New Strategies for Conservation of Gentile di Puglia Sheep Breed, an Autochthonous Capital of Millennial Tradition in Southern Italy

Abstract

Gentile di Puglia (GdP) is an autochthonous sheep breed of Southern Italy included among ovine breeds threatened by genetic erosion and extinction risk, which have been given attention by local and international institutions, thus emphasizing the need for germplasm conservation actions. In the present study, two assisted reproduction approaches, finalized for GdP conservation, were performed: (1) on-farm reproductive efficiency evaluation, expressed as pregnancy rate (PR), twin pregnancy rate (tPR), and body condition score (BCS), for three consecutive breeding cycles and (2) pre-pubertal lambs’ immature cumulus–oocyte complex (COC) retrieval, vitrification, in vitro maturation (IVM), and assessment of meiotic stage and bioenergetic-oxidative status compared with those of other Italian and European commercial breeds. PR and tPR were progressively reduced over time. In all clinical examination times, BCS was significantly lower in nonpregnant ewes compared with pregnant ones. Fresh GdP pre-pubertal lamb COCs achieved meiotic maturation and showed healthy bioenergetic–oxidative status after IVM. Vitrification reduced the oocyte maturation rate in all groups. However, mature oocytes retained their cytoplasmic maturity, expressed as a mitochondria distribution pattern and activity, indicating promising developmental competence. In conclusion, clinical- and biotechnological-assisted reproduction approaches can support conservation strategies of GdP and other local sheep breeds in Southern Italy.

Short- and Long-Term Storage of Non-Domesticated European Mouflon (Ovis aries musimon) Cumulus–Oocyte Complexes Recovered in Field Conditions

“…The ovaries were processed immediately after the ovariectomy in field conditions. For COC retrieval, ovaries were sliced [ 18 ] and follicular contents collected in sterile Petri dishes containing phosphate-buffered saline (PBS) and observed using a WILD Heerbrugg stereomicroscope. Only COCs displaying at least three intact cumulus cell layers and a homogenous cytoplasm were selected for short- or long-term storage and subsequent in vitro culture.…”

“…COCs from other adult animals as well as from the prepubertal female were cryopreserved using the previously established vitrification technique for ovine COCs [ 18 ]. Briefly, selected immature COCs were incubated for 10 min in a 300 μL drop of equilibration solution (ES) consisting of HEPES-buffered TCM 199 medium supplemented with 20% ( v / v ) FCS (base medium, BM) containing 7.5% ( v / v ) ethylene glycol (EG) and 7.5% ( v / v ) dimethyl sulfoxide (DMSO).…”

“…Vitrified COCs were transported in liquid nitrogen at −196 °C to the IVM laboratory where warming and IVM were performed [ 18 ]. The vitrified devices were directly submerged into one 100 µL drop of warming solution (WS) consisting of BM containing 1 mol/L sucrose at 38.5 °C for 1 min.…”

“…IVM methods were adapted from those previously described for domestic sheep ( Ovis aries ) [ 18 ]. The IVM medium was prepared based on TCM-199 medium with Earle’s salts, buffered with 5.87 mmol/L HEPES and 33.09 mmol/L sodium bicarbonate and supplemented with 0.1 g/L L-glutamine, 2.27 mmol/L sodium pyruvate, calcium lactate pentahydrate (1.62 mmol/L Ca 2+ , 3.9 mmol/L Lactate), 50 μg/mL gentamicin, 20% ( v / v ) fetal calf serum (FCS), 10 μg/mL of porcine follicle stimulating hormone and luteinizing hormone (FSH/LH; Pluset ® , Calier, Barcellona, Spain) [ 27 ], and 1 μg/mL 17β estradiol [ 18 ]. The prepared medium was pre-equilibrated for 1 h under 5% CO 2 in air at 38.5 °C, then transferred (400 μL/well) into a four-well dish (Nunc Intermed, Roskilde, Denmark) and covered with pre-equilibrated lightweight paraffin oil.…”

“…In particular, vitrification of immature COCs allows the avoidance of animal hormonal stimulation as an advantage for the field conditions and particularly wild animals. Moreover, importantly, the minimizing of necessary equipment for the procedure (stereomicroscope, cryoprotectant-rich solutions, vitrification devices, and liquid nitrogen) allows flexible scheduling of the IVM day, independent of COC collection and selection time [ 18 ]. On the other side, oocytes are among the most difficult cells to cryopreserve because of their high sensitivity to the mechanical, chemical, osmotic, and thermal stresses imposed during freezing/warming procedures [ 19 ].…”