Ribrio Ivan Tavares Pereira Batista

Dynamics of Recombinant hG-CSF in Transgenic Goat: Preliminary Study in the Founder during Hormonally Induced Lactation

This study aimed to characterize the dynamic of human granulocyte colony-stimulating factor (hG-CSF) during artificial lactation in a transgenic founder goat and to assess its potential ectopic expression and health. The female secreted 93.9 to 1,474.6 µg hG-CSF per mL of milk. Two peaks of serum hG-CSF (3,470 and 7,390 pg/mL) were detected in the first half of the lactation. Outside of the lactation, hG-CSF was absent from serum, indicating no ectopic expression. During the treatment to induce lactation, transgenic female presented increased neutrophil and lymphocyte blood counts when compared to nontransgenic female. Despite transient neutrophilia, serum biochemistry profiles indicated normal liver and renal functions. Thus, transgenic goat expressed hG-CSF in quantities sufficient for a commercial bioreactor and remained clinically healthy.

Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real‐time PCR

Taking into account the importance of goats as transgenic models, as well as the rarity of copy number (CN) studies in farm animals, the present work aimed to evaluate methodological strategies for accurate and precise transgene CN quantification in goats using quantitative polymerase chain reaction (qPCR). Mouse and goat lines transgenic for human granulocyte‐colony stimulating factor were used. After selecting the best genomic DNA extraction method to be applied in mouse and goat samples, intra‐assay variations, accuracy and precision of CN quantifications were assessed. The optimized conditions were submitted to mathematical strategies and used to quantify CN in goat lines. The findings were as follows: validation of qPCR conditions is required, and amplification efficiency is the most important. Absolute and relative quantifications are able to produce similar results. For normalized absolute quantification, the same plasmid fragment used to generate goat lines must be mixed with wild‐type goat genomic DNA, allowing the choice of an endogenous reference gene for data normalization. For relative quantifications, a resin‐based genomic DNA extraction method is strongly recommended when using mouse tail tips as calibrators to avoid tissue‐specific inhibitors. Efficient qPCR amplifications (≥95%) allow reliable CN measurements with SYBR technology. TaqMan must be used with caution in goats if the nucleotide sequence of the endogenous reference gene is not yet well understood. Adhering to these general guidelines can result in more exact CN determination in goats. Even when working under nonoptimal circumstances, if assays are performed that respect the minimum qPCR requirements, good estimations of transgene CN can be achieved.

Overexpression of hyaluronan synthase 2 and gonadotropin receptors in cumulus cells of goats subjected to one-shot eCG/FSH hormonal treatment for ovarian stimulation

Hormonal ovarian stimulation may affect transcripts in somatic cells of cumulus-oocyte complexes (COCs) and affect the resulting oocyte quality. Here, in parallel with morphological classification and in vitro maturation (IVM) rate analysis, we investigated the expression of hyaluronan synthase 2 (HAS2), gonadotropic receptors (FSHR and LHR) and connexin 43 (GJA1) in cumulus cells (CCs) from goat COCs after multi-dose FSH (MD) or one-shot FSH/eCG (OS) treatments, using bovine COCs as control groups. The MD treatment produced more large follicles, and the resulting COCs had a better morphology and IVM rate than were obtained with OS. The OS treatment produced COCs with increased HAS2, FSHR, LHR and GJA1 expression. This gene expression pattern was also observed in the CCs of COCs that showed poor morphological characteristics. On the other hand, the mRNA levels were more similar between groups after IVM; FSHR and LHR were the main genes that showed decreased expression. Some events that occurred in bovine CCs during IVM, such as cell expansion, increased HAS2 expression and decreased GJA1 expression, were less evident or did not occur in goat COCs. In conclusion, increasing HAS2, FSHR, LHR and GJA1 expression in goat COCs does not confer greater meiotic competence to oocytes. Instead, it may result from poor regulation of gene expression in CCs by lower quality oocytes. Finally, cumulus expansion, together with HAS2 upregulation and GJA1 downregulation, seems to be more important for bovine COCs than for goat COCs. Additional studies are needed to investigate the importance of other HAS isoforms and connexins in goat COCs.

