Refinement of a Protocol for the Induction of Lactation in Nonpregnant Nonhuman Primates by Using Exogenous Hormone Treatment

Obtaining sufficient quantities of milk from NHP is necessary for pharmacologic and immunologic studies required for the development and safety assessment of drugs and vaccines to be used in the maternal–infant setting. We previously induced lactation in nonpregnant female rhesus macaques (RM, Macaca mulatta) and African green monkeys (AGM, Chlorocebus sabaeus) for studies of immune responses in milk, but the volume collected was variable. To improve lactation induction protocols for nonbreeding nonhuman primates, we investigated serum hormone levels and collection protocols in AGM and RM. Here, we correlated milk volume with serum levels of endogenous and administered hormones: estradiol, prolactin, progesterone, and medroxyprogesterone in RM and AGM. We also investigated whether age, parity or the timing of milk collections were associated with the volume of milk collected from the AGM and RM in which lactation was induced by using exogenous hormones. We found an inverse correlation with serum estradiol and milk volume in the RM but no significant correlation between milk volumes and the remaining serum hormone levels in the induced RM or AGM. In addition, HIL AGM had higher peak estradiol levels than did naturally lactating AGM. A revised estradiol-sparing protocol increased milk volumes in the AGM. In addition, milk volume in RM was greater in the morning than the afternoon. In conclusion, we have refined a lactation induction protocol in nonpregnant primates, which is a needed alternative to using nursing primates for the assessment of drug levels and immune responses in milk.Abbreviations: AGM, African green monkey; AGM1, first AGM lactation induction; AGM2, second AGM lactation induction; RM, rhesus macaque; NL, naturally lactating; HIL, hormone-inducedThe pharmacokinetics by which drugs and their metabolites or vaccine-elicited immune responses appear in the breast milk of lactating women is currently conducted only in aftermarket studies. Using lactating NHP for these studies would reveal pharmacokinetics, metabolism, and host responses in the milk compartment before the drug was marketed and used clinically in lactating women. However, the resources associated with using breeding female NHP for these types of studies are considerable. A much more time-efficient and cost-effective approach that spares the fetus or infant from exposure to experimental interventions is the induction of lactation in nonpregnant NHP. Moreover, this approach spares additional breeding solely for studies of lactation. In fact, this lactation induction protocol has been used successfully in several studies to explore immunologic and virologic mechanisms of postnatal virus transmission in SIV-infected and HIV1-vaccinated monkeys, with the goal of developing strategies to reduce breast-milk transmission of HIV1.^{1,4,6,11,28,29}We previously described our protocol for inducing lactation in RM and AGM by using exogenous hormone treatment (that is, hormone-induced lactation [HIL]).^22-25,28,29 Increasing doses of depot medroxyprogesterone and estradiol were administered intramuscularly for 2 mo to mimic the hormone fluctuations of natural pregnancy and achieve mammary tissue development. After 2 mo of hormone injections, haloperidol was administered orally to raise serum prolactin levels and induce milk production. Oxytocin was given prior to milk collection to stimulate milk let-down. We also reported a comparison of the cellular and immunoglobulin content in the milk of naturally lactating (NL) and HIL RM. The lymphocyte distribution was similar between the 2 types of milk, yet the IgG content was higher in HIL milk²² but similar to that seen in less mature or early milk. Therefore, we concluded that this method was a good model for the assessment of immunologic parameters but that it may better represent the immunologic milieu of colostrum. The milk volumes obtained from the RM were quite variable (100 µL to 2 mL), and when this protocol was used in AGM, milk volumes collected were much less than expected (30 to 100 μL), often limiting the laboratory assays that could be performed. Therefore, optimizing the HIL protocol in the nonpregnant NHP would further facilitate pharmacologic and immunologic investigations in lactating NHP.The aims of our current study were 1) to determine the relationship between serum hormone levels (progesterone, medroxyprogesterone, estradiol, and prolactin) and other clinical factors to milk volume collected from nonpregnant RM and AGM that underwent HIL and 2) to compare the hormone levels achieved during HIL to those of NL AGM. We hypothesized that age, parity, and serum hormone levels are associated with milk volume in each species after HIL, and we aimed to determine the combination that predicted the highest milk volume. Determining the clinical factors and hormone levels that best predict high milk volume in the HIL protocol would increase the applicability of this technique, obviating the need to use breeding and infant animals in the assessment of drug levels and immune responses in milk.