mun WM This is to certify that the thesis entitled VARIABLES INVOLVED IN GAMETE RECOVERY, IN VITRO FERTILIZATION, EMBRYO CULTURE AND TRANSFER IN THE SQUIRREL MONKEY (SAIMIRI SCIUREUS) presented by Thomas John Kuehl has been accepted towards fulfillment of the requirements for —BhTDT_—degree in lhysiology— 42% Major professor “QM ABSTRACT VARIABLES INVOLVED IN GAMETE RECOVERY, IN VITRO FERTILIZATION, EMBRYO CULTURE AND TRANSFER IN THE SQUIRREL MONKEY (SAIMIRI SCIUREUS) By Thomas John Kuehl These studies were conducted to examine the procedures and variables of an in vitro fertilization system used to provide zygotes for transfer to recipient females. A laparoscopic technique was developed to place a fertilized zygote (in l to 3 pl of fluid) into the ampulla of the oviduct. By use of the laparoscope and a micro- pipette, transfer can be performed to the oviduct in situ without the trauma of laparotomy. Computer programming techniques were used to aid in the col- lection, compilation, and analysis of data from in vitro fertilization experiments. A program was designed for interactive use by the inves- tigator and can produce historical data on individual animals, such as seasonal weight changes of male squirrel monkeys with associated changes in reproductive variables (ejaculate volume and sperm production). These techniques were expanded to a Macaque reproduction data bank and modeling of plasma luteinizing hormone levels in the rhesus monkeys. In the course of in vitro fertilization trials, 74S oocytes were aspirated from 2,l68 follicles (34.4% recovery rate). Of these oocytes, Thomas John Kuehl l37 (l8.4%) were atretic. Thirty-eight percent of the remaining 608 oocytes matured to the metaphase II stage. Differences in aspiration procedures did not affect maturation or in vitro fertility. Male squirrel monkeys used to supply semen were of significantly different fertility. Volumes and cellular composition of follicular fluid were determined for four sizes of follicles. Small (l mm diameter) follicles yielded significantly higher proportions of atretic ova. Five percent CO2 in air and a medium containing both pyruvate and serum proteins yielded the highest fertilization rates. Addition of either 10 or TDD m.i.u. of insulin to this culture medium did not alter in vitro maturation or fertilization, but did significantly reduce the formation of monolayers by follicle cells. Thirteen embryo transfers were performed, but no confirmed pregnancies were found. VARIABLES INVOLVED IN GAMETE RECOVERY, IN VITRO FERTILIZATION, EMBRYO CULTURE AND TRANSFER IN THE SQUIRREL MONKEY (SAIMIRI SCIUREUS) By Thomas John Kuehl A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1976 Love (A the Zight that teada the way a4 we waZh the path 06 Ktfie. --Bonn£e Kuehfl ii ACKNOWLEDGMENTS Sincere thanks and appreciation are expressed by the author to the many people who aided and encouraged him in the research pre— sented in this dissertation, especially Drs. Gail D. Riegle, Satoshi Ariga, Stanislaw Jarosz, graduate students and staff of the Endocrine Research Unit. Special thanks are extended to Greg Priebe and Dan Burke for their help in feeding the marvelous magic monster and to Richard Hall, medical illustrator. The author wishes to extend very special thanks to Dr. w. Richard Dukelow, his major advisor, who has encouraged and supported his scientific efforts to examine ideas and shared in both the delights and frustrations of the outcome. Appreciation is extended to the author's advisory committee and other members of the scientific community for critically evaluating this work, making it and future endeavors more valuable. Most especially, the author wishes to thank his parents for their trust, patience, and support through the years. To the greatest love of my life, only love can express my thanks and appreciation. Its almost time. This work was funded in part by the National Foundation, March of Dimes. iii TABLE OF CONTENTS LIST OF TABLES .......................... LIST OF FIGURES ......................... INTRODUCTION ........................... VARIABLES OF IN VITRO FERTILIZATION AND TRANSFER SYSTEMS IN SAI MI RI S C'I URE' US ........................ Literature Review ..................... In Vitro Fertilization in Primates ........... Embryo Transfer in Primates .............. Materials and Methods ................... Recovery of Gametes from Experimental Animals ..... Culture Environment .................. Transfer of Fertilized Ova to Experimental Animals . . . Experimental Design .................. Results .......................... Discussion ......................... REFERENCES ............................ A LAPAROSCOPIC TECHNIQUE FOR TRANSFER OF EMBRYOS IN NONHUMAN PRIMATES ............................ Abstract ............ ' .............. Introduction ........................ Materials and Methods ................... Animals ........................ Transfer Procedure ................... Results .......................... Discussion ......................... REFERENCES ............................ iv Page UTILIZATION OF COMPUTER TECHNOLOGY FOR REPRODUCTIVE STUDIES IN NONHUMAN PRIMATES ...................... 56 Abstract .......................... 56 Introduction ........................ 56 Materials and Methods ................... 59 Computer Model ..................... 59 Macaque Reproduction Data Bank ............. 6l Computer Record System ................. 6l Results .......................... 68 Computer Model ..................... 68 Macaque Reproduction Data Bank ............. 68 Computer Record System ................. 7l Discussion ......................... 7l REFERENCES ............................ 74 SUMMARY AND CONCLUSIONS ..................... 77 APPENDICES A. PREGNANCY DIAGNOSIS OF SAIMIRI SCIUREUS ........ 79 REFERENCES ....................... 86 B. COMPUTER PROGRAM FOR MAINTENANCE OF INDIVIDUAL ANIMAL RECORDS IN A NONHUMAN PRIMATE COLONY ...... 87 REFERENCES ....................... 93 C. PUBLICATIONS BY THE AUTHOR ............... 94 VITA ............................... 105 TABLE th IO. ll. l2. l3. T4. 15. LIST OF TABLES Time course of early embryonic development in primates (hours) .................... Composition of in vitro fertilization culture media . . . . Fertility of males used as sperm donors ......... Comparison of follicle aspiration techniques ...... Comparison of recovery, maturation, and fertilization of ova from follicles of varying diameter ........ Follicular volume estimates from aspirated fluid and follicle surface diameters ............... Follicular aspirate components from follicles of various diameters .................... Classification of oocyte quality based on initial observation and potential for maturation ........ Classification of oocytes and potential for fertilization ...................... Effect of oxygen tension on ovum maturation and fertilization ...................... Comparison of medium environment of in vitro fertilization cultures ................. Effects of insulin in the in vitro fertilization system . . Time course of development of in vitro fertilized zygotes ......................... Results of embryo transfer in Shimiri sciureus ..... List of variables used in HYPIT simulation program vi Page 15 2l 25 26 27 29 3O 3l 32 33 33 35 36 38 62 TABLE Page l6. List of variables in protocols for Macaque reproduction data bank .................. 63 l7. Period lengths (minutes) of oscillation of simulated LH patterns using different values for THRES ....... 69 l8. Characteristics of gestation in saimiri sciureus ..... 80 l9. Groups of female squirrel monkeys undergoing pregnancy diagnosis ......................... 82 20. Chorionic gonadotropin assays ............... 84 2l. Comparison of diagnostic methods ............. 84 22. CAR system commands .................... 89 vii FIGURE 10. ll. 12. LIST OF FIGURES In vitro fertilized ova with Z-polar bodies (X 200) Ova fertilized in vitro stained with lacmoid to show chromatin of 2-polar bodies and 2 pronuclei (X 300) Diagrammatic presentation of the ovum deposition technique in the squirrel monkey ............ Flow diagram for simulation of plasma LH concentration in the ovarectomized rhesus monkey ........... Form 1 for male squirrel monkey data .......... Form 2 for culture chamber data ............ Form 3 for female squirrel monkey data ......... Results of simulation of pharmacologic blockade of circhoral oscillations of LH by alteration of THRES variable ........................ Seasonal changes in weight and semen characteristics for laboratory maintained male squirrel monkey ..... Frequency distribution of uterine width dimension used for pregnancy diagnosis .............. Sample interactive session with CAR program ...... Sample CAR system form ................. viii Page 37 37 52 6O 65 66 67 7O 72 83 90 9l INTRODUCTION In vitro fertilization of human ova and subsequent transfer of the human blastocyst into the uterus of a woman, either the ova donor or a foster mother, are coming soon. This procedure may even become clinically routine in the near future. Yet, there is considerable controversy surrounding this area of research. With the current interest in birth defect studies, the possibility of screening cultured embryos before they are reimplanted becomes both terrific and terrifying. Before these procedures arrive, studies into their genetic safety are desirable. Most of the current primate research in this area of reproductive physiology involves human material. Suitable nonhuman primate models have not been developed. Recently saimiri sciureus ova have been fertilized successfully in vitro. Techniques are available to develop this species as such a model. The variables involved in obtaining fertilized ova need to be examined with the goal of providing early preimplantation embryos to test the genetic safety of embryo transfer. Because squirrel monkeys are expensive to maintain, procedures must be developed which maximize their use by recording as much data as possible. With the advent of the computer in biomedical research, a powerful tool exists to manage vast amounts of data. Computer routines can be used to manage large data banks for use by investigators in determining basic reproduction values. The objectives of these investigations were: To develop a laparoscopic procedure for placement of fertilized ova into recipient oviducts, To develop and employ computer routines to manage and analyze data on reproductive phenomena, To further refine procedures and identify important variables for production of in vitro fertilized S. sciureus ova, To transfer fertilized ova to recipient animals. VARIABLES OF IN VITRO FERTILIZATION AND TRANSFER SYSTEMS IN SAIMIRI SCIUREUS Literature Review In Vitro Fertilization in Primates Several reviews have been published in recent years documenting the progress of research on oocyte maturation and fertilization in vitro which lead to successful work on embryo transfer (Chang, l968; Thibault, l969; Austin, l970; Brackett, l970; Kennedy, l972; Edwards and Steptoe, 1975). In light of these publications, the present review will be limited to accounts of in vitro fertilization in primates. The history of studies in primate in vitro fertilization can be divided into three periods: (l) prior to 1951; (2) from l95l to l969; and (3) from l969 to the present. The first period was marked by studies such as the report of in vitro fertilization of guinea pig and rabbit uterian oocytes (Onanoff, 1893). Following the discovery of capacitation in l951, a wealth of basic studies were performed. In vitro fertilization was successfully accomplished in a variety of species, but until 1969 few studies were recorded for primate species. In l969, the present period of in vitro fertilization was entered with the first definitive in vitro fertilization of human eggs (Edwards, Bavister and Steptoe, l969). The first period consists of early accounts of in vitro fertilization which were cast in doubt with the discovery of sperm capacitation by Austin (1951) and Chang (1951). During this period human in nitpo fertilization was first reported by Menkin and Rock (1944, 1948). They observed over 800 oocytes and attempted to fertilize 138. After culturing the oocytes with washed spermatozoa, they obtained 2 two-celled and 2 three-celled embryos. Doubt was cast on these studies as one of the three-celled embryos appeared grossly abnormal and no observation of polar body formation or signs of maturation were reported. The second period in the history of studies of primate in vitro fertilization consists of reports of in vitro fertilization of human ova which have been cast in doubt due to inconsistent methodology, lack of adequate evidence of fertilization, the possibility of parthenogenesis and abnormalities of maturation or fertilization. Early in this period, Shettles (1953, 1955) studied over 1,000 oocytes, but only 200 were subjected to insemination. They were cultured with fresh human sperm in a medium of follicular fluid with bits of tubal mucosa added for capacitation. Six oocytes were reported to have undergone cleavage, one to a 32-ce11ed morula by 72 hours post-insemination. Lack of observation of intermediate stages of development of the morula and signs of maturation of the oocytes cultured have placed some doubt on these studies. Petrucci (1961) reported human in vitro fertilization, but signs of maturation and incidence of either successful culture, fertilization or parthenogenesis were not given. This work has never been published in an acknowledged scientific journal. Hayashi (1963) observed fertilization in 20 of 160 follicular oocytes he cultured in vitro in media with human serum proteins, basal salt solution and hormones. The oocytes were cultured with cumulus cells left intact around them. Although penetration of spermatozoa, formation of pronuclei and cleavage to the morula stage were observed, Hayashi stated cleavage to be morphologically indistinguishable from parthenogenesis. Near the end of this period, investigators began studying in vitro maturation of primate oocytes in order to provide more mature ova for fertilization from the limited number recovered. Several investigators reported in vitro maturation of human oocytes (Edwards, 1965; Kennedy and Donahue, 1969) in either defined (F-10 with 4 mg/ml bovine serum albumin) or undefined (TC-199 with 15 to 20% fetal calf serum) media to the metaphase I or II stage. In 1968, Jagiello, Karnicki and Ryan collected oocytes from women pretreated with gonadotropins (pituitary follicle stimulating hormone (FSH) and human chorionic gonadotropin (HCG)). They found that by 22 hours after HCG administration a third of the oocytes were at metaphase I. 0f oocytes obtained in this manner and cultured for an additional 25 to 28 hours, all resumed meiosis and two-thirds reached metaphase II. Suzuki and Mastroianni (1968) examined the fertility of in vitro matured nonhuman primate oocytes. They matured oocytes from Mucaca mulatta in Waymouth medium with 10% rhesus monkey serum and then transferred them to oviducts of inseminated females. Fifteen of 31 oocytes, which had been matured, transferred, and recovered 24 to 48 hours later, were definitely not fertilized. Eight ova were degenerated, six contained two polar bodies or two pronuclei and two contained two degenerating blastomeres each. The authors suggested that a failure of cytoplasmic maturation in vitro may have contributed to reduced fertility of ova matured in vitro. In 1969 the modern period of studies of primate in vitro fertilization was entered as Edwards, Bavister and Steptoe (1969) reported uncontestable fertilization in vitro of human oocytes matured in vitro. Their technique involved culturing follicular oocytes 35 hours in a variety of media containing follicular fluid, inseminating the cultures with washed spermatozoa and monitoring for pronuclei formation. No pronuclei were seen in control ova, but 11 pronucleate eggs were found in inseminated cultures. Some ova were reported to have as many as five pronuclei suggesting polyspermy. They indicated that extra bicarbonate and follicular fluid were added resulting in a higher pH (7.6) than that previously used for in vitro fertilization of human ova. Fertilization was further verified by identification of the midpiece and tail of the fertilizing sperm in the ooplasm of these eggs (Bavister, Edwards and Steptoe, 1969). The onset of the modern period was characterized by the development of several other successful systems for in vitro fertilization of