Clinical safety of intratesticular transplantation of allogeneic bone marrow multipotent stromal cells in stallions.

Although stem cell therapy is a promising alternative for treatment of degenerative diseases, there are just few reports on the use of stem cells therapy in horse's reproductive system. This study aims to evaluate the effect of intratesticular injection of bone marrow mesenchymal stromal/stem cells (MSCs) in healthy stallions, and its outcome on seminal parameters and fertility. In Experiment 1, 24 stallions were divided into treatment group (TG) and control group (CG). In the TG, an intratesticular application of MSC was performed, and in the CG, only PBS was used. Measurements of testicular volume, surface temperature and Doppler ultrasonography were performed 24 and 48 hr after treatments. Fifteen days after application, the testicles were removed and submitted to histological analysis. In Experiment 2, 3 fertile stallions received similarly treatment with MSCs. Physical examination and sperm analysis were performed weekly during 60 days after treatment, and at the end, semen from one of them was used for artificial inseminations of 6 healthy mares. In Experiment 1, clinical examinations showed no signals of acute inflammation on both groups according to the analysed variables (p > .05). Also, no signal of chronic inflammation was observed on histological evaluation. In Experiment 2, stallions presented no physical alterations or changes in sperm parameters, and a satisfactory fertility rate (83%; 5/6) was observed after AI. The results support the hypothesis that intratesticular application of bone marrow MSCs is a safe procedure, and this could be a promising alternative to treat testicular degenerative conditions.

Strategies to improve the fertility of fresh and frozen donkey semen.

Fertility rates of donkey semen in jennies are lower compared to mares. The aims of this study were to evaluate different sperm cryopreservation methods and insemination strategies to improve the fertility of donkey semen in jennies. Three experiments were performed: (1) the comparison of two freezing methods of donkey semen (conventional method and automated method); (2) the determination of a suitable insemination dose of fresh donkey semen for jennies and mares; and (3) the influence of the semen deposition site on fertility of jennies inseminated with frozen donkey semen. For experiment 1, no differences were observed in total motility, angular velocity, curvilinear velocity, straight-line velocity, and plasma membrane integrity between samples frozen with the conventional (Styrofoam box) and the automated method (TK 4000C). However, the automated method provided higher values of progressive motility and rapid cells in frozen-thawed samples in comparison with the conventional method (P < 0.05). For experiment 2, mares were bred using 500 × 10(6) fresh sperm (M); and jennies using 1 × 10(9) (J1) or 500 × 10(6) fresh sperm (J5). Pregnancy rates in M, J1, and J5 were 93% (14/15), 73% (11/15), and 40% (6/15), respectively. When using different insemination doses, 500 × 10(6) or 1 × 10(9) sperm, no significant difference was observed in pregnancy rates of mares (M, 14/15) and jennies (J1, 11/15). Furthermore, there was no significant difference between the two insemination doses in jennies. However, with an insemination dose of 500 × 10(6) fresh sperm, the pregnancy rates were significantly higher in mares (M, 14/15) than in jennies (J5, 6/15; P < 0.05). For experiment 3, the inseminations were carried out in the uterine body (UB) or in the uterine horn of jennies with frozen-thawed donkey semen. No pregnancies were achieved with inseminations performed in the UB (0/12). The pregnancy rate for uterine horn group was 28.26% (13/46) and thus significantly higher than the UB group (0%; 0/12; P < 0.05). In conclusion, the automated method showed higher values on progressive motility and rapid cells parameters compared to the conventional method and can be used as an alternative for freezing donkey semen. The increase in the number of sperm cells per insemination dose using fresh donkey semen improved the fertility rates in jennies. The deep horn inseminations using frozen-thawed donkey semen increased the pregnancy rate in jennies, and the multiple inseminations may be an option to improve the fertility rates of donkey semen in jennies.