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Simple Summary: In recent years, due to industry and market preferences, local animal breeds have been exposed to genetic erosion and extinction risk. Effective strategies for their recovery and conservation are needed. Gentile di Puglia (GdP) is an autochthonous sheep breed, typical of Southern Italy, with an aptitude for wool, meat, and milk production and considerable historical and cultural value. The development of a GdP gamete and embryo cryobank could help to support the numerical reimplementation of this population. Animal germplasm conservation is mainly performed through sperm cryopreservation, whereas strategies on the female side are developed in a limited way. In this study, the dual purpose of monitoring the reproductive efficiency in one pilot GdP farm in the Apulia region and setting up a cryopreservation protocol, by vitrification, of immature cumulus-oocyte complexes (COCs) recovered from pre-pubertal lambs, followed by an analysis of their in vitro maturation potential and bioenergetic-oxidative status, was pursued. The results indicated that traditional reproductive management leads to progressive offspring reduction and that ex situ biotechnological conservation strategies, through immature oocyte vitrification and in vitro maturation, can support in situ conservation, leading to in vitro embryo production and transfer. 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. [ABSTRACT FROM AUTHOR]