Supplementation of 17β-estradiol and progesterone in the co-culture medium of bovine oviductal epithelial cells and ovine spermatozoa reduces the sperm kinematics and capacitation

This study investigated the effect that bovine oviductal epithelial cell (BOEC) and ovine spermatozoa co-culture exposed to different hormonal environments had on ram sperm function over the course of a 24-h incubation period. Ram cooled-stored spermatozoa were selected by swim-up and then co-cultured separately for 24 h at 38.5 °C under 5% CO2 with either: (1) Fert-TALP medium (positive control [POSControl]), (2) Fert-TALP medium supplemented with 17β-estradiol (E2) and progesterone (P4) at concentrations similar to follicular phase (Follicular NEGControl), (3) Fert-TALP medium supplemented with E2 and P4 concentrations similar to luteal phase (Luteal NEGControl), (4) BOEC cultured in the same medium as that of the Follicular NEGControl group (Follicular BOEC group), or (5) BOEC cultured in the same medium as that of the Luteal NEGControl group (Luteal BOEC group). The sperm kinematics, capacitation status, and plasma membrane (PM) integrity were evaluated at different intervals. Sperm PM integrity was not affected (P ˃ 0.05) by BOEC co-culture, regardless of the phase of the estrous cycle. After 4 h of incubation, the Luteal BOEC group presented lower (P <  0.05) progressive motility and total motility than the Luteal NEGControl group while the Follicular BOEC group showed lower (P <  0.05) velocimetric parameters and progressive motility than the Follicular NEGControl group. Throughout the incubation period, both BOEC co-culture groups showed a decrease (P <  0.05) in their capacitation rate in comparison to the POSControl group. Conversely, the Luteal BOEC group presented a higher (P <  0.05) non-capacitated rate than both the POSControl and Luteal NEGControl groups. In conclusion, BOEC co-culture with ovine spermatozoa at either the follicular or luteal phase decreases sperm kinematics and delays sperm capacitation.

Ultrasonographic findings of the mammary gland, liver, gallbladder, spleen, and kidneys in transgenic goats for hG-CSF during induced lactation

In transgenic murine models, the study of certain organs or tissues can be performed after euthanasia of some specimens. However, this practice may not be economically feasible when applied to livestock such as transgenic goats. It is necessary to use minimally invasive methods to perform in vivo studies of organs that may be affected by disorders related to the activity of the transgene, particularly during milk production, when the recombinant protein is secreted. The aim of this study was to describe ultrasonographic findings of the liver, gallbladder, spleen, kidneys, and mammary glands in transgenic goats for evaluating the effect of human granulocyte-colony stimulating factor (hG-CSF) expression in milk during induced lactation. Six female Caninde goats-three transgenic (T) and three non-transgenic (NT)-were subjected to hormone therapy to induce lactation; ultrasonographic examinations of the liver, gallbladder, spleen, kidneys, and mammary gland were performed during both the hormonal therapy and the lactation period at different intervals depending on the organ being examined. On Day 16 (Day 1 = hormonal therapy initiation), all goats were lactating and presented healthy mammary glands, characterized by echogenic parenchyma showing a granular echotexture. Transgenic and non-transgenic goats were compared on the basis of measurements and ultrasound images obtained from each organ. No differences between T and NT animals were observed in the examined area for the liver, gallbladder, spleen, and kidneys. Liver and renal echogenicity and appearance of gallbladder and portal and hepatic veins were similar in all females. Ultrasonographic findings of the mammary gland, liver, gallbladder, spleen, and kidneys in transgenic goats did not show any difference from those in non-transgenic goats.Thus, these results suggest that the presence and expression of the transgene had no effect on the ultrasonographic findings of mammary gland and abdominal organs in the transgenic goats. Moreover, the findings shows that ultrasonography is a useful screening tool for clinical examination of transgenic goats; this allows the investigation of possible disorders and avoids the unnecessary use of invasive techniques.