevertheless, commercial reporter mRNAs are far less available than plasmids for such purpose. Thus, in this work, we aimed at the optimisation of an easily performed protocol to produce a suitable eGFP mRNA. By utilising molecular biology kits customarily employed in molecular biology laboratories working with RNA-based techniques and starting from any eGFP coding vector, we produced four mRNA molecules: (1) eGFP mRNA (non-polyadenylated); (2) Kozak-eGFP mRNA (non-polyadenylated, produced from the Kozak-containing amplicon); (3) eGFP-PolyA mRNA (polyadenylated); (4) Kozak-eGFP-PolyA mRNA (containing both signals, Kozak sequence and poly(A) tail). These mRNA molecules were transfected into HEK 293 FT cells, readily transfectable, and into the MDBK bovine lineage, which has been observed as difficult-to-transfect DNA constructs. eGFP expression could be detected both by flow cytometry and by fluorescence microscopy after transfection with the polyadenylated mRNAs. Upon cytometric analysis, we noted a marked difference among the mRNA groups (p < 0.01), both in fluorescent population percentage and in florescence intensity. We showed here the necessity of the polyadenylation step in order to achieve cell expression of the eGFP observable under fluorescence microscopy. The presence of the Kozak sequence, as a 5' element, seems to augment significantly the level of protein produced upon mRNA transfection. We presented here an easy protocol to allow production of functioning mRNAs from any DNA construct. The molecules produced may aid in the standardisation and controlling most of the RNA-related experiments as well as it gives proper guidance for researchers performing expression of other proteins through mRNA transfection.

Post implantation development reveals that biopsy procedure can segregate "healthy" from "unhealthy" bovine embryos and prevent miscarriages.

Embryo biopsy has been performed in bovine in vivo produced embryos for the last twenty years, but little could be done with few embryonic cells in the past. Recently, advances in single cell analysis enabled a wide range of applications using embryo biopsy, from morphology to genetics analysis and different omics-techniques, which are promising for in vitro-fertilized (IVF) embryos. The aim of this study was to address if biopsy procedure would affect post implantation development of IVF blastocyts. Here we show that blastocyst stage do not affect re-expansion of biopsied embryos (regular blastocyst: 73.7%; expanded blastocyst: 73.1%), but affects (p<0.05) implantation (regular blastocyst: 37.8%, expanded blastocyst: 61.0%), so ideally biopsy should be performed in expanded blastocysts. No detrimental effect of biopsy procedure was detected for post-implantation development (calving rates, Biopsy: 47.1%, Control: 41.9%), and normal calves were born (Birth weight, Biopsy: 32.10±7.20kg; Control: 30.95±5.43kg). Surprisingly, we found interesting results suggesting embryo survival can be increased with aggressive procedures (such as embryo biopsy), and this is highly associated with early pregnancy loss (Biopsy: 0%, Control: 17.4%). This finding also suggests morphological classification of day 7 blastocysts is far from ideal, and supposedly, unhealthy embryos can implant but are bound to miscarriage during the first trimester (non-biopsied embryos). Our results show biopsy procedure is safe for bovine IVF embryos, and shed new light into the importance of conceptus in early pregnancy loss in cattle.

Sperm pretreatment with heparin and l-glutathione, sex-sorting, and double cryopreservation to improve intracytoplasmic sperm injection in bovine.

In bovine, intracytoplasmic sperm injection (ICSI) remains inefficient partially due to low levels of sperm decondensation. The aim of this study was to determine whether the injection of normal size sperm pretreated with heparin (Hep) and l-glutathione (GSH), the use of sex-sorted sperm, the double round of sperm freezing/thawing (re frozen), or the combination of these approaches can improve sperm decondensation and embryo development after ICSI in cattle. Cleavage and blastocyst rates were evaluated on days 2 and 7 post ICSI. Quality of ICSI blastocysts was analyzed by the relative expression of four genes by qPCR and the DNA fragmentation index by TUNEL assay. For all assays, semen samples were co-incubated with pCX-EGFP 50 ng/µl before ICSI. GFP expression, which can be detected by fluorescence microscopy, was used as a tool to estimate the success of sperm decondensation after ICSI. The use of normal size sperm pretreated with 80 µM Hep-15 mM GSH for 20 h (Hep-GSH) increased cleavage, blastocyst and EGFP + blastocysts rates (60.8, 19.4 and 61.9%) compared to control ICSI (35, 4.9 and 20%) (p < 0.05). Moreover, HMGN1, GLUT5, AQP3 and POU5F1 transcription levels did not differ between ICSI Hep-GSH and IVF embryos. The use of sex-sorted sperm (X, Y) improved cleavage rates and EGFP expression at day 4 (83 and 30.2% for ICSI Y and 83.2 and 31.7% for ICSI X) compared to non-sorted group (50.9 and 15.1%), not showing differences at the blastocyst stage. Finally, sex sorting (X) was combined with Hep-GSH and/or re frozen treatments. The use of Hep-GSH diminished cleavage rates from ICSI X re frozen group (80.4% vs. 94.2%) and blastocyst development from ICSI X group (3.3% vs. 10%), compared with their controls (p < 0.05). While Hep-GSH pretreatment induced lower transgene expression at day 4, no differences were found at the blastocyst stage between ICSI groups (from 58.3 to 80%). TUNEL assay showed higher DNA fragmentation indexes for all ICSI treatments (p < 0.05), except for ICSI X Hep-GSH, which did not differ from IVF X control. In conclusion, the use of normal size sperm pretreated with Hep-GSH, combined or not with sex-sorting by flow cytometry could improve ICSI outcomes in cattle.