human ova. In 1969 Jacobson, Sites, and Arias-Bernal reported such a system for in vitro maturation and fertilization of human follicular oocytes. They cultured oocytes in a medium of Hank's solution, 20% fetal calf serum and 5% human follicular fluid. Forty- one oocytes were cultured singly in microdrops, some with about 50 motile sperm. More than 50% of the ova inseminated cleaved and most were found to contain the normal diploid chromosome number. Eight of 11 were female embryos (46XX) and three were male embryos (46XY). All had cleaved to at least the four-celled stage. In 1970 Steptoe and Edwards were able to recover in vivo matured oocytes by laparoscopy from preovulatory follicles of women treated with human menopausal gonadotropin (HMG). Based on this study HMG and HCG were used to stimulate in vivo maturation of human oocytes (Edwards, Steptoe and Purdy, 1970). Laparoscopies were performed 30 to 32 hours after the final HCG injection and 393 oocytes were obtained for culture. The oocytes were cultured l to 4 hours, inseminated and and examined for evidence of fertilization at varying intervals. Several media were tried but Ham's F-lO medium modified to pH 7.6 gave the best results. Forty-five of the pronucleate ova that resulted from insemination went on to cleave. These investigators speculated that follicular fluid or granulosa cells were important for fertiliza- tion. Steroids in the follicular fluid or synthesized by granulosa cells were thought to affect the sperm acrosome and provide for capac- itation in vitro. Seitz et a1. (1971) went a step further by adding sodium estrone sulfate (Premarin) to their medium of Ham's F-lO and heat-inactivated human serum. Their system used human oocytes matured in vitro. Ova were matured for 24 hours in culture, then inseminated with either washed spermatozoa or sperm which were preincubated in a rhesus monkey uterus. Eight of 50 ova in culture with preincubated spermatozoa were fertilized and ranged in development from the two- celled stage at 40 hours to a twelve-celled stage by 72 hours after insemination. None of 15 ova exposed to washed spermatozoa cleaved, but through use of time lapse cinematography, the authors observed penetration of the zona pellucida. They concluded that if capacitation were required for penetration of the human zona pellucida, it could be accomplished in vitro without exposure to fluids from the female tract. In 1972 studies of primate in vitra fertilization were expanded to nonhuman primate species. Following the model of Seitz et al., investigators added estrone to their culture medium in an attempt to in vitra fertilize squirrel monkey oocytes (Johnson et al., 1972; Johnson, 1972). They reported that spermatozoa attached to the zona pellucida of follicular oocytes cultured in tissue culture medium (TC-199) plus 25 pg/ml estrone sulfate with either 20% heat-inactivated squirrel monkey serum or agamma fetal calf serum. 0f four oocytes cultured with spermatozoa, one showed the sperm attachment and two had sperm in their surrounding cumulus cell masses. In another study, Cline et al. (1972) reported fertilization in vitra of in viva matured S. sciureus oocytes. They used a medium of TC-l99 with 20% heat- inactivated agamma newborn calf serum plus bovine serum albumin. 0f 16 mature oocytes recovered from squirrel monkeys pretreated with pregnant mare's serum (PMS), 7 appeared fertile, 5 of which developed to the two-celled stage. In an expanded study, 22 mature ova were utilized in an in vitra fertilization system employing preincubation of ejacu- lated spermatozoa in follicular contents plus medium prior to insemi- nation of test cultures (Gould et al., 1973). Eleven of the 22 ova showed sperm in the perivitelline space, extrusion of the second polar body or pronuclei formation. Six ova went on to cleave to the two-celled stage. Twenty-four oocytes matured in vitva and cultured in this system showed no signs of fertilization, further suggesting a reduced fertility of ova in vitra matured. Kraemer (1973) described in vitra systems for maturation and fertilization of oocytes in yet a third primate, the baboon (Papia cynocephalus). Baboon follicular oocytes were cultured in a variety of media including TC-l99 with 15% calf serum, TC-l99 with baboon serum, follicular fluid, Brinster's medium and 50% follicular fluid with Brinster's medium. The highest percentage of maturation to metaphase II after 48 hours of culture was obtained with 50% follicular fluid- 50% Brinster's medium but no attempt was made to fertilize these oocytes. Instead, follicular oocytes recovered from the ovaries of baboons primed with gonadotropins were cultured in a variety of fer- tilizing media including follicular fluid, Brinster's medium and Bavister's medium. They received sperm which were either fresh, incubated in the uterus or in the cervical mucus. Incubation with sperm lasted 8 hours followed by transfer of the ova to Ham's F-10 with 20% heat-inactivated calf serum for 72 hours. Most of the oocytes were still in the germinal vesicle stage after 72 hours of culture. Four oocytes were fertilized in Brinster's and Bavister's media and were found to have two polar bodies and two pronuclei. One six-celled ovum was recovered after fertilization in Bavister's medium by sperm which had been incubated in cervical mucus. All six nuclei were visible. No other primate species have been successfully in vitra fertilized to date. 10 Recently some investigators of primate in vitra fertilization have more closely examined the in vitra maturation of follicular oocytes and the capacitation requirement of primate sperm. Dukelow and Chernoff (1969) found indirect evidence for a need of capacitation by human and rhesus monkey sperm. This requirement could be at least partially accomplished in a foreign uterine environment (the hamster uterus being most suitable. Soupart has examined both in vitra maturation and sperm capacitation in the development of a system for in vitra fertilization of human oocytes. Soupart and Morgenstern (1973) obtained in vitra penetration by sperm through the zona pellucida and fertilization of oocytes that were recovered from ovaries of gonadotropin-treated women. They used a two-media culture system. The oocytes were first cultured for 38 to 48 hours in a maturation medium containing 75% follicular fluid and 25% modified Bavister's medium (used successfully by Edwards et al., 1970) with or without exogenous gonadotropic hormones (FSH, LH, and HCG). Control ova were cultured in the same fertilization medium without exogenous hormones. The results showed no effect of exogenous gonadotropins in the maturation culture on the proportion of oocytes maturing. Overall, 34% of the oocytes matured to the metaphase 11 stage. The authors found that gonadotropins in the fertilization media did significantly increase sperm penetration into or through the zona pellucida. More oocytes showed sperm penetration than matured, leading the authors to conclude that sperm penetration is not dependent on oocyte maturation but may require sperm capacitation. The enhancement of sperm penetration was thought to be mediated by the gonadotropin ll stimulation of the follicle cells present among the oocytes in the fertilization medium. The authors also observed that the tail of the fertilizing sperm or its remnants could not be found in oocytes recovered 28 hours after insemination, either in the fresh state or after fixing and staining. Bavister et al. (1969) had previously reported observing the midpiece and tail of spermatozoa in human fertilized ova 11 to 14.5 hours after insemination of oocyte cultures with washed sperm. Soupart and Strong (1974) later found bits of degenerating sperm tail in oocytes of the late pronucleate stage, but these were only visible by means of electron microscopy. They also found microscopic evidence that sperm cells passing through the zona pellucida had undergone the acrosomal reaction which may have taken place during contact with follicular cells in the culture medium. Soupart and Strong did not observe sperm penetration through the zona until 5 hours after sperm were exposed to ova and follicular cells in the culture. They pointed out that this delay was consistent with other investigator's reports of not observing sperm presence in the perivitel- line space until 7 to 7.5 hours after insemination (Edwards et al., 1970). The authors concluded that 5 to 7 hours are required for human sperm capacitation in the in vitra fertilization environment. In his most recent paper, Soupart (1975) described a two-stage. maturation culture system for immature human oocytes. He hypothesized that immature oocytes transplanted into a culture medium, enter an environment in which their satellite cells (corona radiata and cumulus oophorus) are much less numerous than in their original follicles. To 12 remedy a possible lack of estrogen and progesterone production which might normally act in regulation of maturation, the oocytes were cultured sequentially in Ham's F-10 medium containing these steroids. First, oocytes were primed in culture with estradiol-l7B (0.1 pg/ml) for 4 hours. Then the oocytes were incubated in the presence of 17a- hydroxyprogesterone (0.1 ug/ml) for 40 to 44 hours. At the end of the 44 to 48 hours of culture, 45% of the immature oocytes were in the metaphase 11 stage. This system may not yet be optimal for in vitra maturation of human oocytes, as Jagiello et a1. (1975) have found maturation of human oocytes in vitra can be stimulated by ovine LH, estradiol-17B (with LH added later), and prostaglandin E]. They also examined rhesus monkey oocytes and found stimulation of in vitra maturation by ovine LH and prostaglandin E1. However, the fertility of these oocytes (Jagiello et al., 1975) was not examined. Soupart was able to achieve 20% fertilization in vitra of oocytes matured by the two-stage method. At the present, these studies seem to indicate that both in viva and in vitra matured primate oocytes can be fertilized in vitra. Contents of ovarian follicles, such as follicular fluid and cumulus cells, which are aspirated along with the oocytes seem to play a role in the capacitation process in vitra. Other variables, such as hydrogen ion concentration, oxygen tension, osmotic pressure, and composition of the culture media (including additions), are probably important to the success of a given in vitra fertilization system. These will be described in the discussion of the results of the present research. 13 The modern period of primate in vitva fertilization with its predominate human orientation, has brought with it the possibility of providing embryos by methods other than in situ fertilization for gestation in infertile women. The relative lack of nonhuman primate models to study this process (and especially the teratogenic potential) first led the author to develop such a system with the squirrel monkey (Kuehl, 1974; Kuehl and Dukelow, 1975). The examination of the vari- ables in this system following the initial development is one subject of this work. Embryo Transfer in Primates Several reviews of embryo transfer as well as a discussion of transfer techniques will be provided in a later portion of this dis- sertation (page 49). This section will be limited to discussion of the specific application of embryo transfer techniques to primates. To date, there are no scientific reports where a primate (human or nonhuman) embryo has been successfully transferred to yield a pregnancy. There have been three published accounts of successful embryo transfer in the popular press. The first was a Wire Services' story of July 17, 1974, quoting Dr. Douglas Bevis of the University of Leeds, England, as stating that 3 of 30 embryo transfers attempted in women had resulted in the birth of live, normal young. The details and subsequent scien- tific publication of this study have not been forthcoming. In another account, Dr. P. C. Steptoe described an ectopic pregnancy following transfer of a human blastocyst to the uterus. The blastocyst was grown in culture from an in vitra fertilized human oocyte. Finally, Dr. D. C. l4 Kraemer (Personal communication, 1975) has transferred an in viva fertilized baboon embryo, by surgical technique, to the uterus of a recipient baboon. A live, normal infant was born at term following this transfer and work is currently underway to verify the parentage. Other than these three accounts, a primate uterus has yet to implant an embryo placed there artificially. Three factors can be differentiated which may affect the outcome of an embryo transfer. These are (l) the stage of development of the transferred embryo, (2) the route of transfer, and (3) the implantation and fetal growth after transfer. All three factors must be considered in getting the birth of a live, healthy infant. Ova and early embryos in various stages of development have been successfully transferred in a number of nonprimate species (reviewed by Chang and Pickford, l969; Dzuik, 1969; Adams and Abbott, 1971; Edwards and Steptoe, 1975). The particular stage of development was not important to success as long as synchrony between the stage of development of the embryo and the endocrine status of the recipient was attained. Table 1 compares the early embryonic development of three species of nonhuman primates and man. While implantation varies from 7 days after ovulation in the human (Hertig, 1975) to 9 days in the rhesus monkey (Heuser and Streeter, 1941) and baboon (Hendrickx and Houston, 1971), the time course of early cell division in these species is the same. Recently, Eddy et a1. (1975) have shown the oviductal transport time of rhesus monkey ova to be 72:12 hours. Thus as the fertilized zygote reaches the uterus, it is in the eight-celled stage, 15 .mmmp . ._.0 HO ocwflcm>gmmao mm; mmmum ovcoxcnso msp cowumcweomcw cmumm mesa; cw we?» mg» mpmowncw enema em sow mmcwucmaa .mzcou: soc» cwcmapw oxcnsm mumowvc_ Acucm ucaocm mmmwzucmcma .uozup>o soc; umgm=_w mm: macaw omcoxgasm mg» cowpmpa>o Lopwm meson =_ ms_u on» oumowccw on»: r» so» mm_gu=m« muep on mmp mmm. cu PF_ ammuv mmm cu Pm mow ou mm mmm v mm.pp osgwa e» euaompvv efiom. as may euAva own com sass as expense 059m qump vv ofimep op ompv ofiowp op mmv UANN op may owe oem cams 2» mzNoxmooemo ommsm gem on ms nu“ o» we awe ou om gem o» «N *wowuma 2» afiwmv am.m~ on»: 2» Bu. 9 UN E 60606: chomp op omv «cams 2» mxmsxwom wgwsvnm umxuoumapm apnea: spgaom ug_;h vacuum “meme anon mm_umam capo; mcowmw>ru mmo>oopo uzouwm Amgaogv mouwspga cw acman—m>mu owcoxgnsm xpgmm eo omgaou «Ewe .p o—amh 16 which is in agreement with the in viva observations shown in Table 1. To be in synchrony, primate transfers of two-polar-body, two-celled and four-celled zygotes should be made to the oviduct of the recipient by 24, 36, and 48 hours, respectively, after ovulation. Embryos of eight or more cells should be transferred to the uterus at or slightly before the time they would normally be found in viva. Transferring a given stage of embryo to the appr0priate site and at the proper time following ovulation, will ensure synchrony of the embryo to the endocrine state of the recipient. The route of transfer refers to the technique used in depositing an embryo in the female reproductive tract. Both surgical and non- surgical techniques have been used in laboratory and domestic animals (see discussion, page 49). In baboons, Kraemer (1975) used a surgical procedure to expose the uterus. Passage through the uterine wall to the lumen was done by needle puncture. A fine catheter was passed through the needle to the uterine lumen. Embryos of more than eight cells were flushed into the lumen in a small volume of medium. Edwards and Steptoe (1975) preferred the cervical approach in women. Their method required only local anesthesia and no puncture of the uterine wall. A catheter was passed directly through the cervix and a blasto- cyst was injected into the uterine lumen. No oviductal transfers have been described in primates. The important consideration with regard to route of transfer is to select the pathway resulting in the least damage to both embryo and the recipient. 