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Oocyte vitrification allows for the storing of endangered breed female gametes. Cryoprotectant (CPA) concentration and exposure time should ensure cell protection with minimal toxicity. In the present study, a high concentration-rapid exposure (HC-RE) and a low concentration-slow exposure (LC-SE) vitrification protocol, using dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as permeating CPAs, were evaluated on meiotic competence and bioenergetic-oxidative status of pre-pubertal lamb immature COCs after in vitro maturation (IVM). For each protocol, COCs vitrified through a traditional protocol and fresh ones were used as controls. Both protocols allowed COC morphology preservation after vitrification-warming (V-W) and cumulus expansion after IVM. The maturation rate (7% and 14%) was comparable to the vitrified control (13% and 21%) but not satisfactory compared to fresh ones (58% and 64%; p < 0.001). The rate of mature oocytes displaying a perinuclear/subcortical (P/S) mitochondrial distribution pattern, an index of cytoplasmic maturity, was comparable between vitrified and fresh oocytes. The LC-SE vitrification protocol did not affect quantitative bioenergetic-oxidative parameters compared to both controls whereas HC-RE protocol significantly reduced intracellular reactive oxygen species (ROS) levels, indicating cell viability loss. In conclusion, to improve pre-pubertal lamb immature COC vitrification, the combination of low CPA concentrations with prolonged exposure time could be more promising to investigate further.

Reproductive biotechnologies can be used as a supporting tool, through gamete conservation and in vitro embryo production, in the preservation of invaluable and irreplaceable animal genetic resources. In the present study, immature mouflon cumulus-oocyte complexes (COCs) collected from ovariectomized female ovaries underwent short- or long-term conservation (24 h maintained in Earle's/Hank's (EH) medium or vitrification) under field conditions and afterwards transported to the laboratory where they were cultured for in vitro maturation (IVM) and assessed for oocyte meiotic competence and bioenergetic-oxidative status. Utilization of both storage techniques led to COC morphology preservation, as well as cumulus expansion and oocyte meiotic resumption after the IVM procedure. Quantitative bioenergetic-oxidative parameters were reduced in vitrified oocytes compared with EH ones. Immature COC storage needs to be optimized in both domesticated and non-domesticated sheep as a part of the strategy to avoid the loss of valuable genotypes of these animal species.

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.

The meagre, Argyrosomus regius , is a valued fish species of which aquaculture production might be supported by the development of a stem germ cell xenotransplantation technology. Meagre males were sampled at a fish farm in the Ionian Sea (Italy) at the beginning and end of the reproductive season. Small and large Type A undifferentiated spermatogonia were histologically identified in the germinal epithelium. Among the tested stemness markers, anti-oct4 and anti-vasa antibodies labeled cells likely corresponding to the small single Type A spermatogonia; no labeling was obtained with anti-GFRA1 and anti-Nanos2 antibodies. Two types of single A spermatogonia were purified via density gradient centrifugation of enzymatically digested testes. Testes from fish in active spermatogenesis resulted in a more efficient spermatogonial stem cell (SSC) yield. After cell seeding, meagre SSCs showed active proliferation from Day 7 to Day 21 and were cultured up to Day 41. After cryopreservation in dimethyl-sulfoxide-based medium, cell viability was 28.5%. In conclusion, these results indicated that meagre SSCs could be isolated, characterized, cultured in vitro, successfully cryopreserved, and used after thawing. This is a first step towards the development of a xenotransplantation technology that might facilitate the reproduction of this valuable species in captivity.