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Abstract.The objective was to examine the effect of seminal plasma on cryopreservation of sperm from rhesus macaques. Sperm cryosurvival was evaluated by sperm motility and acrosomal integrity. Compared with slow cooling (−0.4 C/min) from 37 C (body temperature) to 4 C, rapid cooling (−16 C/min) caused cold shock in rhesus macaque sperm. The cryosurvival of sperm was decreased regardless of the presence or absence of seminal plasma (P<0.05). However, the presence of seminal plasma during cold shock at a rapid cooling rate improved sperm motility and acrosomal integrity in individual monkeys. Male-to-male variation in sperm cryosurvival was observed after cryopreservation (P<0.05), and the presence of seminal plasma during sperm cryopreservation improved sperm motility and acrosomal integrity in individual monkeys (P<0.05). Furthermore, by adding seminal plasma from monkeys with good sperm cryosurvival to sperm freezing extender, the frozen-thawed motility and acrosomal integrity of sperm from monkey with poor cryosurvival were improved (P<0.05). The present study indicated that seminal fluid is beneficial to sperm undergoing cold shock or cryopreservation in individual monkeys. The cryosurvival of sperm from rhesus macaques with poor sperm freezability could be improved by the presence of seminal plasma from males with good sperm cryosurvival. This finding provides a useful method for genetic preservation in this important species.Key words: Cryopreservation, Rhesus macaque, Seminal plasma, Sperm (J. Reprod. Dev. 57: [737][738][739][740][741][742][743] 2011) T he rhesus macaque is one of the most important animal models in biomedical research because of the genetic and physiological similarities to humans. Meanwhile, the population of the wild rhesus macaque is declining due to the habitat degradation and genetic isolation resulting from human activity. Maintenance and breeding of rhesus macaques are costly. Sperm cryopreservation can provide an efficient tool to preserve valuable genetic resources. With the combination of assisted reproductive technologies, such as artificial insemination (AI), in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and embryo transfer (ET), animal offspring can be produced using cryopreserved sperm at a relatively low cost and high efficiency.Information on the cryopreservation of rhesus macaque sperm is limited. Only one research group reported live birth by AI using frozen-thawed sperm [1]. Similar to other animals, such as boars, bulls and stallions [2][3][4], rhesus macaque sperm also showed male-to-male variation in post-thaw survival [5,6]. Therefore, the development of an optimal freezing protocol that is universally suitable for rhesus macaque sperm cryopreservation is challenging. Individual variation in seminal plasma composition among animals was demonstrated to have a significant role in post-thaw sperm motility [7]. Studies have indicated that seminal plasma benefits the postcryopreservation survival of human, boar, bull, ram and stallion sperm [8...

ABSTRACT. Ethylene glycol (EG) has been speculated to be the most appropriate penetrating cryoprotectant for cryopreservation of rhesus macaque sperm due to its higher permeability coefficient. The present study aimed to determine the optimal EG concentration, freezing rate and holding time in liquid nitrogen (LN 2 ) vapor for rhesus sperm cryopreservation. Among six tested EG concentrations (0, 0.18, 0.35, 0.7, 1.4 and 2.1 M), 0.7 M EG showed the most effective cryoprotection (P<0.05). Sperm frozen with 0.7 M EG at -183C/min showed higher post-thaw motility than sperm frozen at -10, -67 or -435C/min (P<0.05). Sperm frozen in LN 2 vapor at -183C/min with 0.7 M EG and a holding time of 10 min showed higher post-thaw motility compared with a holding time of 5 or 15 min (P<0.05). The function of sperm cryopreserved at the optimized EG concentration, freezing rate and holding time was further evaluated by in vitro fertilization. Of the 36 oocytes collected from gonadotropin-stimulated rhesus macaques, 61.1% were fertilized, and 61.1, 44.4 and 36.1% of the oocytes developed to 2 cells, morulae and blastocysts, respectively. Our findings provide an alternative penetrating cryoprotectant and optimal protocol for genetic preservation purposes in this important species. The rhesus macaque has become an endangered species due to the habitat loss and degradation resulting from human activity. Meanwhile, it has been one of the most widely used laboratory animals in medical and biological research. Sperm cryopreservation can be the most effective method for preserving valuable genetic resources and relieve the heavy financial burden of maintaining colonies. 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.The cryoprotective agent (CPA) is one of the most important factors that affect the cryosurvival of sperm. Glycerol (Gly) is the widely used penetrating CPA for nonhuman primate sperm cryopreservation [15]. The optimal Gly concentration for rhesus sperm cryopreservation is 5% (0.7 M) [20], which is consistent with the results for cynomolgus macaque sperm cryopreservation [19]. In addition to Gly, other types of penetrating CPAs, including dimethyl sulfoxide (DMSO), propylene glycol (PG) and ethylene glycol (EG), have been applied for cryopreservation of nonhuman primate sperm [2,3,7,11,20]. However, study of the fundamental cryobiology of rhesus macaque sperm indicated that EG is the most appropriate CPA for cryopreservation of rhesus macaque sperm due to its higher permeability coefficient compared with Gly, DMSO and PG [1]. The freezing rate during cryopreservation is another important factor that affects sperm survival. During ...