17 A final consideration for successful embryo transfer is implantation and development of a normal pregnancy. If the embryo has been transferred in synchrony with the recipient and the route of transfer has not been damaging, a pregnancy should result. Failure of a pregnancy in primates may be due to embryonic death. Recently, Edwards and Steptoe (1975) have described a phenomenon in women which is of particular importance to primate systems of embryo transfer. They reported that histological examination of the uteri of women treated with gonadotropins for the recovery of oocytes revealed the endometrium to be secretory. They suggested that this uterine stimulation may be inadequate for implantation because the interval between administration of HCG and menstruation was shorter in treated women than the interval between the LH surge and menstruation in the normal cycle. They sug- gested that the recipients, which receive gonadotropins for stimulation of follicular growth, be given additional steroid hormones or HCG (to support the corpora lutea) for two weeks following transfer. This procedure might prevent failure of the pregnancy due to a luteal deficiency. This consideration is important as primate recipients are often primed with gonadotropin to provide in viva matured ova or to synchronize them with the embryo to be transferred Materials and Methods Recovery of Gametes from Experimental Animals Both male (7) and female (96) adult squirrel monkeys (S. sciureus) of either Brazilian (Tarpon Z00, Inc., Tarpon Springs, 18 Florida) or Bolivian (Primate Imports Corp., Port Washington, New York) type were used in these studies. The maintenance of these animals has been previously described (Kuehl and Dukelow, 1975). In brief, animals were housed in groups of two to five in stainless steel, flush type cages or in a large group (30 to 50) in a colony cage (Harrison, 1973). Fresh water was supplied ad Zibidvm. The diet consisted of commercial monkey feed (Wayne Monkey Diet, Allied Mills, Chicago, Illinois). The animal quarters had controlled fluorescent lighting on a 12:12 light- dark cycle and was temperature-controlled at 21t2°C. Relative humidity ranged from 40 to 60% dependent on season. Oocytes were recovered from gonadotropin treated females just prior to the time of expected ovulation. Female squirrel monkeys were induced to ovulate without regard to their natural cycle using a regime previously reported (Dukelow, 1970) with modifications during the anovulatory season (Kuehl and Dukelow, 1975). The regime consisted of four daily intramuscular injections of follicle stimulating hormone (FSH) (1 mg, FSH-P, Armour-Baldwin Laboratories, Omaha, Nebraska) with an intramuscular injection of 500 iu of human chorionic gonadotropin (HCG) (A.P.L., Ayerst Laboratories, New York, New York) on the morning of the fifth day. From June 15 to October 15 of each year (anovulatory season) a fifth FSH injection was added. Neither progesterone nor estrogen injections were used in this regime. Oocytes were recovered by aspiration from the follicles. Two aspiration procedures were used. The first was a surgical procedure, laparotomy, used to expose the ovaries for aspiration using a 1 ml tuberculin syringe containing 19 0.05 to 0.10 ml of culture medium. As described previously (Kuehl and Dukelow, 1975), all visible follicles were punctured laterally with a 25 gauge needle (bevel side down) and the contents drawn into the The second procedure used the laparoscope to observe the syringe. Follicles observed in this way were ovaries (Dukelow et al., 1971). measured and classified according to stage of develOpment. Aspiration was as stated above, except that the needle was inserted through the skin and abdominal wall to reach the ovaries. Follicles were aspirated as early as 4 hours and as late as 56 hours after HCG administration but usually 8 to 12 hours after HCG. Spermatozoa were recovered from male squirrel monkeys by electroejaculation using the method and restraint system described by Kuehl and Dukelow (1974). The ejaculate, in the form of coagulum, was incubated in 1 to 2 m1 of culture medium at either room temperature (23°C) or 37°C for 0.2 to 2 hours. The resulting sperm suspension was aspirated from the sample tube containing the coagulum and used to inseminate oocyte cultures. M Environment The culture environment consisted of four components: (1) CUT ture vessel, (2) temperature, (3) atmosphere, and (4) medium. One Cu ‘I ture vessel, a chamber-slide (tissue culture chamber/slide, No. 4808, Lab-Tek Products, Naperville, Illinois) with eight chambers, was Used throughout the course of these studies. The incubation te"‘PE\"a‘t‘.ure was maintained at 37 to 38°C. The area used for gamete conection and culture examination was maintained at or above 30°C 20 during all procedures. Five culture atmospheres were examined. All test atmospheres contained 5% carbon dioxide, with variable proportions of moist oxygen (0, 10, 20, 30, and 95%). Nitrogen was used to complete the balance. Based on these results, an atmosphere of 5% carbon dioxide in air (20% oxygen) was used in all the other studies. Five different media were tested in the culture system. Table 2 compares the composi- tion of these media. Gametes were incubated in only one medium throughout a trial period. Transfer of Fertilized Ova to Experimental Animals Mature oocytes, fertilized in vitra, were transferred to the oviducts of recipients using the procedure described on page 51. The recipients were synchronized with the embryo donors by the ovulation induction procedure. All transferred embryos (2 polar body--2 pro- nucleate to four—celled zygotes) were deposited in the oviduct of the recipient ipsilateral to the ovary with a corpus luteum. Following the transfer, some recipients received either HCG (50 iu) or proges- terone (5 mg, A 4-pregnen-3, 20-dione, Sigma Chemical Company, St. Louis, Missouri). A11 recipients were dianosed for pregnancy using the procedures discussed in Appendix A. Experimental Design Variables of the in vitra fertilization system were examined by conducting multiple trials. A trial consisted of the recovery of male and female gametes, the incubation period with several samples, and a transfer procedure when ova were fertilized and a recipient 21 Table 2. Composition of in vitra fertilization culture media Media composition Ingredient TJK-l TJK-2 TJK—3 TJK-4 TJK-5 TC-l99*a 80% 80% 100% 100% Ham's F-10*b 80% Serum*c 20% 20% 20% BSA”d 6 mg 6 mg Pyruvate**e 72 ug 72 ug 72 ug Penicillin**f 50 units 50 units 50 units Pen-strep“g 100 units 100 units Heparin**h 1 unit 1 unit 1 unit 1 unit 1 unit *Percent of total volume. **Units per ml. aMedium 199 with Earle's salts and L-glutamine, North American Biologicals, Inc., Rockville, Maryland. bHam's F-lO with L-glutamine, North American Biologicals, Inc., Rockville, Maryland. cAgamma newborn calf serum, North American Biologicals, Inc., Rockville, Maryland. dCrystallized albumin from bovine serum, Sigma Chemical Co., St. Louis, Missouri. eSodium pyruvate, Sigma Chemical Co., St. Louis, Missouri. fPotassium penicillin G, Calbiochem, San Diego, California. gPenicillin-dyhydrostreptomycin mixture, North American Biologicals, Inc., Rockville, Maryland. hSodium heparin, Wolins Pharmacal Corp., Melville, New York. 22 female was available. Variables were altered one at a time over the course of several trials. A series of trials was conducted for each variable with all the values of the variable to be compared. The series of oxygen tensions, for example, had five trials per series. Within a trial, treatments were examined, with each treatment occupying a separate chamber. Chambers with medium alone, medium plus spermatozoa suspension, and medium plus follicular aspirate (no sperm) served as controls for pH measurements, sperm motility and fibroblasting of cumulus cells noted during an incubation period. During the course of these studies, other investigators suggested that addition of insulin to the culture medium would improve oocyte maturation and fertilization. The three levels tested (0, 10, and 100 m.i.u./m1, crystalline bovine pancreatic insulin, Sigma Chemical Co., St. Louis, Missouri) were added to chambers within a trial in 5 ul of 0.15 molar sodium chloride, thus constituting three treatments within a trial. The females used for trials were randomly assigned and treated with an equivalent ovulatory regime of gonadotropins throughout the year to minimize seasonal effects. Males were not controlled for seasonality, but sperm concentrations were maintained at 2.0:1.0 X 106 spermatozoa per ml for the 0.5 m1 culture volume. Effects due to male differences in fertility were minimized by using sperm from only one male per series of trials. Treatments within a trial, of course, received the same sperm suspension. The data from each trial was placed on computer file (see discussion of DAS system, page 61) by experimental unit. For example, 23 an appr0priate data form was completed for each male, female, and culture chamber. Listings were compiled from this data bank. As maturation, fertilization, and fibroblasting of cumulus cells are dichotomous measurements, Chi-square with Yates' correction for discrete data was used to analyze the results (Spiegel, 1961). Culture effects on maturation were isolated from other effects by examination of the number of ova maturing in vitra. Stage of development of an ovum was determined before and during in vitra culture by microscopic observation of the chamber slide. At the end of the culture period (50 to 80 hours), the stage of development was confirmed by staining with lacmoid (Iwamatsu and Chang, 1972). For ova to be selected for transfer, the following criteria for fertilization were used: 1. Two or more polar bodies present in the perivitelline space and two pronuclei within the ooplasm. 2. Two or more polar bodies present in the perivitelline space; two equal sized blastomeres. 3. Two or more polar bodies present in the perivitelline space; four equal sized blastomeres. 4. Five or more blastomeres of sizes that could be associated with cleavage type divisions. Criteria two through four had to be verified by several observations to ensure that fragmented ova were not mislabeled as multicelled embryos. Where a culture chamber contained more than one oocyte, the relative positions of each of the oocytes were noted. Thus an individual oocyte could be followed throughout the culture period. 24 Results A total of 745 oocytes were recovered from 2,168 follicles for a 34.4% recovery rate. Of these oocytes 137 (18.4%) were classified as atretic. These oocytes showed either darkening, vesicularization and F shrinkage of the ooplasm or uneven division of the ooplasm (fragmen- tation) when first observed in culture. They were not considered capable of maturation, leaving 608 oocytes with the possibility of reaching the metaphase II stage. Thirty-eight percent did, and 78 of these were fertilized in vitra by spermatozoa from one of the five males listed in Table 3. There was a significant (p<:0.05) difference in the fertility of the males. 0f the 46 mature ova not exposed to sperm, none showed any nuclear formations suggestive of parthenogenesis and none cleaved. A comparison of the techniques of ovum recovery is shown in Table 4. No significant differences (p<:0.05) were found for any of the comparisons between laparoscopy and laparotomy. While there were no differences in oocyte recovery, atresia, and maturation between aspiration techniques, considerable differences were found among groups of ova from follicles of different sizes. These results are shown in Table 5. There was a significant (p<<0.05) differ- ence in the recovery rates from the various follicles, with 1 mm diam- eter follicles yielding a significantly higher proportion of atretic ova. The fertility of oocytes recovered from the various sized follicles was similar except for those from the 1 mm diameter follicles. There was also a tendency for larger follicles to yield oocytes that would mature to the metaphase I stage or beyond in culture. Other 25 Table 3. Fertility of males used as sperm donors Number of Number of ejaculates mature ova Number Male used inseminated fertilized* Percentage DUKE 41 100 40 40.0 MICK 11 36 24 66.7 1399 7 13 1 7.7 1400 11 34 12 33.3 HIRO 1 4 1 25.0 Total 71 187 78 41.7 *Significant difference (p<<0.05) when analyzed by X2 method for male. 26 m.mm mmm e.m_ NmF v.em men mop.m Pouch “.mm Nep w.m_ mm o.¢m mow oo¢._ zeopocmqmo m.m¢ Fm N.m_ mm ~.mm mum mom xaoumoemaoo mmuxooo .oz mmuxuoo .oz mmpuwFFow .oz umpmswamm mzcwcnum» owpmcpmco: eo N we R we & m_UWPPoe eo .oz m>o umezpmz mmpxooo o_umsu< >L¢>oomc mpxuoo mmzcwcgomp cowpmewamm m_uPF_oe we com_emneou .e e_nee 27 use: we um>emm umezums NmF po mpv sewam op cmmoaxm m>o umgzpme xpco mmuzpucw cmgzpms eo “cause; .mmem mPowFPoe cacao com agammms mEmm ace» Amo.ouvav pavememwc xppcmquwcmwmas .mNEm m_uw__om to» nocams Nx xa nm~»_m:m can: Amo.ouvav wucmgmmewu ucmuwewcmwma .Ampocucou noumcw5mmcw n .uwssume new m:_gapme soon mmuz_och “.mp m_\m _.m¢ mm ato.mm mm “.mm omm\m~ F o.om om\mF «.mm mm m.up em m.mm F~m\um_ N ¢.o¢ om\m~ _.mm mm N.mp om N.P¢ mom\mmp m Ameou goes as «-mv _.Nm u\¢ o.mn NF _.PP N P.Nm mm\mp xsoumpz>owca vacuums 2:32. 059:? .oz mmuzooo .oz .93 . Pow 37: F Pom A55 mo a \mpwasmm ico: mo & mo & »o N \mwuxuoo cmqumen empauwF_ou m>o memea m>o mcwczumz mwuzooo owpmsp< exem>oumc muzuoo 1.. . e emumEmwu mcwxcw> mo mmFQWFFom soc» m>o mo cowumNTprgow can .cowumgzums .xgm>oome mo comwgmaeou .m mFQMH 28 differences between these follicles are considered in Tables 6 and 7. Only data from successful aspirations (yielding ova) were included in these tables. Other than follicle surface diameter, the actual physical appearance of the oocyte can be used for classification. Soupart and Morgenstern (1973) postulated the classification system shown in Table 8, which groups oocytes by the appearance of their cellular investments. Three mature ova were classed as atretic, however lacmoid staining revealed metaphase II chromatin configuration. The proportion of oocytes beginning to mature (metaphase I and beyond) was found to vary significantly (p<:0.05) between the classes of ova. The predominant ovum type seen in culture was termed "probable nonovulatory." Table 9 examines in vitra maturation and fertility of these oocytes. Again, there were significant differences between classes of ova for both in vitra maturation and fertilization. In all categories the probable preovulatory oocytes rate the highest. The title "preovulatory" assigned by Soupart and Morgenstern may be a misnomer as these ova were recovered from all four follicle sizes described earlier. The effects of changes in the components and environment of the culture are best examined by considering in vitra maturation and fertil- ization. Table 10 shows the results of the oxygen tension trials. In vitra maturation was highest for cultures maintained in the 20 and 30% oxygen atmosphere. Although numbers were low, fertilization was found to occur in all but one atmosphere. Studies of oxygen tension were conducted using medium TJK-2. In different trials, the five media were compared (Table 11) in the 20% oxygen atmosphere (5% carbon dioxide 29 Table 6. Follicular volume estimates from aspirated fluid and follicle surface diameters Follicular Calculated Fluid aspirated Diameter diameter fluid pl/follicle calculated (mm) volumea (N)b (mm)c Preovulatory 33.51 26.0 (11) 3.7 (3-4 mm with dome) 3 14.14 11.7 (59) 2.8 2 4.19 5.0 (46) 2.1 1 0.52 1.8 (31) 1.5 aIn ul/follicle: assuming sphere formula (Volume==4/3 n radius3) using aspirated follicle diameter (4 mm diameter used for preovulatory calculation). b(N) = number of aspirates. cAssuming sphere formula, given aspirated fluid volume. 30 Table 7. Follicular aspirate components from follicles of various diameters Cell indicesa Aspirates Follicular containing gel diameter Follicle Red blood (mm) cells cells No./total (%) Preovulatoryb 3.0C 6.2 5/11 (45) 3 3.5 5.9 24/59 (41) 2 3.3 5.5 7/46 (15) l 2.4 4.9 1/31 (3) aLogarithm to base 10 of number of cells (i.e., 103 cells, index==3.0). bThree to 4 mm with dome (preovulatory formation). cMean per follicle aspirated. .muxooo mo maxu Low uocpms Nx xn vm~zpmcm cog: Amo.ouvav mocmgmeewn pcmowmw:m_me .mmuxooo vacuums ecu mcwczuwe soon mmuzpoch 31 m.me vow 0.00, va Peach umWFQQm >meoop ccm mFFmo comm AFchowmmooo umuczoc mFmem> chwEme umwpagm .xxowpm >L0pmpz>omsa m.qo om “.mm eqP .m_nm>cmmno cog: xpmmooo .m>wm:mpxm mpnmaoca “commea mpowmm> chwscmm mmewpmeom xcopm—:>o:oc m.om __F o.nq mm_ mpnm>cmmno can: memxmp pomasou mcoz mpnmnosa covampcmEmmcw Lo mmwxcwczm Lo\ucw Fouxo maocmmoeo; -co: .cowuonempzo ovumcum Lo m.m m m.m_ om iwmm> .mc_cmxsmo 2mm so mcoz mcoz mumsmcmmma “xv .oz Faye» mmpxuoo mamepw> mF—mo convene mmms mpxuoo «mmpxuoo mmcwsspmz mo N mcocou m=_=E:o mo max» cowpmcapme com mepcmpoa ncm cowum>cmmno pmmuwcw co comma xuw—mzc mHXUoo wo cowpmoww_mmm_u .m open» 32 Table 9. Classification of oocytes and potential for fertilization Type of Matured Matured Fertile/ oocyte No.a in vitra* Percentage matured* Percentage Degenerative or atretic 3 O 0 0/3 0 Probable nonovulatory 80 43 53.8 20/66 30.3 Probable preovulatory 75 69 92.0 35/68 51.5 Total 158 112 70.9 55/137 40.1 aTwenty-one matured ova not inseminated. *Significant difference (pi<0.05) when analyzed by X2 method for type of oocyte. 