Live Rhesus Offspring by Artificial Insemination Using Fresh Sperm and Cryopreserved Sperm1

Abstract

Artificial insemination (AI) and the cryopreservation of sperm with full reproductive capabilities are vital in the armamentarium of infertility clinics and reproductive laboratories. Notwithstanding the fantastic successes with AI and sperm cryopreservation in numerous species, including humans and cattle, these assisted reproductive technologies are less well developed in other species of importance for biomedical research, such as genetically modified mice and nonhuman primates. To that end, AI at high efficiency in the rhesus macaque (Macaca mullata) and the successful cryopreservation of rhesus sperm is presented here, as are the complexities of this primate model due to differences in reproductive tract anatomy and gamete physiology. Cryopreservation had no effect on the ability of sperm to fertilize oocytes in vitro or in vivo. Post-thaw progressive motility was not affected by cryopreservation; however, acrosome integrity was lower for cryopreserved (74.1%) than for fresh sperm (92.7%). Fertilization rates did not differ when fresh (58.1%; n = 32/55) or cryopreserved sperm (63.8%; n = 23/36) were used for in vitro fertilization. Similarly, pregnancy rates did not differ significantly after AI with fresh (57.1%; n = 8/14) or cryopreserved sperm (62.5%; n = 5/8). Seven live rhesus macaques were born following AI with fresh sperm, and three live offspring and two ongoing pregnancies were obtained when cryopreserved sperm were used. Cryopreservation of rhesus sperm as presented here would allow for the cost-effective storage of lineages of nonhuman primates with known genotypes. These results suggest that either national or international centers could be established as repositories to fill the global needs of sperm for nonhuman primate research and to provide the experimental foundation on which to explore and perfect the preservation of sperm from endangered nonhuman primates.

Delay of Ovulation Due to Diets Containing Levonorgestrel in Cynomolgus Monkeys (<i>Macaca fascicularis</i>)

“…Although reproductive technology for monkeys, such as artificial insemination and embryo transfer has been improved [4,5,[10][11][12], control of the ovarian cycle has not been established. There are a few reports showing that progestins are useful for control of ovulation in monkeys.…”

Optimization of Ethylene Glycol Concentrations, Freezing Rates and Holding Times in Liquid Nitrogen Vapor for Cryopreservation of Rhesus Macaque (Macaca mulatta) Sperm

“…To our knowledge, this is the first case of rhesus oocytes fertilized in vitro using sperm cryopreserved with EG. In contrast, sperm cryopreserved with 0.7 M Gly have already been successfully used for IVF [4,20,21] and AI [4] in rhesus macaques. Our findings will facilitate research studies of gamete biology and preimplantation embryo development in nonhuman primates.…”

“…Rhesus offspring can be efficiently rederived using frozen sperm with the assistance of artificial insemination (AI), in vitro fertilization (IVF) and embryo transfer (ET) at low cost. However, there has been only one report of live birth of rhesus offspring resulting from AI using cryopreserved sperm [4]. Therefore, an optimal protocol for cryopreservation of rhesus macaque sperm with complete retention of fertilizing ability is still needed.…”

Paternal Mitochondrial DNA Transmission During Nonhuman Primate Nuclear Transfer

“…We have confirmed through pedigree records that 16426 was of Indian origin (data not solely associated with NT or a consequence of interspecific crossing in nonhuman primates, we analyzed two shown). Consequently, we subcloned 14609 and sequence analysis revealed the expected variation in hooffspring generated through artificial insemination (AI; Sánchez-Partida et al 2000). These two AI offspring, mology to that of 14893 and 16426.…”