ABSTRACT
This study aims to evaluate the effects of adding alpha lipoic acid (ALA) to the in vitro ovarian tissue culture medium, either fresh or after vitrification/warming. For this purpose, 10 ovaries from five adult sheep were used. Each pair of ovaries gave rise to 16 fragments and were randomly distributed into two groups: fresh (n = 8) and vitrified (n = 8). Two fresh fragments were fixed immediately and considered the control, while another six were cultured in vitro for 14 days in the absence; presence of a constant (100 µM/0-14 day) or dynamic (50 µM/day 0-7 and 100 µM/day 8-14) concentration of ALA. As for the vitrified fragments, two were fixed and the other six were cultured in vitro under the same conditions described for the fresh group. All the fragments were subjected to morphological evaluation, follicular development and stromal density (classical histology), DNA fragmentation (TUNEL), senescence (Sudan Black), fibrosis (Masson's Trichome), and endoplasmic reticulum stress (immunofluorescence). Measurements of the antioxidant capacity against the free radicals 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and estradiol (E2) levels in the culture medium was performed. The results showed that in the absence of ALA, in vitro culture of vitrified ovarian fragments showed a significant reduction (P < 0.05) in follicular morphology and increased the presence of senescence and tissue fibrosis (P < 0.05). Dynamic ALA maintained E2 levels unchanged (P > 0.05) until the end of vitrified ovarian tissue culture and controlled the levels of ABTS and DPPH radicals in fresh or vitrified cultures. Therefore, it is concluded that ALA should be added to the vitrified ovarian tissue in vitro culture medium to reduce the damage that leads to loss of ovarian function. To ensure steroidogenesis during in vitro culture, ALA should be added dynamically (different concentrations throughout culture).
Subject(s)
Thioctic Acid , Tissue Culture Techniques , Animals , Female , Thioctic Acid/pharmacology , Sheep , Tissue Culture Techniques/veterinary , Ovary/drug effects , Ovarian Follicle/drug effects , Antioxidants/pharmacology , Vitrification , Cryopreservation/veterinaryABSTRACT
Milk production of transgenic does was evaluated by ultrasound measurements of the mammary gland. Two Canindé goats, which were nine months of age were used in the trial, one non-transgenic or other transgenic for hG-CSF. For hormone-induced lactation, animals were given estradiol (0.25mg/kg, IM), progesterone (0.75mg/kg, IM), and prednisolone (0.4mg/kg, IM). Ultrasonographic exams were carried out during milking, using a Falcon 100 ultrasound equipment with a 5MHz convex probe and were performed by the same operator. The results were expressed as mean±standard error. The maximum greater length and shorter length of the cistern were respectively 5.14cm and 1.36cm for the transgenic animal and 7.28cm and 2.25cm for non-transgenic, which is consistent with the maximum milk volume produced. The relationship between the average area of cisterns and milk yield was expressed as a linear correlation curve, with a correlation coefficient significantly positive for both transgenic (Y=-1.1314+10.8538*x; r=0.97) and non-transgenic (Y=-21.7551+18.3634*x; r=0.97) animals. In conclusion, the ultrasound is a practice and appropriate technique to evaluate the cisterns in ruminant udders in transgenic animal.