33 Table 10. Effect of oxygen tension on ovum maturation and fertilization Percentage Ovum maturation Fertilization 02 1" Mature/ Fertilized/ atmosphere nonatretic Percentage mature Percentage 0 4/18 22 1/4 25 10 4/12 33 1/4 25 20 41/87 47 18/41 44 30 6/13 46 0/6 0 95 1/9 11 1/1 100 Table 11. Comparison of medium environment of in vitra fertilization cultures In vitra matured Fertilized Total Total Number/ Fertilized/ Medium cultures oocytes testa Percentage maturedb Percentage TJK-l 46 76 16/51 (31.4) 2/11 (18) TJK-2 63 135 37/93 (39.8) 19/38 (50) TJK-3 41 58 26/46 (56.5) l7/23 (74) TJK-4 28 44 11/26 (42.3) 6/13 (46) TJK-5 22 26 7/17 (41.2) 3/6 (50) aNonmature, nonatretic oocytes at culture start. bMatured (metaphase II) ova exposed to motile sperm. 34 in air). Medium TJK—3 gave the greatest in vitra maturation and fertilization. Osmolarity of this medium at 38°C after equilibration with the 20% oxygen atmosphere was 311 m Osm/kg. The pH of all five media remained between 7.3 and 7.5 throughout the culture period. Medium TJK-3 was used in the study of insulin in the medium. The results are shown in Table 12. While insulin did not increase in vitra maturation or fertilization, it did significantly depress the formation of fibroblast monolayers in culture chambers with more than 1,000 follicle cells. In the course of these experiments, the serial observations of individual fertilized ova were used to construct a time course of early embryonic development in vitra (Table 13). Observations of individual zygotes immediately before and after a change in developmental status were pooled by two-hour intervals following insemination. The median intervals for each stage through the eight-celled zygote are shown. Only 23% of the 2-polar body, 2-pronucleate zygotes cleaved during the in vitra culture. Lacmoid staining of noncleaving fertilized zygotes showed the expected chromatin configurations. Figure 1 shows such a 2-polar body, 2-pronuc1eate zygote. At the end of the culture period, this zygote was stained (Figure 2) and both polar bodies and pronuclei were found. Thirteen zygotes were transferred to recipient females prior to the end of culture. Table 14 provides a summary of these procedures. Recipient 1321 received a 2-polar body, Z-pronucleate zygote 34.1 hours after an ovulatory injection of HCG. On day 20 (18.5 days after the transfer) the corpus luteum was still present 35 .cwpamcm eo muomwwm toe cosams Nx xa umechm cog: Amo.onvgv mocmsmewwu uchwewcmwma .mcapPau cw mpwuoe mew: Esmgm open: vwgapme was“ m>o apco cmuapucH a .Aumuapucw #0: ago; weep _mwuwcw an awesome m>ov m>o owpmgumco: xpco umuzpoch 2 2: mm 25 3 a; :53... 8: =w_:m=H mm 23 8 m: om 83 253:. o: =PF=mcH mm mm\mm em m~\mp mm c¢\mm Focpcoo mumpcmocma «quop\gmampocoe mmmucmogwa weaves mmmucmugma moppmspm mewscom meansmgu 9 \.oz icoc\.oz mppmu mcwumm_nocnwa xuwpwusmm cowumsapme Ez>o Empmxm cowpmNVFPucmw capes me any cw :w_:mcw we muommmm .~_ mpnme 36 Table 13. Time course of development of in vitra fertilized zygotes Developmental stage of 50% of zygotes (hours after insemination) No. of Stage zygotes Earliest Observed 2-polar body- 2-pronucleate 78 6 22 2-celled 18 20 40 4-celled 8 46 52 8-celled 6 52 72 aThe earliest possible time after insemination that 50% of the zygotes could be at the given stage. bThe observed time, after insemination, that 50% of the zygotes had reached the given stage. 37 Figure 1. In vitro fertilized ova with 2-polar bodies (X 200). Figure 2. Ova fertilized in vitro stained with lacmoid to show chromatin of 2-polar bodies and 2 pronuclei (X 300). r. u. {i 0.. o I . . 1 .v .0 I. s. in a. . t o \ . . . . .a.:. . . C . 9m 1 . w . .1 l .w . . .k f.v-I ~. v Table 14. 38 Results of embryo transfer in Saimiri sciureus Genetic Parent S Recipient Time of Stage Female Male female transfer transferred Ovary Treatment 1393 DUKE 1308 9.8 2PB—2PN PRE CS 1378 MICK 1404 10.6 2PB-2PN PRE HCG 1378 MICK 1399 11.5 2PB-2PN PRE HCG 1321 MICK 1366 22.5 2PB-2PN CH None 1384 DUKE 1321 34.1 2PB-2PN CL None 1367 MICK 1367 35.6 2PB-2PN CL None 1374 MICK 1374 35.9 2PB-2PN CL None 1391 MICK 1362 38.5 2PB-2PN CL None 1374 MICK 1360 36.1 2-ce11 CL HCG 1374 MICK 1312 36.4 2-ce11 None PROG 1361 DUKE 1312 37.7 2-ce11 CL None 1361 DUKE 1323 56.2 2-cell CL None 1303 DUKE 1302 71.6 4-ce11 CL None aHours after Key: PB PN ('3 r 11 II II II II II II II ovulatory HCG administration to recipient. Polar body Pronucleus Preovulatory follicle Corpus hemorrhagicum Corpus luteum Cervical stimulation 50 i.u. injection on day four 5 mg progesterone daily. 39 and the uterus was ballooned but measured only 6 mm in width. A mouse uterine-weight assay, performed on the plasma, was negative for gonad- otropin. Five days later a confirmatory laparoscopy was conducted and the corpus luteum was not observed. While at least two other recipients presented signs of pregnancy, none were confirmed. Discussion With the use of in vitra fertilization system described above, fertilized squirrel monkey ova have been obtained. This system can provide a supply of early preimplantation embryos to permit the exam- ination of the genetic safety of embryo transfer procedures in a primate model. These embryos could also be used in teratological testing to examine the hypothesis that preimplantation embryos are relatively resistant to teratogenic agents (Austin, 1973). However, ova fertilized in vitro are the result of a multi-step process that starts with an ovarian follicle and ends with the fertilized ovum in culture. Attrition during the procedure is high. The present results should provide for improvement of this condition in the future. By the extensive use of laparoscopic procedures, one can maximize the usage of valuable animals, a point especially pertinent with the current shortage of nonhuman primates for research. Laparo- scopic recovery of oocytes does not alter the normal development of the ovum. This finding confirmed the related human work of Edwards et a1. (1969), and Morgenstern and Soupart (1972). Such procedures avoid extensive adhesions while providing for greater comfort to the animals. 40 An added advantage is that the aspiration procedures can be repeated many more times with laparoscopy, at least providing for an increase in the number of follicles aspirated and confirming similar observation in the Cynomologus Macaque (Dukelow and Ariga, 1976). The size of the follicles (surface diameter) aspirated does affect the recovery. The larger follicles have more follicular fluid and more loosely confined oocytes, enhancing the recovery rate. Pre- ovulatory follicles offer an exception to this rule. A higher propor- tion of aspirations from preovulatory follicles contain gelatinous or viscous fluid which blocks the needle making recovery of the oocyte more difficult. The maturity and fertility of these ova is high as would be expected if they were to be ovulated. The data presented for collection of red blood cells in the aspirated fluid indicates an increased vascularity and fragility of capillaries in the vicinity of the follicle as it increases in size. These cells do not affect the fertility in vitra (Kuehl, 1974) but do make observation of oocytes more difficult during the culture period. Follicle cells are diluted by a factor of ten when placed in the culture chamber. Smaller chamber volumes would decrease this dilution and perhaps improve maturation and fertility as Brinster (1970) has reported for mouse ova. Soupart (1975) has suggested that dilution of follicle cells is a cause of decreased ovum maturation in vitra. These results indicate in vitro maturation of squirrel monkey oocytes is at the same level as that achieved by Soupart's use of a two-stage maturation medium, so extra steroid supplementation of the medium may not be necessary to achieve maturation. 41 Soupart and Morgenstern (1973) proposed a classification system for human oocytes (shown in Table 8). They estimated potentials of nil, good, or excellent to describe oocytes that initially appear degenerate, nonovulatory or preovulatory, respectively. The results for the squirrel monkey confirm their estimates and further suggest similar potentials for the in vitra fertility of aspirated ova. The majority of the ova that mature in the present system do so in vitra. This further emphasizes the importance of the culture environment for maturation of oocytes. Two requirements for mammalian fertilization (Soupart and Strong, 1975) are the availability of mature oocytes (those having achieved the metaphase II stage of meiotic division) and the availability of capacitated sperm. The present system supports oocyte maturation as demonstrated by the finding of lacmoid-stained chromatin in the first polar body and a metaphase spread in the ooplasm. Spermatozoa have also been observed in the perivitelline space. Thus, the system supports in vitra capacitation. Soupart and Strong (1974) define three criteria as convincing evidence of fertilization short of a successful transfer of an in vitra fertilized embryo and birth of an offspring. These are (1) the presence of two or more polar bodies in the perivitelline space, (2) the presence of two pronuclei within the ooplasm, and (3) the presence of remnants of the fertilizing sperm midpiece and tail within the ooplasm. The first two of these criteria have occurred in this system. Furthermore, the development of the fertilized squirrel monkey ovum follows a similar time course through the first, second, and third cleavage divisions to the human in vitra 42 fertilized embryo in culture (Edwards, 1973). However, we were unable to find conclusive evidence of the fertilizing sperm tail and midpiece in these fertilized ova. Soupart and Strong (1974) have reported a progressive dismantling of the midpiece and tail of the fertilizing sperm in the late pronucleate stages of human in vitra fertilized ova, so that remnants were visible only by means of electron microscopy. As the ova in this system were cultured for 48 hours or more before fixation and staining, a similar phenomenon may account for the inability to observe the remnants of the fertilizing sperm. The culture environment may affect both in vitra maturation and fertility in this culture system. The results of study of the effect of oxygen tension on maturation and fertilization are in contrast to the rabbit work of Brackett (1966) who found optimal results in cul- ture media having a low oxygen tension but the difference is perhaps explained by the very moist atmosphere of our culture incubator. Brackett and Williams (1965) did report a high (73%) rate of fertil- ization when the relative humidity was 96 to 97%. Very high levels of oxygen (95%) restricted maturation although the one ovum maturing was ultimately fertilized. Edwards (1975) recommends a low level of oxygen (5%) for human in vitra fertilization as does Soupart (1975). This lower level is not required in the squirrel monkey system in the 0.5 ml chamber. The medium which gave the best results contained serum with added pyruvate (TJK-3) using a medium 199 base. Ham's F-lO medium contains pyruvate in the formulation, but in these trials gave the poorest results. When insulin was added to the medium, no enhancement 43 of fertilization was observed. Soupart's (1975) contention that additions of insulin (10 m.i.u./m1) to the medium favor follicle cell maintenance is contraindicated by the decrease in monolayer formation shown in Table 12. The results from the transfer experiments have not, as yet, been positive. Squirrel monkeys have supported normal pregnancies following induction of ovulation (Jarosz and Dukelow, 1976). The technique of placing the zygote in the oviduct has proven to be successful. Three females were given a low dose of HCG to stimulate luteal tissue (Surve et al., 1973). Another received cervical stimu- lation and one was given progesterone. None of these treatments resulted in a pregnancy. REFERENCES Adams, C. E., and M. Abbott. 1971. Bibliography on recovery and transfer of mammalian eggs and ovarian transplantation. Reproduction Research Information Service Ltd., Cambridge. Ariga, S. 1976. Personal communication. 1976. Austin, C. R. 1951. Observations on the penetration of the sperm into the mammalian egg. Austr. J. Sci. Res. B4: 581-597. Austin, C. R. 1970. Sperm capacitation, biological significance in various species. In Advances in the Biosciences, 4th Schering Symposium on mechanisms involved in conception. Edited by G. Raspe. Pergamon Press and Viewing, New York. Austin, C. R. 1973. Embryo transfer and sensitivity to teratogenesis. Nature 244 : 333 . Avendano, S., H. D. Croxatto, J. Pereda, and H. B. Croxatto. 1975. A seven-cell human egg recovered from the oviduct. Fertility Sterility 26: 1167-1172. Bavister, B. 0., R. C. Edwards, and P. C. Steptoe. 1969. Identification of the midpiece and tail of the spermatozoan during fertilization of human eggs in vitra. J. Reprod. Fart. 20: 159-160, 1969. Brackett, B. G. 1966. Biochemistry of in vitra capacitation and in vitra fertilization, Ph.D. dissertation, University of Georgia, Athens. Brackett, B. G. 1970. In vitra fertilization of mammalian ova. In Advances in the Biosciences, Fourth Schering Symposium on Mechanisms Involved in conception. Edited by G. Raspe. Pergamon Press and Viewing, New York. Brinster, R. L. 1970. In vitra cultivation of mammalian ova. In Advances in Bioscience, Fourth Schering Symposium on Mechanisms Involved in Conception. Edited by G. Raspe. Pergamon Press and Viewing, New York. Chang, M. C. 1951. Fertilizing capacity of spermatozoa deposited in the Fallopian tubes. Nature 168: 697-698. 44 45 Chang, M. C. 1968. In vitra fertilization of mammalian eggs. J. Anim. Sbi., suppl. 1, 27: 15. Chang, M. C., and S. Pickford. 1969. Egg transfer in the laboratory animal. In The Mammalian Oviduct. Edited by E. S. E. Hafez and R. J. Blandau. University of Chicago Press, Chicago. Cline, E. M., K. G. Gold, and C. W. Foley. 1972b. Regulation of ovulation, recovery of mature ova of the squirrel monkey (saimiri sciureus). Fed. Prac. 31: 360. Croxatto, H. B., S. Diaz, 8. Fuentealga, H. D. Croxatto, D. Carrillo and C. Fabres. 1972. Studies on the duration of egg transport in the human oviduct. I. The time interval between ovulation and egg recovery from the uterus in normal women. Fertility Sterility 23: 447-458. Dukelow, W. R., and H. N. Chernoff. l969. Primate sperm survival and capacitation in a foreign uterine environment. Am. J. Phys. 216: 682-686. Dukelow, W. R. 1970. Induction and timing of single and multiple ovulations in the squirrel monkey (Saimiri sciureus). J. Reprod. Part. 22: 303-309. Dukelow, W. R., S. J. Jarosz, D. A. Jewett, and R. M. Harrison. 1971. Laparoscopic examination of the ovaries in goats and primates. Lab. Animal Science 21: 594-597. Dukelow, W. R., and S. Ariga. 1976. Laparoscopy in nonhuman primates. In Laparoscopy (in press). Williams and Wilkins. Dzuik, P. J. 1969. Egg transfer in cattle, sheep and pigs. In The Mammalian Oviduct. Edited by E. S. E. Hafez and R. J. Blandau. University of Chicago Press, Chicago. Eddy, C. A., R. G. Garcia, 0. C. Kraemer and C. J. Pauerstein. 1975. Detailed time course of ovum transport in the rhesus monkey (Macaca mulatta). Biol. Reprod. 13: 363-369. Edwards, R. G. 1965. Maturation in vitra of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature (London) 208: 349-351. Edwards, R. G., B. D. Bavister, and P. C. Steptoe. 1969. Early stages of fertilization in vitra of human oocytes matured in vitra. Nature (London) 221: 632-635. Edwards, R. G., P. C. Steptoe, and J. M. Purdy. 1970. Fertilization and cleavage in vitra of preovular human oocytes. Nature (London) 227: 1307-1309. 46 Edwards, R. G., and P. C. Steptoe. 1975. Physiological aspects of human embryo transfer. In Progress in Infertility. Edited by S. J. Behrman and R. W. Kistner. Little, Brown and Company, Boston. Gould, K. G., E. M. Cline and W. L. Williams. 