Animal-derived products are widely used in sperm cryopreservation for their cryoprotective properties. These components, however, tend to be replaced because of sanitary risks. STEMALPHA.CRYO3 (Ref. 5617; Stem Alpha, Saint-Genis-l'Argentière, France), called "CRYO3," is a chemically defined preservation medium currently used for freezing human tissue and adult stem cells. The aim of this study was to evaluate the effect of a CRYO3-based medium on ram sperm freezing regarding in vitro parameters and in vivo fertility. Semen from nine Charolais rams was collected using an artificial vagina, then split and frozen using two media: a CRYO3-based medium or a control medium containing egg yolk (10%) and milk (45%). Sperm membrane integrity (propidium iodide [PI]/SYBR-14 and calcein AM/ethidium homodimer-1), acrosome integrity (FITC-PNA/PI), and mitochondrial membrane potential (JC-1) were assessed using flow cytometry, while functional membrane integrity was assessed using a hypo-osmotic swelling test and motility parameters, evaluated by computer-assisted sperm analysis. Pregnancy rates, prolificacy, and the average daily weight gain (DWG) of lambs were evaluated after performing 195 laparoscopic inseminations. The control medium showed significantly higher results than CRYO-based medium for all in vitro parameters, except for linearity and straightness (motions parameters). Conversely, field trials showed no significant difference between the control medium and the CRYO3-based medium for pregnancy rates (72.2% and 67.9%, respectively), prolificacy (1.8 and 1.6, respectively), and the DWG (0.34 and 0.35 kg/d, respectively). This preliminary study showed that CRYO3 cannot replace egg yolk and milk in freezing extenders for commercial purposes. However, as laparoscopic inseminations allowed a 67% pregnancy rate, CRYO3-based medium remains an option for international transport or long-term storage of genetic diversity.
Keywords: animal-derived products; hyaluronic acid; pregnancy rate; protein-free; ram sperm; semen freezing.