Developmental competence and expression of the MATER and ZAR1 genes in immature bovine oocytes selected by brilliant cresyl blue.

The objective of this work was to evaluate the selection of immature bovine oocytes by brilliant cresyl blue dye (BCB) and expression of transcripts MATER and ZAR1. Cumulus-oocyte complexes (COCs) from slaughterhouse ovaries were exposed to BCB diluted in mDPBS and incubated for 60 min at 38.5 degrees C in humidified air. After exposure those COCs were distributed in two groups, according to their cytoplasm colour: BCB+ (coloured cytoplasm) or BCB- (colourless cytoplasm). The control group was submitted to in vitro maturation (IVM) immediately after morphological selection and holding control group COCs were exposed to mDPBS without BCB but in the same incubation conditions of BCB+ and BCB- group. The COCs of all groups were submitted to IVM, in vitro fertilization (IVF) and in vitro culture (IVC). Cleavage rate (72 h post-insemination) was similar between control (65.3%) and BCB+ (64.4%) groups, but greater than (p < 0.05) holding control (49.8%) and BCB- (51.3%) groups. Blastocyst rate (192 h post-insemination) was not different between BCB+ (18.5%) and control (16.3%) groups, but greater (p < 0.05) than BCB- (8.4%) group. No difference was found for blastocyst rate between holding control group (14.2%), control and BCB+ groups. The relative expression of MATER and ZAR1 genes was evaluated by real-time PCR in immature oocytes collected from the control, holding control, BCB+ and BCB- groups. Despite the relative expression of MATER in holding control, BCB+ and BCB- were down regulated in comparison to control group there was no statistical difference (p > 0.05) in the relative expression of MATER and ZAR1 transcripts among groups. The results indicate that the BCB dye detects immature oocyte populations with different developmental competence, although no improvement in in vitro embryo production using oocytes exposed or not to BCB was observed. Development competence of immature oocytes exposed to BCB does not seem to be associated with variations in the expression of MATER and ZAR1 transcripts.

Short intervals between ultrasonographically guided follicle aspiration improve oocyte quality but do not prevent establishment of dominant follicles in the Gir breed (Bos indicus) of cattle.

The aim of the present study was to evaluate oocyte recovery and embryo yield using two different ovarian follicular aspiration schedules in donor cows of the Gir breed. Pluriparous, non-lactating Gir cows (n = 14) were randomly allocated to one of two groups, one of which had aspirations of ovarian follicular contents conducted once a week (TVFA-1x) and the other twice weekly (TVFA-2x), for nine consecutive weeks. Before follicle aspiration, follicular population was recorded in three classes according to size (> 6 mm, 6-9 mm and > 9 mm). The cumulus-oocyte complexes (COCs) recovered were identified, morphologically classified and in vitro matured, fertilized with Gir sperm and cultured in CR2 medium for 7 days. There was no difference (P > 0.05) in the size of the largest follicle, number of follicles identified or follicular content aspirations between TVFA-1x and TVFA-2x groups. Large follicles (> 9 mm) were observed for all the aspiration intervals considered (3, 4 or 7 days). More oocytes were recovered per session in TVFA-1x as compared with TVFA-2x (8.9 +/- 0.8 versus 7.0 +/- 0.7, P < 0.01), resulting in a greater recovery rate in this group (74.3% versus 58.7%, P < 0.01). More COCs of Grade I were recovered from TVFA-2x (22.6% versus 13.3%, P < 0.01). There was no difference in cleavage rate between groups, but the percentage of embryos that reached the blastocyst stage was greater in TVFA-2x as compared with the TVFA-1x (31.8% versus 21.6%, P < 0.01). The greater in vitro performance qualities of TVFA-2x oocytes compensates for the greater oocyte recovery rate in TVFA-1x, demonstrating a greater embryo production potential. Despite showing uncommon follicular dynamics characteristics when subjected to follicular aspiration, Gir cows can be successfully used as oocyte donors for in vitro embryo production.