1973. Observations on the induction of ovulation and fertilization in vitra in the squirrel monkey (Saimiri saiureus). Fertility and Sterility 24: 260-268. Harrison, R. M. 1973. Ovulation in saimiri sciureus induction, detection, and inhibition. Ph.D. Thesis, Michigan State University. Hayashi, M. 1963. Fertilization in vitra using human ova. Prac. VII Conference Inter. Planned Parenthood Fed. Singapore, pp. 505-510. Hendrickx, A. G., and M. L. Houston. 1971. Description of stages IV, V, VI, VII, and VIII. In Embryology of the Baboon. Edited by A. G. Hendrickx. University of Chicago Press, Chicago. Hertig, A. T. 1975. Human stages in implantation. In Progress in Infertility. Edited by S. J. Behrman and R. W. Kistner. Little, Brown and Company, Boston. Heuser, C. H., and G. L. Streeter. 1941. Development of the macaque embryo. Cbntr. Embryal. Garney. Instn. 29: 17-228. Iwamatsu, T., and M. C. Chang. 1972. Sperm penetration in vitra of mouse oocytes at various times during maturation. J. Reprod. Fert. 31: 237-247. Jacobson, C. B., J. G. Sites and L. F. Arias-Bernal. 1970. In vitra maturation and fertilization of human follicular oocytes. Inter. J. Fert. 15: 103-114. Jagiello, G., J. Karnicki and R. J. Ryan. 1968. Superovulation with pituitary gonadotrophins. Method for obtaining meiotic menaphase figures in human ova. Lancet. 1: 178. Jagiello, G., M. Ducayen, W. Miller, J. Graffeo and J. S. Fang. 1975. Stimulation and inhibition with LH and other hormones of female mammalian meiosis in vitra. J. Reprod. Fert. 43: 9-22. Jarosz, S. J., and W. R. Dukelow. 1976. Temperate season outdoor housing of saimiri sciureus in the northern United States. J. Med. Prim. (in press). 47 Johnson, M. P. 1972. Oviduct fluid and its effect on the fertilizing ability of capacitated rabbit sperm and studies on in vitra fertilization. M.S. Thesis. Michigan State University, East Lansing, Michigan. Johnson, M. P., R. M. Harrison, and W. R. Dukelow. 1972. Studies on oviductal fluid and in vitra fertilization in rabbits and nonhuman primates. Fed. Prac. 31: 369. Kennedy, J. F., and R. P. Donahue. 1969. Human oocytes: maturation in chemically defined medium. Science 164: 1292-1293. Kennedy, J. F. 1972. Maturation and fertilization of human oocytes in vitra. In Oogenesis. Edited by J. D. Biggers and A. W. Schuetz. University Park Press, Baltimore. Kraemer, D. C., and A. G. Hendrickx. 1971. Description of stages I, II, III. In Embryology of the Baboon. Edited by A. G. Hendrickx. University of Chicago Press, Chicago. Kraemer, D. C. 1973. Personal communications. Kraemer, D. C. 1975. Personal communication. Kuehl, T. J., and W. R. Dukelow. 1974. A restraint device for electroejaculation of squirrel monkeys (Saimiri sciureus). Lab Anim. Sci. 24: 364-366. Kuehl, T. J. 1974. In vitro fertilization in Saimiri sciureus. M.S. Thesis. Michigan State University. Kuehl, T. J., and W. R. Dukelow. 1975. Ovulation induction during the anovulatory season in saimiri sciureus. J. Med. Primatalogy. 4: 23-31. Kuehl, T. J., and W. R. Dukelow. 1975. Fertilization in vitra of saimiri sciureus follicular oocytes. J. Med. Primatalagy 4: 209-216. Lewis, W. H., and C. G. Hartman. 1933. Tubal ova of the rhesus monkey. Cantri. Embry0., No. 180, pp. 9-14. Menkin, M. F., and J. Rock. 1948. In vitra fertilization and cleavage of human ovarian eggs. Am. J. Obstet. Gynecol. 55:440-452. Onanoff, M. J. 1951. C. R. see. Biol., Paris, 5: 749, 1893 (as cited by S. U. Smith); Fertilization in vitra of the mammalian egg. Biochem. Soc. Symp. 7: 3. Petrucci, D. 1961. Producing transplantable human tissue in the laboratory. Discovery 22: 278 (as cited by Kennedy, 1972). 48 Rock, J., and M. F. Menkin. 1944. In vitra fertilization and cleavage of human ovarian eggs. Science 100: 105-107. Seitz, H. M., G. Rocha, B. G. Brackett and L. Mastroianni. 1971. Cleavage of human ova in vitra. Fertility and Sterility 22: 255-262. Shettles, L. B. 1953. Observation on human follicular and tubal ova. Am. J. Obstet. Gynecol. 66:235-247. Shettles, L. B. 1955. A morula stage of human ovum developed in vitra. Fertility and Sterility 6: 677-682. Soupart, P., and L. L. Morgenstern. 1973. Human sperm capacitation and in vitra fertilization. Fertility and Sterility 24: 462-478. Soupart, P., and P. A. Strong. 1974. Ultrastructural observations on human oocytes fertilized in vitra. Fertility and Sterility 25: 11-44. Soupart, P. 1975. In vitra maturation and fertilization of the human oocytes. La Fé candatian; Cbllaque da la Saciété Nationale pour L'Etude do la Sterilite et de la Fé candite, pp. 81-93. Spiegel, M. R. 1961. Schaum's outline of theory and problems of statistics. McGraw-Hill Book Co., New York. Steptoe, P. C., and R. G. Edwards. 1970. Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotrophins. Lancet. 1: 683. Surve, A. H., F. E. Harrington and R. L. Elton. 1973. Effect of chorionic gonadotropin on corpus luteum of the monkey. Prac. Sbc. Exp. Biol. Med. 144: 963-968. Suzuki, 5., and L. Mastroianni, Jr. 1968. The fertility of in vitra cultured monkey ovarian follicular oocytes. Fertility and Sterility 19: 500-508. Thibault, C. 1969. In vitra fertilization of the mammalian egg. In Fertilization: Comparative Morphology, Biochemistry, and Immunology. Vol. 2. Edited by C. B. Metz and A. Monroy. Academic Press, New York. A LAPAROSCOPIC TECHNIQUE FOR TRANSFER OF EMBRYOS IN NONHUMAN PRIMATES Abstract A laparoscopic technique has been developed for placement of zygotes (early preimplantation embryos) into the fimbriated end of the squirrel monkey oviduct. This technique allows for transfer of l to 3 pl of fluid to the oviduct in situ without the trauma of laparotomy. Introduction The first successful transfer of a fertilized ovum was by Heape (1890). He used a surgical technique to transfer two four-celled zygotes from an Angora rabbit to the oviduct of a mated Belgian rabbit. Both zygotes implanted and the young were born alive. Since that time, successful transfers have been reported for many laboratory and domestic species and several reviews have been published (Adams and Abbott, 1971; Chang and Pickford, 1969; Dzuik, 1969). Generally ova to the two-celled stage are transferred to the ampulla of the oviduct, whereas zygotes in later stages of development are transferred to the uterus of the recipient. Synchrony of the recipient to the ovum donor is necessary to obtain a viable pregnancy (Edwards and Steptoe, 1975). Most techniques used for ovum and zygote transfer require surgical procedures to expose the oviduct. Techniques for transfer 49 50 to the uterus are either surgical (requiring laparotomy to expose the uterus) or nonsurgical (entering to the uterus via the vagina). All species in which successful transfers have been reported tend to have a flacid type uterus as compared to the muscular primate uterus. No primate species (human or nonhuman) has yet borne the product of a transfer. The techniques used in the primate have so far involved transfer to the uterus by puncture through the uterine wall or the cervix (Edwards and Steptoe, 1975). Transfers to the oviduct have not been attempted. The goal of the present study was to develop a method for transfer of small volumes of fluid containing fertilized ova to the oviduct of a recipient host using a laparoscopic procedure to avoid the trauma of major abdominal surgery. Materials and Methods Animals Squirrel monkeys (saimiri sciureus) of Colombian and Bolivian origin were maintained as previously described (Kuehl and Dukelow, 1975). Mature females were assigned to ovum donor-recipient host pairs, induced to ovulate using a regime previously reported (Dukelow, 1970) with modifications during the summer anovulatory season (Kuehl and Dukelow, 1975), and synchronized with the ovulating dose of HCG (500 iu, A.P.L.; Ayerst Laboratories, New York, N.Y.). Techniques for ova recovery, in vitra fertilization and zygote culture have been described elsewhere (Kuehl and Dukelow, 1975; Kuehl et al., 1976). 51 Transfer Procedure Recipient females were anesthesized with sodium pentobarbital (16.2 mg intraperitoneally) and laparoscoped using a technique we have previously described (Dukelow et al., 1971; Harrison and Dukelow, 1974). A second accessory probe was inserted ventrally on the side opposite the manipulatory probe (Figure 3). The ovaries were examined for ovulation points and transfers were made to the oviduct ipsilateral to the ovary with a corpus luteum. The accessory probe was withdrawn from the con- tralateral side and replaced with a Hartman alligator forceps (Roboz Surgical Instrument Company, Inc., Washington, D.C.). These forceps were used to manipulate the fimbria so that the opening to the ampulla was visible through the laparoscope. The zygotes were rinsed three times in fresh medium to remove motile spermatozoa. The zygotes were taken up individually in a 1 p1 volume with a micropipette (Micro/pettor A, (Range: 1 to 5 p1); Scientific Manufacturing Industries, Emeryville, California). The accessory probe was withdrawn from the ipsilateral side and replaced with the micropipette (Figure 3). The tip of the micropipette was inserted into the ampular opening of the fimbria at least 1 mm deep. The plunger was pressed, forcing the zygote and 1 p1 volume out of the pipette into the oviduct. The pipette was withdrawn from the ampullar Opening with the plunger depressed. Following comple- tion of the procedure, the recipient was maintained in a recumbent position for five minutes. Three females were laparoscoped and received transfers of a Trypan Blue (0.05%) solution in l, l, 2, 3, 4, and 5 pl volumes (one transfer per oviduct) using the procedure ""’—Figu e 3.T Diagrammatic presentation of the ovum—deposition technique in the squirrel monkey. .3185 \ ' F ‘2’ it i 4' '. ~. . . .. an "in r. \ 4 3‘3} ‘1 . C . "3.43 ' ‘ n . l - » e: L»: » Hit-i9. ‘ ’- - v! ".‘m‘ . - . T‘sfi‘. 1' J ‘7 £5 - "i .4 .._ 1i. 53 described above. Following the dye transfers, the fimbria were examined for signs of leakage of the dye. Results Twelve zygotes have been transferred. Five two-celled and seven 2-polar body, 2-pronucleate in vitra fertilized zygotes were transferred to twelve synchronized recipients. Although some gesta- tional changes (uterine size increases, weight gain, and increased water consumption) were noted in two animals receiving two-celled zygotes, no proven pregnancies have yet been found. In the transfers of Trypan Blue solution, 1, 2, and 3 pl of dye could be deposited at the ampullar site without leakage from the ampulla. Small amounts of dye solution were visible on the fimbria following the 4 and 5 p1 volume transfers. Within fifteen seconds of the removal of the miCro- pipette from the oviduct, all traces of dye had moved to the ampullar opening and disappeared into the oviduct. Thus the dye transfers demonstrated that small volumes used to transfer zygotes can be placed at the ampullar opening of the fimbria without loss to the peritoneal cavity. Discussion This report constitutes the first use of an ancillary laparoscopic procedure to deposit either fertilized ova or fluids into the oviduct in situ. The successful conclusion of a transfer with the birth of a live infant requires that each step of the 54 multiphase procedure be successful. While a variety of both laboratory and domestic species have yielded to such experimental manipulations, primates have not. This oviductal transfer technique represents one step of this multiphase procedure in the squirrel monkey to recover ova from ovarian follicles, mature and fertilize them in vitra, and deposit them into a foster mother. REFERENCES Adams, C. E., and M. Abbott. 1971. Bibliography on recovery and transfer of mammalian eggs and ovarian transplantation. Reproduction Research Information Service Ltd., Cambridge. Chang, M. C., and S. Pickford. l969. Egg transfer in the laboratory animal. In The Mammalian Oviduct. Edited by E. S. E. Hafez and R. J. Blandau. University of Chicago Press, Chicago. Dukelow, W. R. 1970. Induction and timing of single and multiple ovulations in the squirrel monkey (Saimiri sciureus). J. Reprod. Fert. 22: 303-309. Dukelow, W. R., S. J. Jarosz, D. A. Jewett, and R. M. Harrison. 1971. Laparoscopic examination of the ovaries in goats and primates. Lab. Animal Science 21: 594-597. Dzuik, P. J. 1969. Egg transfer in cattle, sheep, and pigs. In The Mammalian Oviduct. Edited by E. S. E. Hafez and R. J. Blandau. University of Chicago Press, Chicago. Edwards, R. G., and P. C. Steptoe. 1975. Physiological aspects of human embryo transfer. In Progress in Infertility. Edited by S. J. Behrman and R. W. Kistner. Little, Brown and Company, Boston. Heape, W. 1890. Preliminary note on the transplantation and growth of mammalian ova within a uterine foster-mother. Prac. Ray. sac. 48: 457-458. Harrison, R. M., and W. R. Dukelow. 1974. Morphological changes in the saimiri sciureus ovarian follicles as detected by laparoscopy. Primates 15: 305-309. Kuehl, T. J., and W. R. Dukelow. 1975. Ovulation induction during the anovulatory season in saimiri sciureus. J; Med. Primatalagy 4: 23-31. Kuehl, T. J., and W. R. Dukelow. 1975. Fertilization in vitra of saimiri sciureus follicular oocytes. J. Med. Primatalagy 4: 209-216. Kuehl, T. J., D. E. Wildt, and W. R. Dukelow. 1976. Nonhuman primate in vitra fertilization and embryo transfer: Their relationship to birth defect research. Adv. Med. Primatology (in press). 55 UTILIZATION OF COMPUTER TECHNOLOGY FOR REPRODUCTIVE STUDIES IN NONHUMAN PRIMATES (CODATA Bulletin (accepted), 1976) Abstract Computer programming techniques were used to (1) aid raw data collection, handling and compilation into data banks, (2) analyze banked data for basic reproductive values and specific experimental designs, and (3) utilize reproductive values for construction and simulation of computer operated models. Raw data from in vitra fertilization exper- iments with squirrel monkeys was compiled in a master file for statis- tical analysis. A bank of reproduction data for Macaque monkeys was established and examined for menstrual cycle characteristics. A com- puter simulation model was constructed to simulate the circhoral pattern of plasma LH concentrations observed in ovariectomized rhesus monkeys. A control point of the model was found and manipulated for expansion of the model to include drug induced changes in these circhoral patterns. Introduction The computer has served as a useful tool in biomedical research (Lindberg, 1974). Applications in two areas, computerized record keep- ing and simulation modeling, are of particular interest to investigators (If nonhuman primate reproduction. The value of computer simulation of 56 57 complex physiological systems has been discussed by several investigators (Schwartz, 1968; Riggs, 1970; Apter, 1974). The logistics for handling such computer simulation models have evolved along two lines. One approach has been to create new language for specific systems. Garfinkel (1968) used this approach in developing a machine- independent language for analyzing and simulating biochemical systems. This program was used to analyze experimental data from studies of rat liver cytosol gluconeogenesis (Achs et al., 1971), construct a simula- tion model (Anderson et al., 1971) and verify the model in later studies of rat liver dicarboxylic acid distribution (Anderson and Garfinkel, 1971). The other approach has been to use more general packaged pro- grams. For example, the recent study by Phair et a1. (1975) used the SAAM computer program (1967) to construct and modify a preliminary model of human lipoprotein metabolism. The latter approach was used to model the ovarian-pituitary interactions of the rat estrous cycle (Schwartz and Waltz, 1970) using CSMP (an IBM packaged simulation program). Plasma concentrations of estrogen and luteinizing hormone (LH) and the timing of ovulation were simulated for the rat. Other investigators have proposed and simulated similar models for the human menstrual cycle using both approaches (Vande Wiele et al., 1970; Shack et al., 1971; Cargille and Dixon, 1973). While these investigators have increased the number of variables included in their simulations (by adding other reproductive hormones, indices of follicle maturity and corpus luteum functions), none have provided for variability between experimental subjects or between cycles of a single subject. Such considerations 58 are needed to construct a simulation model for predicting reproductive phenomena of an individual subject. This study will present a simula- tion model for hypothalamic pituitary interactions in individual ovariectomized rhesus monkeys. To expand this model to include intact females, physiological data for individual animals are needed. The nonhuman primate literature does not contain much of this type of data: a problem discussed by Terry and Morrow (1972). They suggested the establishment of data banks as one possible solution. This disser- tation will describe a bank of Macaque reproduction data. The estab- lishment of this data base led the author to consider a data record system for studies of squirrel monkey reproduction. Computerized data record systems are relatively new to biomedical research. These systems are of two general types: those for processing animal status and cost expenditures (Warren et al., 1972) and those for handling research data (Munro et al., 1972; Ransil, 1974). The major drawback to wide acceptance of these data systems has been their specificity for the originator's facilities. Recently, two systems, Primate Information Retrieval System (PIRS) (Sciabbarrasi and London, 1974) and Primate Information Management Experiment (PRIME) (PRIME Manual, 1972), have been developed to handle both research data and animal status information at primate research facilities. They are complete and flexible to cover the variety of input data, but they produce only preprogrammed status tables. While status and performance data are important to studies of reproduction, specific research projects often require one-time looks at experimental variables. Also, computer facilities large enough to handle these programs 59 are stocked with packaged statistical programs requiring special data structures. A program which manipulates a complex data file to produce data structures compatible with such packaged statistical programs would prove more useful. This was the direction followed in constructing the data record system described below. Materials and Methods All programming was written in FORTRAN (version 4.5) for use on a Control Data Corporation 6500 computer (Michigan State University HUSTLER 2 system). The programs were designed for interactive execution through a remote terminal (model 33 ASR teletype). Programs and data files were stored centrally on magnetic disk, while locally maintained card files provided security from system failure. MSU STAT system (1974) provided packaged statistical programs. Plotting was done on a Cal Comp 963 incremental pen plotter using software provided on the HUSTLER 2 system (Plotting and Graphics, 1974). The programs described below (HYPIT and DAS) are available from the Endocrine Research Unit, Michigan State University. Computer Model A mathematical model was formulated for hypothalamic-pituitary interactions leading to circhoral oscillation in the plasma LH concen- tration of an ovarectomized rhesus monkey. Literature values used in modeling and for simulation of pharmacologic blockade of these circhoral patterns were from work by Knobil and his coworkers (Atkinson et al., 1970; Dierschke et al., 1970; Bhattacharya et al., 1972). Figure 4 Figure 4. 6O ”mi... ~43- «ei- eel—— SWM MLTIPLKATIOI NV ISM IE r * 11.2.1 (1'70 9.: o Tut QWRM -nnm imam: Mm! DCMVOTOOH IE1 ”0|!“me Flow diagram for simulation of plasma LH concentration in the ovariectomized rhesus monkey. 61 shows a flow chart for the computer simulation program (termed HYPIT). Variables used in the simulation are listed in Table 15 along with their initial values. Simulation was carried out in two modes. The first was to initiate variables and determine the sensitivity of the output (plasma LH concentration) to changes in control variables (THRES, RFILL, and gain factors). The second mode was to simulate an individual animal's response to a neuroleptic drug. Macaque Reproduction Data Bank Protocols were developed consisting of the variables shown in Table 16. These protocols were used to structure data from a Macaca fascicularis colony located at the Endocrine Research Unit, Michigan State University, and a Macaca arctaides colony of Sandoz, Ltd., Basel, Switzerland. A line of data (values for the protocol) was recorded for each menstrual cycle of every female Macaque. MSU STAT routines were used to analyze these data for cycle and menses characteristics. Computer Record System An 823 line FORTRAN program (termed Data Analyzer Series, OAS) was developed to manage data collected during the course of in vitra fertilization experiments with squirrel monkeys. DAS employs a sub- routine structure which contains algorithms to (1) produce raw data forms, (2) edit the master file, (3) search the master file for user specified entries and (4) list selected data in user specified format. The master file consists of entries of data (termed cells). Three 62 Table 15. List of variables used in HYPIT simulation program .a. Symbol Variable Initial value RFILL Net rate of filling of GNRH stores -1.0 %/lOO/hour T Time interval from previous GNRH release 0.0 hour PFILL Amount GNRH available for release (i.e., GNRH store) 0.0 %/100 HOLD Amount GNRH in transit to pituitary receptors 0.0 %/100 61 Gain for GNRH signal effect on synthesis of LH 300 ug/%-hour PKl “Basal" LH synthesis 0.0 pg/hour GZ Gain for GNRH signal effect on release of LH 300 ug/%hour PK2 "Basal" release of LH 0.0 ug/hour ALHS Amount LH available for release 0.0 ug RLHR Net rate of LH release cal. pg/hour RLHS Net rate of LH synthesis cal. pg/hour ANIMAL WEIGHT Mass of animal to be simulated 5.0 kg PVPAW Plasma volume per animal weight 40.0 m1/kg VSYS Plasma volume 200.0 ml CLLH Clearance of LH from plasma volume 110.0 ml/hour DT Increment of time, plasma is cleared before next iteration of simulation 0.02 hour F1 Gain of CLH due to clearance of volume of plasma calculated CLH Plasma concentration of LH 50.0 mg/ml GHLHR Gain hypothalmus LH release -l.0 ml-hour/mg THRES Threshold for “firing" of GNRH neuron 12.0 REF Reference point of threshold "firing mechanism" 0.0 TEST A test value used to trigger "firing" of GNRH calculated GNRH Gonadotropin releasing hormone LH Luteinizing hormone 63 Table 16. List of variables in protocols for Macaque reproduction data bank Type of protocol (location of colony) Female monkey number Menses data Menses length and type of flow Ovulation confirmation (laparoscopy, laparotomy, or pregnancy) Day of ovulation and side of ovulation ovary Male monkey number (if breeding involved) Mating date Semen quality Day of pregnancy test and test results Implantation bleeding date Length of implantation bleeding and type of flow Mode of pregnancy termination Date of termination Offspring monkey number Sex of offspring Laboratory notebook cross-reference 64 types of cells are used, corresponding to the three experiment units of the in vitra fertilization system (the female, ova donor; the male, sperm donor; and the culture chamber containing the gametes). The structure of the cells are shown by the data forms used to record cell values (Figures 5, 6, and 7). All cells have several common features. The first line (cell descriptor line) contains information on the cell type and composition as well as a unique cell number for cross- referencing (IC). Each cell has a line for integer data and a line for decimal data unique to that type of experimental unit. These are followed by from two to five optional groups of lines providing time series or repeated observations for a treatment. All cells contain a laboratory notebook reference and entry date (year-day) which provides a reference point. A form was prepared for each experimental unit in the in vitra fertilization system and entered as a data cell from the remote terminal. The DAS program prompts the user during an interactive session to respond with a command (LIST, EDIT, SEEK, HELP, STOP and FORM) which directs program execution through the pr0per subroutines. The LIST and EDIT commands provide management of files used by the program including updating and correcting of the master file. The SEEK allows the user to filter desired information from the master file and place it in a format for use by packaged routines or for immediate consumption as a list or table. HELP is used to remind the user of program operation during the interactive session and STOP terminates the program. The FORM statement generates new raw data forms which are printed at the 65 ill” ANALVIER SERIES 1'1"] FOR“ 1- 1 I 0 fl 0 0 11 I I v o 0 can -0 o-o 0-0 0-0 . .vQ-O-Ocv ’ .-' 'I C III 9 IL IC IICT NVOT NIIL NSCL N‘. UNI. NCCL NHIL ”LL NIL v-o-v-o o-v-v IO ' 9-' DH '6"? L18 800K REF SPERN NEOIUH TE”? ‘7" . . . . 9-9-0-9-9 » -o o-v-o-o-o "EIGHT SIT! KIN CON; VOL BIL VOL VIRIIVPE I HEIGHT REF VINE FCC! ID I OUT "2 E665 FEDY "A". I V-F-F-F-F-F-F F-F-F-F-F F-‘oF-F-F l-A-l-A I I I-I I-I IF TYPE-IIIAYUllflJgTNEI I v u - ISDIVS RATING 2 . o o JIVAGINIL SPERM 3 . . . IZHII 0R 3!!! VIVIDMTWEI I. u a u I!!! . SPERM A! 5 . u . JIPOSITION A! 6 . a u I . . . I o u u 9 - c o 111 . . . H, - . . 12 . . . VIRIREF REF IIHE PC Pl! PC HORN PM RE“) OSHOLIL SPERM Ball: ‘66 ICIDI INT 3! 52 S! l ”SCI. I F-F-FOF F-F-F-F F-F-F-F F~F-F-F-F F-F-F‘F-F F-F-F-F-F-F I I-I I-I I-I I-I I . o - o a Z n . . . - o J c . . o o o t c . . o u I S . . . . . . 6 o . o o o o 7 . . . o . o I . . . u . o 9 u . o n n a ll 0 u o o o - Vll-SOUICE P00 KARVO PMEIO SE! OLD ID LEIG'IHTOF-SEIT MIL F001 CA! I III. I-I Z-I l-l I-I-I-I I I l-A'A F-F-F-F F-FoF-F F-F-F-F For-For o a . a Z . a u o .l a a . . In. "I SPACES 0F LETYEIIIIG PER LINE. 1 Z J h 5 , ”7 7 I Figure 5. Form 1 for male squirrel monkey data. DIYI 2 0 o o—o-o—o-o o 0-9 It TC TIC! NTOT 0-9-9-o-o-o o-ooo-o—o-o OVUN "0”"79 CULTURE In . . VOLIFOL FLUID FEOIUM 66 I‘tALVIFP SFUIFS DITA FFR'O .7. n 0 9-9 G 0 0 0 9-0 0-9 9.0 M111 NQCL ‘01 V9". 9-.-.-. .O. . FINALE FSIT. SIDE {H 98C SUN ‘5' P‘FOIUN REF IE1? ITH SWCIIL VIC VIS PLLQ XC PIN HOE. L33 326‘ D”. 9-. 0 0 0 O O B 9-. 9-. 0-9 ’IECL NNIL NLL NM. 9 O Q .0. .-’-. O 0-. 0-. .0. O 0 9-0-9-.-. 0 .0. OO'O.OO-O-O-Q O o-o-o-o-o—o o-o-o—o-o-o o-o-o-o-e-o 0-9-9-0-9-0 coo-4-0-9-9 NHL .70 SPICFS OF L'TTFRINfi 9N SPF? H SUS PFP LINF) OS" urea. r-r-r-r-r-r r-r-r-r r-r-r-r-r r-F-r-r—r I 2 3 ‘ S 'IRI 9" TIME PC PH 3 o o 2 o o 3 . . ~ 0 o 5 o o 6 o o 7 o o O o o 9 o o I. a o 33 o o I? o 0 I3 0 o R‘ o o 15 o o ‘6 o o 17 o o I. o o 19 o o ?0 o c Figure 6. ) SPF-2H CONC OVA! CIM CR SPFRHIKIN BIND AGGI F180 SACT OVA( Z [-1 (FOL ASP SPERM FRET INSEHINITE) IIVE IN «RS 3 h 5 6 I I I I I I I I 1*! I I 1°! I I I-I I I 1.1 I I I-I l I Form 2 for culture chamber data. 67 5.". A‘I.Vlt9 S"Il'- )I'I ’C“ I. I : a . t I C II o ooo.o-v-U v 0-9 0-0 0.. '00 0-9 9.0 0-9 0.0 9.. It l'. “L! up! “It ~'.‘.L '1" In, «CL hulk 'nL Ill 0...... o o I v o 0 900-. 0-9 I-.-‘/On.’.-. . g g H IJ'TIJH!’ 4' ‘11 «Ct. L" IS!” it Hit-CUP)!“ MV "A! I no use! In "vac-u: A: Inn-,1 o .OOOO-OO. DU ”If "gnome-J arm nun ”my an on.) 1‘) uucvuflcD-HH Inn root In I nut-t l-I l-l I-l l-I-l-t I I-l-A-I F-'-'-' '-'-'-' 64.7.: r-t-I-v ‘ O D t C : O I O I ’ I I O l In. on Svnus W Lilli-1m. 'H LIhU I 2 I § § I‘D-OPUG v.2. v.0. SIC! 'IOSFIIN "55' I” I)“ I‘ IJ | 'I‘f‘oIVSU “IL I‘I I I I F F'FJ". '-'-‘O'O' F-F070'07 '.'-'-'-'-F I o o u o 2 o o o o ’ I O I . Q t 0 a 0 ’ C . . . ‘ . . . . I I Q C I O c o o 0 Q C O O . I. o o o I 53 o o o u ‘2 . . C C l’ O . O . I! a o n o I‘ o o o o I. I o I o [7 a a o o I. t o o o 19 o o o o t. . C U . VIII 'I'E'CJ‘VS) IEIG"I LINGVHIII'U '1 L0 .0 I!" UID UTC 0" S'L 'l L'L U'L C'l 'm CHI. CLI. 3'. N‘ L" V" a. '0' “I a. FILL F-FOF-FOFOF I’d-'0' F-F-F-F I I I 191 I‘I I'I I-I I'I I'I I'I I'I I'I I'I I'I I°I I'I IOI I‘I I'I I'I T'I I'I I'I ‘ . . . 2 o o o s C . . ~ I C . s . . . ° . U ' F n o o . I . U a D . . x. C . . VIDIOVAIV 0"le IS‘ 0“ CULTUII ID "IL 70!. FLUID ‘1. '0. “AL I I P! I-I I-I-I-I'I-I F-F-F-F-F-F I-I COOVOvfloo-cgu- 00.0.10... p Figure 7. Form 3 for female squirrel monkey data. 51' W6 pa ha tug FE; "Er 68 central site by a high-speed line printer. The program was tested by a sample filtering of cells to collect body weight and semen qualities for a colony male, DUKE. Results Computer Model A computer model (HYPIT) was used to simulate circhoral patterns of plasma LH in ovariectomized rhesus monkeys. When simu- lation variables were initiated to the values shown in Table 15, the period length between peaks of plasma LH concentration was 75 minutes. The THRES variable (a threshold setting in the model) was found to be a convenient control point for the period length (Table l7). In a simulation of the action of haloperidol, a neuroleptic drug, THRES was modified to produce the simulated pattern shown in Figure 8. This pattern agrees with that experimentally derived using 0.5 to 1 mg/kg haloperidol in ovariectomized rhesus monkeys (Bhattacharya et al., 1972). Macaque Reproduction Data Bank The data bank was established using reproduction protocols for two species of Macaques. Each colony has contributed five years of reproduction data to the bank. A total of 2,309 reproductive cycles were included for the two species. Specific results, especially con- cerning menstrual cycle characteristics and ovulation time in M. fhscicularis have been described elsewhere (Dukelow, l975). 69 Table 17. Period lengths (minutes) of oscillation of simulated LH patterns using different values for THRES Period THRES (minutes) 3 204a 5. 150 8 112 9 102 10 93 11 84 12 75 13 67 14 60 15 54 16 49 19 35 21 30 aMeasured as the mean for five cycles from 10 minute sampling intervals. 70 15.00 UE o O. 4'. V9 T RES Ito 0 00 E L 80.00 1 64.00 19.00 3g.oo PLRSHH LH IN NO PER HL 18.00 J cp.00 T T .00 10.00 12.00 2100 4100 6100 6100 SIMULRTION TIME IN HOURS Figure 8. Results of simulation of pharmacologic blockade of circhoral oscillations of LH by alteration of THRES variable. 7l Computer Record System The computerized record system (DAS) was established with 550 cells of data from in vitro fertilization studies. The system was utilized to examine observed seasonal changes in a male used to provide sperm. Male squirrel monkeys have been shown to undergo seasonal weight change, in wild and semi—natural conditions, which have been correlated to testis weight and changes in spermatogenesis (DuMond and Hutchinson, l967; Baldwin, 1970; Nadler and Rosenblum, 1972). In our laboratory one particular male, DUKE, has been electro- ejaculated and weighed regularly for approximately two years. The program was used to filter from the master file a listing of the weight, volume of ejaculate, and number of sperm collected throughout DUKE's history. The listing was structured for use in a plotting routine and the results are shown in Figure 9. A listing of the data was also made. Other results using this system to compile oocyte data are found on page 24 of this dissertation. Discussion A simulation model has been presented for the hypothalamic- pituitary interaction responsible for the circhoral LH plasma concen- tration pattern in ovariectomized rhesus monkeys. A simulated drug interaction yielded results similar to those seen experimentally. The pattern of depressed circhoral surges of LH release was also observed experimentally with the infusion of estradiol into ovariectomized rhesus monkeys (Yamaji et al., l972). Using the THRES variable in 72 .O’) 69.70 81 I x x TOTRL SPERH IN ”ILLIQNS 29.00 43.00 * x x x x x x x 1:00 0:00 I I x I: x x x x 9:50 3 o 3 o CORGULUM VOL IN HL 0:00 o 1000.00 _1 I 890.00 8§0.00 090.00 9§0.00 —i I II I II I I I II BODY HEIGHT IN GMS 750.00 J. I RECORD FDR DUKE IN DRYSbe‘ 9700.00 Figure 9. Seasonal changes in weight and semen characteristics for laboratory maintained male squirrel monkey. 73 the simulation model as a control point for this action of estrogen adds another dimension to this model. The data bank of Macaque data has been used to exchange data between two laboratories. While the Endocrine Research Unit colony is used predominantly in laparoscopic studies to determine ovulation times, the Sandoz Ltd. colony is used in mating studies for production of timed pregnancies. These types of data compliment each other and can be combined to increase understanding of Macaque reproduction. The computerized banking of data was extended to research data. Placing complex in vitro fertilization data in such a system provides three advantages: (1) masses of data can be quickly available for compilation of tables and listings, (2) algorithms can be provided that interactively form data structures for statistical treatment, and (3) compilation of historical information can easily be made (i.e., information on the entire research life of a given animal can be obtained in a matter of minutes). This latter advantage is particularly important for nonhuman primate reproduction research. Due to the high cost of these animals and the nonterminal nature of the research, experimental subjects are often maintained for several years and used in many experiments. Also some phenomena, such as seasonal weight changes, may only become apparent after individual animals have been studied for several years. REFERENCES Achs, M. J., J. H. Anderson and D. Garfinkel. l97l. Gluconeogenesis in rat liver cytosol. I. Computer analysis of experimental data. Cbmputers Biomed. Res. 4: 65-106. Anon. 1974. Plotting and Graphics. Computer Laboratory User‘s Guide. Vol. VII. Michigan State University. Anon. 1974. The MSU STAT System. Computer Laboratory User's Guide. Vol. VIII. Michigan State University. Anon. l972. The PRIME System. Oregon Regional Primate Research Center, Beaverton. Anderson, J. H., M. J. Achs and D. Garfinkel. l97l. Gluconeogenesis in rat liver cytosol. II. Computer simulation of control properties. Cbmputer Biomed. Res. 4: 107-125. Anderson, J. H., and D. Garfinkel. l97l. Calculated intracellular distributions of dicarboxylic acids in rat liver. Cbmputer Biomed. Res. 4: 43-64. Atkinson, L. E., A. N. Bhattacharya, S. E. Monroe, D. J. Dierschke and E. Knobil. l970. Effects of gonadectomy on plasma LH concentra- tions in the rhesus monkey. Endo. 87: 847-849. Apter, J. T. l974. Models and mathematics in medicine. In Medical Engineering. Edited by C. D. Ray. Yearbook, Chicago. Baldwin, J. D. 1970. Reproductive synchronization in squirrel monkeys (saimiri). Primates 11: 317-326. Berman, M., and M. F. Weiss. 1967. SAAM’ManuaZ. U.S. Public Health Service Publication No. 1703. Government Printing Office, Washington, D.C. Bhattacharya, A. N., D. J. Dierschke, T. Yamaji and E. Knobil. l972. The pharmacologic blockade of the circhoral mode of LH secretion in the ovariectomized rhesus monkey. Endo. 90: 778-786. Cargille, C. M., and J. K. Dixon. l973. Servoendocrinology: A system dynamics model of corpus luteum function in the normal cycle and in pregnancy. Sixth Ann. Mtg. Society fer the Study of’Reproduction. Abstract 72. 74 75 Dierschke, D. J., A. N. Bhattacharya, L. E. Atkinson and E. Knobil. l970. Circhoral oscillations of plasma LH levels in the ovariectomized rhesus monkey. Endo. 87: 850-853. DuMond, F. V., and T. C. Hutchinson. 1967. Squirrel monkey reproduction: The "fatted" male phenomenon and seasonal spermatogenesis. Science 158: l467-l470. Garfinkel, D. l968. A machine-independent language for the simulation of complex chemical and biochemical systems. Cbmputers Biomed. Res. 2: 31-44. Lindberg, D. A. B. l974. Conference on the computer as a research tool in the life sciences. Fed. Proc. 33: 23ll-2312. Munro, I. C., S. M. Charbonneau and R. F. Willes. 1972. An automated data acquisition and computer based computation system for applica- tion to toxicological studies in laboratory animals. Lab. Animal science 22: 753-756. Nadler, R. D., and L. A. Rosenblum. l972. Hormonal regulation of the "fatted" phenomenon in squirrel monkeys. Anat. Rec. 173: 18l-188. Phair, R. D., M. G. Hammond, J. A. Bowden, M. Fried, W. R. Fisher and M. Berman. 1975. A preliminary model for human lipoprotein metabolism in hyperlipoproteinemia. Fed. Proc. 34: 2268-2270. Ransil, B. J. 1974. Use of table file structures in a clinical research center. Fed. Proc. 33: 2384-2387. Riggs, D. S. l97l. Control Theory and Physiological Feedback Mechanisms. Williams and Wilkins Co., Baltimore. Schwartz, N. B. l968. Newer concepts of gonadotropin and steroid feedback control mechanisms. In Textbook to Gynecologic Endocrinology. Edited by J. J. Gold. Harper and Row, New York. Schwartz, N. B., and P. Waltz. l970. Role of ovulation in the regulation of the estrous cycle. Fed. Proc. 29: 1907-1912. Sciabbarrasi, S. A., and S. T. London. 1974. A computerized primate information retrieval system (PIRS). Lab. Animal Science 24: 773-782. Shack, W. J., P. Y. Tam and T. J. Lardner. l97l. A mathematical model of the human menstrual cycle. Biophysical J. 11: 835-838. Terry, M. W., and A. C. Morrow. 1972. "Normal" values: A critique and a proposal. Proc. Third Cbnfl Exp. Med. Surg. Primates, pp. l48-l5l. 76 Vande Wiele, R. L., J. Bogumil, I. Dyrenfurth, M. Ferin, R. Jewelewicz, M. Warren, T. Rizkallah and G. Mikhail. 1970. Mechanisms regulating the menstrual cycle in women. Recent Progr. Harm. Res. 26: 63-103. Warren, W., R. W. Yurt, J. A. Porter, Jr., W. R. Canaday, Jr., and B. S. Linn. l972. Computer processing of animal housing status and expenditures. Lab. Animal Science 22: 731-733. Yamaji, T., D. J. Dierschke, A. N. Bhattacharya and E. Knobil. 1972. The negative feedback control by estradiol and progesterone of LH secretion in the ovariectomized rhesus monkey. Endo. 90: 77l-777. SUMMARY AND CONCLUSIONS Saimiri sciureus was used as a model nonhuman primate for investigations of variables involved in in vitro fertilization and embryo transfer. Associated problems of development of a transfer technique and data handling were also studied. The following con— clusions are indicated by the data obtained: 1. A laparoscopic technique was developed which allows placement of from 1 to 3 ul of fluid into the ampulla of the squirrel monkey oviduct. A computer simulation model can be constructed and used to simulate circhoral patterns of plasma LH in ovariectomized rhesus monkeys. A computerized data bank can be established for exchange of Macaque reproduction data. A computer program can be used to compile and examine in vitro fertilization data for specific experimental designs, or historical data. Fertility of male squirrel monkeys varies between individuals. There are differences between follicles in the proportions of oocytes recovered and in proportions of recovered oocytes that are atretic. Oocytes can be classified by cellular investments with regard to potential to mature and be fertilized in vitro. 77 10. 78 Insulin added at the l0 m.i.u. or 100 m.i.u. per ml level does not alter maturation or fertilization in vitro. The time course for in vitro development of fertilized zygotes is similar to that of rhesus monkeys and humans. Transfer of ova from the squirrel monkey, fertilized in vitra, has not resulted in a pregnancy. APPENDIX A PREGNANCY DIAGNOSIS OF SAIMIRI SCIUREUS APPENDIX A PREGNANCY DIAGNOSIS OF SAIMIRI SCIUREUS A reliable determination of pregnancy is necessary in experiments involving transfer of fertilized ova, so that recipients aborting or resorbing embryos are not misclassified as nonpregnant. Table 18 summarizes previously reported characteristics of gestation (squirrel monkey) which have been used to diagnose pregnancy. The first two, uterine size increase and chorionic gonadotropin con- centration in the plasma or urine, were found to occur early in the first trimester at the time the embryo is in the primitive streak stage (embryonic stage VIII) (Goss et al., l968). In the course of their embryological studies, Goss et al. (l968) reported the most objective indication of pregnancy in the squirrel monkey to be an enlarged (approximately 20 mm) and slightly hyperemic uterus with an ovary having a corpus luteum. These observations were made using laparotomy 20 to 25 days after conception. Nathan et al. (l966) used a rat-uterine-weight assay for chorionic gonadotropin to diagnose pregnancy from 20 to l05 days after conception. There were two false positive reactions of 23 tests and no false negative reactions. Castellanos and McCombs (l968) were successful in early diagnosis of pregnancy using a mouse-uterine-weight assay to detect elevated urinary gonadotropins. They diagnosed ll of 17 females pregnant. Only two 79 80 Table 18. Characteristics of gestation in saimiri sciureus Days after Characteristic conception References Uterine size increase 20 to 25 Goss et al., 1968 (by laparotomy) Chorionic gonadotropin: (a) plasma 20 to 105 Nathan et al., 1966 (b) urine 28 Castellanos and McCombs, l968 Uterine size increase 42 to 56 Rosenblum, 1968 (by palpation) 49 Hopf, 1967. 61 Nathan et al., 1966 Diabetes insipidus 60 Clewe, I969 60 to 135 Travis and Holmes, 1974 Maternal weight gain 60 to 95 Hopf, 1967 67 to 147 Travis and Holmes, 1974 Visual abdominal enlargement 74 to 102 Hopf, 1967 Fetal skeleton by x-ray 81 Nathan et al., 1966 84 McKim et al., 1972 102 Hopf, 1967 81 pregnancies went to term; the remaining nine females resorbed their embryos before the ninth week of pregnancy. Female squirrel monkeys in each of five groups (Table 19) were tested for pregnancy using at least one of four methods. The first was examination of the reproductive tract with the laparoscope. Pregnancy was diagnosed as positive when the uterus was found to be enlarged, hyperemic, and with a corpus luteum on an adjacent ovary. Figure 10 provides a comparison of uterine width dimensions for pregnant and nonpregnant animals. The width was taken as the distance between tubal-uterine junctions measured to the nearest millimeter. With this measure alone, pregnancy could be accurately diagnosed 100% of the time if the width was greater than 10 mm. Animals with uteri 8 to 10 mm in width were subjected to a confirmatory laparoscopy. The remaining three methods involved examination of either urine, collected by suprapubic puncture, or plasma, collected by femoral venipuncture, for chorionic gonadotropin with one of the three assays shown in Table 20. A preliminary test of the three methods was performed with (1) squirrel monkey urine collected from females 8 to 12 hours following a 500 iu intramuscular injection of HCG (A.P.L., Ayerst Laboratories, New York, N.Y.). (2) urine collected 8 and 10 days after the HCG injections, and (3) 0.15 molar sodium chloride solution with l iu of HCG per ml. The results in Table 20 show that the commercial human pregnancy test did not detect the HCG in any of three early urine samples that gave positive reactions by the other two methods. Table 21 provides a comparison based on the number of incorrect pregnancy 82 Table 19. Groups of female squirrel monkeys undergoing pregnancy diagnosis Category Time of testa Nonpregnant 1 to 56 days post-HCG injection Nonpregnant at random intervals for non-HCG injected females Possibly pregnant Possibly pregnant Possibly pregnant at 30 day intervals during natural mating 20 to 29 days post-HCG injection combined with either: artificial insemination, natural mating, or embryo transfer 21 days after the end of natural mating aHCG injection: 500 iu of Human Chorionic Gonadotropin (A.P.L., Ayerst Laboratories, New York, N.Y.) injected as an ovulating dose as part of ovulation induction regime used in this species (Dukelow, 1970; Kuehl and Dukelow, l975). 83 8 8 6 a". 8 9 NUNPREGNRNT ’_. 5% . 20 T0 30 0an PREGNANT c2: LAJo 0 EC: ° 'Zm. é cfi~ "1 Z x: P- 333 23. 3:358. 23. z {:3 a: D O. D O 05c; :0 5.3.;- I: :3 z 3 /’*\_ /A\. 8 c11.00 s'.00 ' I — '2'0.'00 “23:00 “30.00 10.00 '1's.00 1 UTERINE NIDTH IN HM Figure 10. Frequency distribution of uterine width dimension used for pregnancy diagnosis. 84 Table 20. Chorionic gonadotropin assays Detection of HCG in Urinea Saline Method 8-12 hrs 8 and 10 day l iu/ml Bioassay b Mouse Uterine Weight Positive Negative Positive Immunoassay 0 Commercial Human Test d Negative Negative Positive Sub-Human Primate Test Positive Negative Positive aUrine of squirrel monkey injected with 500 iu HCG collected 8 to 12 hours or 8 and 10 days later. bWith 20 day Swiss-Webster mice using method of Wilson et al., 1970. cPrognosticon Immuno-Diagnostic Pregnancy Qualitative Test, Organon Inc., H. Orange, N.J. Table 21. Comparison of diagnostic methods Number of tests False a False Method Total P051t1ve Negative Laparoscopy 220 0 0 Mouse Uterine Weight 18 l 0 Commercial Human Pregnancy Test 8 3 0 Sub-Human Primate Test 13 2 0 aAll false positive reactions were from 20 and 22 day plasma. 85 diagnoses for each of the four methods. The false positive reactions found with plasma samples might be due to a cross reaction of squirrel monkey pituitary gonadotropin. No false positive reactions resulted from tests of squirrel monkey urine. Laparoscopic examination of the reproductive tract clearly is the method of choice. Ten pregnancies were detected within a month of conception with this method. REFERENCES Castellanos, H., and H. L. McCombs. 1968. The reproductive cycle of the new world monkey. Fertility Sterility 19: 213-227. Clewe, T. H. 1969. Observations on reproduction of squirrel monkeys in captivity. J. Reprod. Fert., Suppl. 6: 151-156. Goss, C. M., L. T. Popejoy II, J. L. Fusiler and T. M. Smith. 1968. Observations on the relationship between embryological development, time of conception and gestation. In The Squirrel Monkey. Edited by L. A. Rosenblum and R. W. Cooper. Academic Press, New York, N.Y. Hopf, S. 1967. Notes on pregnancy, delivery, and infant survival in captive squirrel monkeys. Primates 8: 323-332. McKim, 0., Jr., T. C. Hutchinson and J. A. Gavan. 1972. Prenatal growth of long bones in rhesus and squirrel monkeys (Macaca mulatta and Sdimiri sciureus). Am. J. Phys. Anthrop. 36: 353-358. Nathan, T. S., L. A. Rosenblum, G. Limson and J. H. Nelson, Jr. 1966. Diagnosis of pregnancy in the squirrel monkey. Anat. Rec. 155: 531-535. Rosenblum, L. A. 1968. Some aspects of female reproductive physiology in the squirrel monkey. In The Squirrel Monkey. Edited by L. A. Rosenblum and R. W. Cooper. Academic Press, New York. Travis, J. C., and W. N. Holmes. 1974. Some physiological and behavioral changes associated with oestrus and pregnancy in the squirrel monkey (saimiri sciureus). J. Zool. (London) 174: 41-66. Wilson, J. G., R. Fradkin and A. Hardman. 1970. Breeding and pregnancy in rhesus monkeys used for teratological testing. Teratology 3: 59-72. 86 APPENDIX B COMPUTER PROGRAM FOR MAINTENANCE OF INDIVIDUAL ANIMAL RECORDS IN A NONHUMAN PRIMATE COLONY APPENDIX B COMPUTER PROGRAM FOR MAINTENANCE OF INDIVIDUAL ANIMAL RECORDS IN A NONHUMAN PRIMATE COLONY The nonhuman primate colony at the Endocrine Research Unit consists of animals used in a variety of nonterminal studies. Their responses to a variety of treatments as well as basic health statistics (especially body weights) are recorded as citable entries in laboratory notebooks by the investigators using them. A computer program was developed to maintain a file of animal records for this nonhuman primate colony. The program and associated data file comprise the Colony Assign- ment Record system (CAR). Like the Primate Information Retrieval System (PIRS) (Sciabbarrasi and London, 1974) and the Primate Information Man- agement Experiment (PRIME) (PRIME Manual, 1972), the CAR system utilizes the concepts of a permanent master file, to contain all animal records, and unique animal identification numbers, to allow access to individual animal records. The CAR system was designed to be used by the investi- gators in conjunction with a laboratory notebook numbering system used for the past six years. The objectives of the CAR system were (1) to maintain a listing, by animal, of all laboratory notebook entries at a common site, readily accessible to all investigators and (2) to store both current and historical information on each animal. 87 88 The program consists of 165 lines of FORTRAN, version 4.5, and contains no subroutines. The master file of animal histories is stored as an alphanumeric permanent file. Both the program and master file are stored on magnetic disks at the central site, a CDC 6500 computer located at the Computer Center of Michigan State University. The CAR program is operated by HUSTLER 2, MSU's operating system, in conjunction with MISTIC 2 (The Michigan State Interactive Computing System). The investigator communicates with MISTIC 2, by telephone lines, from the remote site, a model 33 ASR teletype located at the Endocrine Research Unit. Both the program and master file are attached for use by the investigator at the beginning of each interactive session. MISTIC 2 then loads the program and begins execution. The program was designed for interactive use and responds to the six CAR commands shown in Table 22. A sample interaction with the program is shown in Figure 11. The investigator responses are underlined. Using the FILE command, new data can be added to the master file from a previously supplied tape. In this case, no data was to be filed. The LIST command can be used to provide the investigator with a complete listing of the master file and the current project assignment of every squirrel monkey in the colony. Each project is assigned a number which is termed a pool. There are currently nine pools, but more can be added to a maximum of 99. The SEEK command is used to provide a remote site printout of the history of any animal on file. The illustration is for BONN, an infant female. Each of the seven lines is an entry containing the animal's name, sex code, species code, pool code, location code, date 89 Table 22. CAR system commands Command Special user actiona Program action FILE Supply new status data Adds new data to master file LIST Dispose copy to central Copies master file at terminal site, if required or central site SEEK None Asks for animal identity and provides complete history HELP None Provides brief explanation of program operation FORM Dispose copies to Copies data forms for use in central site structuring data for filing STOP None Terminates current interaction with program aAny actions other than answering questions posed by program during interactive session. ._-....__.‘ .—-~-... --—4_ __ 90 UELCOHE T3 CAR.C3LGNY ASSIGNHENT RECQRDS.HAVE A NICE STAY. DC YOU NEED REL”? YES THIS DRQCRAH KEERS TABS 9N MSNKEY RESEARCH ASSIGN‘ENT. ANIRALS ARE RLACED INT! PGZLS AND LISTINGS CF THESE PGQLS CAN BE PRODUCED INTERACTIUELY.THE PRSGRAH RESPZNDS T2 THE FQLLSNING CZNRANDS FILE.LIST.SEEK.WEL°.STZPofflRH. FOR FURTHER REL? SEE T31 KUEWL OR TUE CAR RANUEL. YOUR "154 IS RY C3WWAND.JUST ENTER YQUR DESIRE. T3 FILE DATA YOU RUST HAVE ENTERED A TARE2 UITR CORRECTLY FORMATTED DATA IN 3RDER .HAVE YOU? YOUR VISH IS MY CGNRAND.JUST ENTER YZUR DESIRE. SHOULD GUTDUT BE DISCONVECTED? YES — PGZL I I 0.903L 2 I! 41.9031. 3 - SpPQZL A - 2.90’3L 5 3 O. OQZL 6 ' IS.°98L 7 ' I7.PGGL S ' 0.990L 9 - A.PZZL IO ' O. Y3UR VISR IS WY CZYHAND.JUST ENTER YQUR DESIRE. NZULD YZU LIKE A CONRLETE CURRENT STATUS LIST? ZRHIASTERIIIIIICH ANIMAL S‘IALL IT BETIO'MI‘JNE") 1 IO/ 9/75 BORN T3 I406 AND JUAN EBNN 4 I I I BGNN A I 9 2 II/I2/7S BROUGHT INSIDE¢L03KS BZLIVIAN) BBNN A I 9 2 II/I2/75 UEISRT!I79 GRS BBNN A I 9 2 II/I9/75 VEIGHIV TJK-IA-ISC205 CNS) MN.“ 0 I 9 2 II/26/75 VEIGHIN TJ‘I-IS-33(2I9 G‘IS) 339V 4 I 9 2 I2/ 3/75 VEIGRIN TJK-IS-33(235 GHS) EDNN A I 9 2 I2/IO/TS UEIGHIN TJK-IS-33I24I GUS) 9R.‘ASTERIVRICW ANIMAL SHALL IT BE?(O-”N3NE") NZVE YOUR WISH IS RY CSHVAND.JUST ENTER YOUR DESIRE. FDR” “Ba “ANY FURRS? (IZIIQI FQRMS 3V SUTI’UT. T1 :ET. "DI 593 SE. QUTDUT. 9R. ” YOUR "IS“ IS WY C3M1AND.JUST ENTER YOUR DESIRE. STOP THAT CINCLUDES CAR. HAVE A NICE DAY. END CAR Figure 11. Sample interactive session with CAR program. 91 comm Assxcmcut RECORD roan snooceme RESEARCH uun.nsu THE CAR PROGRIH REQUIRES THE USE OF CODES T0 INPUT SOHE BASIC STATUS DATA, UNILE ALLOWING A CONNENT SECTION FOR INVESTIGATOR USE. IT IS SUGGESTED THAT A LAB BOOK PE‘ERENCE AND BRIEF (1 0R 2 “090) COHHENT OR DATUH BE PLACED IN THIS SECTION FOR LATER USE IN COMPILATION OF THE ANIMAL HISTORY. ONE ANIMAL FER FOR" PLEASE. INPUT OF DATA HILL RE "ADE FASTER. IAHE SE! SPECIES POOL LOCATION DATE COMHEMTIQO SPACES) I I 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 I 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 I I 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 I I 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 I I 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 I I 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 I I 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 I I 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 I I 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 I I 9 9 9 O 9 O O O Q 0 O O O O O FOLLOIING IS CONFLETE LIST OF CODES SPECIESI1=S. SCIUREUS.28H. FASICULARISI SEIIISHALE9ZSFEHALE.3=INFANT-HALEgbaINFANT-FENALEI POOLIIIOUTSTDE ENVIRON PROJECT 28X" VITRO FERTILIIATION PROJECT 3'LAPAROTOHV AOHESIONS btPfiEGNANT StNATUPAL CYCLE PROJECT 6xPESTING POOL 7=UTE9INE FLUSN PPOJECT B'QUARANTIIE 9=LACTATIONI LOCATIONII'CUTSTDE COMPOUN0923INSIOE STEEL CAGE, J’INSIOE GANG CAGE) IF NO POOLpINOICATE HITH lERO.NEH POOLS TO BE ADDED BY TJK ON DEMAND " Figure 12. Sample CAR system form. 92 of the entry and investigator comment. The HELP command generates a brief description of the program and the legal commands. The FORM command generates the data forms to be used by the investigator to add entries to the master file. A sample form is shown in Figure 12. The codes for each coded category are given at the bottom of the form for easy investigator use. When enough entries are gathered by the investigator, they are typed onto a paper tape (TAPE 2) the way they appear on the data form. These entries are then filed as described above. The final command is STOP. This will terminate the interactive session with the program. The investigator can then dispose the output, a list or group of forms, to the central site for printout by the high speed line printer. The CAR system went into use on May 20, 1975, and holds records for all the squirrel monkeys at the Endocrine Research Unit. REFERENCES Anon. 1972. The PRIME system. Oregon Regional Primate Research Center, Beaverton. Sciabbarrasi, S. A., and W. T. London. 1974. A computerized primate information retrieval system (PIRS). Lab. Animal Science 24: 773-782. 93 APPENDIX C PUBLICATIONS BY THE AUTHOR APPENDIX C PUBLICATIONS BY THE AUTHOR Full Papers . A Restraint Device for Electroejaculation of Squirrel Monkey (Saimiri sciureus), by Thomas J. Kuehl and W. Richard Dukelow. Laboratory Animal science 24: 364-366, 1974. 9 Ovulation Induction During the Anovulatory Season of saimiri sciureus, by T. J. Kuehl and W. R. Dukelow. JOurnaZ ofNMedical Primatology 4: 23-31, 1975. . Fertilization In Vitro of Squirrel Monkey (saimiri sciureus) Follicular Oocytes, by T. J. Kuehl and W. R. Dukelow. Journal of'MedicaZ Primatology 4: 209-216. 0 Nonhuman Primate In Vitro Fertilization and Embryo Transfer: Their Relationship to Birth Defect Research, by T. J. Kuehl, D. E. Wildt and W. R. Dukelow. Advances in Medical Primatology (in press), Plenum Press, 1976. 0 In Vitro Fertilization of Nonhuman Primates, by W. R. Dukelow and T. J. Kuehl. La Fécondation; Cblloque de la Sbciété Nationale pour L'Etude de la Stérilité et de la Fécondité, pp. 67-80, 1975. 9 A Laparoscopic Technique for Transfer of Embryos in Nonhuman Primates, by T. J. Kuehl and W. R. Dukelow. Jaurnal of’Medical Primatology (in press), 1976. Abstracts - See the following pages for texts of abstracts. 94 Fed. Proc. 32:283A, 1973 OVULATION-MENSTRUAL CYCLE RELATIONSHIPS IN MACAO/1 FASCICULARISI J. M. R. Rawson, T. J. Kuehl, and W. R. Dukelow Endocrine Research Unit, Michigan State University East Lansing, Michigan 48823 Using laparosc0py in nonhuman primates facilitates the observation of follicular development to the moment of rupture and cumulus mass extrusion and also the study of such temporal relationships as the lengths of the follicular and luteal phases of the cycle. 115 cycles were observed in 17 regularly cycling ML fascicularis. The mean cycle length was 30.81:l.0 day with a median and mode of 30 days. In 78 cycles where follicular development was seen, 38 (48.7%) occurred on the left ovary and 40 (51.3%) on the right. In 19 cases where the time of ovulation was defined within a 24 hr. period, the follicular phase was 14.0::l.l days and the luteal phase was 15.61:l.8 days. In 5 animals where ovulation was observed during paired consecutive cycles, 63.6% had ovulations occurring on opposite ovaries compared with 36.4% on the same ovary. In 3 animals where ovulation was observed, adhesion of the cumulus mass to the ovarian surface was noted. Laparoscopy and Sernylan anesthesia did not affect normal ovulation. One animal, sub- jected to anesthesia and venapuncture for 36 consecutive days and 1Presented at the Fed. of Amer. Soc. for Exp. Biol. meeting, Atlantic City, New Jersey, April 15-20, 1973. 95 96 several laparoscopies, ovulated, conceived on Day 16 of the cycle (Day 3 of anesthesia and v.p.), and delivered normally on Day 169 of gestation. A RESTRAINT DEVICE FOR SEMEN COLLECTION OF SQUIRREL MONKEYS (SA IMIRI SCUIREUS) 1 F. E. Howe, T. J. Kuehl, and W. R. Dukelow Center for Laboratory Animal Resources Michigan State University East Lansing, Michigan 48823 A restraint device designed to allow a technique to capture, restrain and collect semen from a squirrel monkey without assistance. Male squirrel monkeys are effectively restrained using this device with very little danger of biting the handler and no danger of injury to the animal during the electroejaculation procedure. Other procedures such as oral dosing, collecting blood, tuberculin testing and taking rectal temperature can be performed. The construction of this device is simple and inexpensive. Material such as wood and foam rubber are used. A more permanent material such as acrylic plastic could be used in place of the wood. (Supported by NIH Career Development Award No. l-KY-HD35, 306-01.) 1Presented at the 24th Annual Mtg. Amer. Assoc. for Lab. Anim. Sci., Miami Beach, Florida, October 1-5, 1973. 97 Fed. Proc. 33, 281A, 1974 OVULATION INDUCTION AND IN VITRO FERTILIZATION IN SQUIRREL MONKEYSl T. J. Kuehl and W. R. Dukelow Endocrine Research Unit, Michigan State University East Lansing, Michigan 48823 Captive squirrel monkeys (saimiri sciureus) show a seasonal response to a subtle ovulation induction regime of 5 days progesterone, 4 days FSH (1 mg) and a dose of HCG (500 i.u.), the minimal response occurring from July to September. A 3)(3 factorial design with 18 adult female monkeys was used to determine the effects of increasing doses of HCG (500, 1000 or 1500 i.u.) and increasing dose and duration of FSH (1 mg for 4 days, 2 mg for 4 days, or 1 mg for 5 days) on ovula- tion between early July and mid-October. An increase in either dose or duration of FSH significantly increased the ovulation. Increasing HCG was ineffective. For in vitro fertilization adult monkeys were induced to ovulate without regard to natural cycle. Laparotomies were 4 to 12 hr after HCG and follicular oocytes were recovered in 80% TC-l99 and 20% fetal calf serum. Semen ejaculates were mixed in the medium and the sperm suspension and oocytes cultured at 37°C in a 5% COZ/air environ- ment. Of 654 follicles aspirated, 155 oocytes were recovered. Fifty 1Presented at the Fed. of Amer. Soc. for Exp. Biol. meeting, Atlantic City, New Jersey, April 7-12, 1974. 98 99 developed to the first polar body stage and 14 of the 50 were fertilized (28%). Seven of the 14 fertilized ova reached the 2-polar body, 2 pronucleate stage and the rest were 2 or more cells. NONHUMAN PRIMATE IN VITRO FERTILIZATION AND EMBRYO TRANSFER: THEIR RELATIONSHIP TO BIRTH DEFECT RESEARCH T. J. Kuehl, D. E. Wildt, and W. Richard Dukelow Endocrine Research Unit, Michigan State University East Lansing, Michigan 48823, USA Because of the adaptability of Saimiri sciureus to laboratory procedures and the background of information available on this species relating to ovulation and semen collection, we have developed a system capable of allowing in vitro fertilized S. sciureus ova to develop to at least the four-cell stage. We collect semen by electroejaculation and divide the seminal coagulum for dissolution in culture-slide compartments. The fertiliza- tion medium (TC-199) includes 20% agamma fetal calf serum. To date we have been able to culture sperm and ova for periods up to 48 hours with strong sperm motility either in solution or when attached to the zona pellucida. Follicular growth is stimulated by a regime of 1 mg FSH daily for 4 days followed by 500 i.u. HCG, i.m. This is an adaptation of our previously published regime for ovulation in this species during the period from October through June. During the other 3 months it is necessary to either double the amount of FSH injected or extend administration to 5 days. 1Presented at the Jerusalem Conference on Primatology, Jerusalem, Israel, Feb. 24-26, 1975. 100 101 For 38 animals, 273 follicular oocytes were recovered by aspirating 654 follicles for an average recovery of 42% or 7.2 oocytes per animal. 0f the aspirated oocytes, 20% were found to be fragmented. The remaining 247 oocytes were incubated in 65 cultures and 50 developed to the first polar body stage. Fourteen of the 50 matured ova (28%) were determined to be fertilized and ranged in development between two polar body, two pronucleate and four-cell embryos. In vitro fertilization offers the research worker many unique opportunities for investigation of drug effects on the sperm, ovum and embryo during the preimplantation stages of pregnancy. Since it is at this time that cytogenetic damage frequently occurs, the technique has special teratological application. Recent discoveries relating to different karyotypes 0f the subspecies of scimiri, coupled with successful in vitro fertilization and embryo transfer, provide valuable tools for studies of genetic damage and developmental effects. SQUIRREL MONKEY FOLLICULAR OOCYTE RECOVERY, IN VITRO FERTILIZATION, AND EMBRYO TRANSFER VIA THE LAPAROSCOPE1 T. J. Kuehl and W. R. Dukelow Endocrine Research Unit, Michigan State University East Lansing, Michigan 48823 The squirrel monkey (Saimiri sciureus) was chosen for this study of in vitro fertilization and embryo transfer in nonhuman primates because of the background of information available on this species relating to ovulation and semen collection. Semen was collected by electroejaculation and the seminal coagulum was incubated to obtain a sperm suspension for distribution to culture/slide compartments. The culture medium (TC-199 or Ham's F-lO) included 20% agamma calf serum. Follicular growth was stimulated in females by a regime of 1 mg FSH daily for 4 days followed by 500 iu. HCG, i.m. During the summer months FSH administration was extended to 5 days. Oocytes were recovered for culture 8 to 12 hours after HCG administration by follicular aspiration techniques employing either laparotomy or laparoscopy. The oocyte along with the associated cumulus mass and follicular fluid were distributed to culture/slide chambers according to their follicular sources. The ova in culture were observed at intervals for evidence of fertilization. At varying stages following fertilization the ova were removed from 1Presented at the 8th Ann. Mtg. of the Soc. for Study of Reprod., Fort Collins, Colorado, July 22-25, 1975. 102 103 culture and transferred to the oviducts of synchronized recipient females using a laparoscopic technique. The recipient females with transferred embryos were then monitored for pregnancy and gestational changes. Oocytes recovered from follicles of gonadotropin-treated females and cultured in vitro with spermatozoa resulted in fertilized ova capable of developing to the eight-cell stage in culture. In total 559 follicular oocytes were recovered from 1617 follicles for a 34.6% average recovery. Of these ova 19.7% were atretic at the beginning of the culture period. Of the remaining 449 ova, 34.7% matured to the metaphase 11 stage and 51 of these 156 mature ova were fertilized in vitro. Ova retaining cumulus and corona radiata vestments showed a higher tendency to mature (62% maturing) when compared to ova without such vestments (7% maturing). Two of 3 females receiving embryo transfers at the 2-cell stage had an enlarged uterus 60 days after transfers. However, at 90 days the females were nonpregnant. IN VITRO FERTILIZATION OF NONHUMAN PRIMATES1 W. R. Dukelow and T. J. Kuehl Endocrine Research Unit, Michigan State University East Lansing, Michigan 48824 A total of 604 oocytes from 1754 squirrel monkey ovarian follicles were recovered (34.4%) for use in in vitro fertilization trials. Of these, 119 (19.7%) were atretic. Of the remainder, 36.1% matured to the metaphase II stage and 58 (33.1%) were fertilized in vitro. Cleavage was noted to the eight-cell stage. Two-cell ova were transferred to the fimbria of recipient females with two animals showing preliminary signs of pregnancy but failing to complete pregnancy. All procedures of ovum recovery and transfer of two-cell embryos were done laparoscopically to avoid major surgery with the animals. 1Presented at the Colloquium of the French Nat. Soc. for the Study of Sterility and Fertility, Paris, France, December 11, 1975. 104 Name: Born: Birthplace: Formal Education: Degrees Received: Member of: Honors: VITA THOMAS JOHN KUEHL February 7, 1950 Marshfield, Wisconsin Rockford West High School Rockford, Illinois Michigan State University East Lansing, Michigan Bachelor of Science (with honors) Michigan State University, 1972 Master of Science Michigan State University, 1974 International Primatological Society American Association for the Advancement of Science Recipient of the Sigma Xi Award for Outstanding Graduate Research, 1975. 105 1111111 88 111111111111 111111111111 0 5 4 1 3 o 3 9 312